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Supporting Information

Conditional Guide RNAs:Programmable Conditional Regulation of CRISPR/Cas Function

in Bacterial and Mammalian Cells via Dynamic RNA NanotechnologyMikhail H. Hanewich-Hollatz†,§, Zhewei Chen†,§, Lisa M. Hochrein†, Jining Huang†, and Niles A. Pierce†,‡,¶,⇤

ContentsS1 Methods S4

S1.1 Rational design of libraries of orthogonal cgRNAs using NUPACK . . . . . . . . . . . . . . . . . . . . . . . S4S1.1.1 Target test tube specification for terminator switch mechanism . . . . . . . . . . . . . . . . . . . . . . S5S1.1.2 Target test tube specification for splinted switch mechanism . . . . . . . . . . . . . . . . . . . . . . . S7S1.1.3 Target test tube specification for toehold switch mechanism . . . . . . . . . . . . . . . . . . . . . . . S9

S1.2 Methods for bacterial studies in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S11S1.2.1 Plasmid construction and molecular cloning for bacterial cgRNA studies . . . . . . . . . . . . . . . . S11S1.2.2 Bacterial culture and silencing assay for cgRNA studies . . . . . . . . . . . . . . . . . . . . . . . . . S11S1.2.3 Flow cytometry for bacterial cgRNA studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S11

S1.3 Methods for mammalian studies in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S13S1.3.1 Plasmid construction and molecular cloning for mammalian cgRNA studies . . . . . . . . . . . . . . . S13S1.3.2 Mammalian cell culture and induction assay for cgRNA studies . . . . . . . . . . . . . . . . . . . . . S14S1.3.3 Flow cytometry for mammalian cgRNA studies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S15

S1.4 Quantitative fluorescence analysis for E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S16S1.4.1 Measuring signal in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S16S1.4.2 Fold change for constitutively active cgRNAs (ON!OFF logic) with silencing dCas9 in E. coli . . . . S16S1.4.3 Dynamic range for constitutively active cgRNAs (ON!OFF logic) with silencing dCas9 in E. coli . . . S16S1.4.4 Fold change for constitutively inactive cgRNAs (OFF!ON logic) with silencing dCas9 in E. coli . . . S17S1.4.5 Dynamic range for constitutively inactive cgRNAs (OFF!ON logic) with silencing dCas9 in E. coli . . S17S1.4.6 Fractional dynamic range for cgRNAs with silencing dCas9 in E. coli . . . . . . . . . . . . . . . . . . S17S1.4.7 Crosstalk for orthogonal cgRNAs in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S17

S1.5 Quantitative fluorescence analysis for HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S18S1.5.1 Measuring signal in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S18S1.5.2 Fold change for constitutively active cgRNAs (ON!OFF logic) with inducing dCas9 in HEK 293T cellsS18S1.5.3 Dynamic range for constitutively active cgRNAs (ON!OFF logic) with inducing dCas9 in HEK 293T

cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S19S1.5.4 Fractional dynamic range for cgRNAs with inducing dCas9 in HEK 293T cells . . . . . . . . . . . . . S19S1.5.5 Crosstalk for orthogonal cgRNAs in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . S19

S2 Sequences S21S2.1 Sequences for cgRNAs, triggers, and control gRNAs . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S21S2.2 Transcriptional promoter and terminator sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S23S2.3 Genomically incorporated gene sequences (E. coli Ec001) . . . . . . . . . . . . . . . . . . . . . . . . . . . . S23

S3 Plasmids S25S3.1 Constitutively active terminator switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S25S3.2 Constitutively active splinted switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S28S3.3 Constitutively inactive toehold switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S31S3.4 pdCas9+lacI in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S34S3.5 Constitutively active terminator switch in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . S36†Division of Biology & Biological Engineering, California Institute of Technology, Pasadena, CA 91125, USA. ‡Division of Engineering & Applied

Science, California Institute of Technology, Pasadena, CA 91125, USA. ¶Weatherall Institute of Molecular Medicine, University of Oxford, Oxford OX3 9DS,UK. §Authors contributed equally. ⇤Email: [email protected]

S1

S4 Schematics of putative ON and OFF states S43S4.1 Constitutively active terminator switch cgRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S43S4.2 Constitutively active splinted switch cgRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S43S4.3 Constitutively inactive toehold switch cgRNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S44

S5 Flow cytometry replicates S45S5.1 Constitutively active terminator switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S45

S5.1.1 ON state, OFF state, and conditional response (cf. Figure 2c) . . . . . . . . . . . . . . . . . . . . . . . S45S5.1.2 Orthogonal library studies (cf. Figure 2d) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S45

S5.2 Constitutively active splinted switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S46S5.2.1 ON state, OFF state, and conditional response (cf. Figure 3c) . . . . . . . . . . . . . . . . . . . . . . . S46S5.2.2 Orthogonal library studies (cf. Figure 3d) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S46

S5.3 Constitutively inactive toehold switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S47S5.3.1 ON state, OFF state, and conditional response (cf. Figure 4c) . . . . . . . . . . . . . . . . . . . . . . . S47S5.3.2 Orthogonal library studies (cf. Figure 4d) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S47

S5.4 Constitutively active terminator switch in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . S48S5.4.1 ON state, OFF state, and conditional response (cf. Figure 5b) . . . . . . . . . . . . . . . . . . . . . . S48S5.4.2 Orthogonal library studies (cf. Figure 5c) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S49

S5.5 Quantifying ON state, OFF state, fold change, and dynamic range . . . . . . . . . . . . . . . . . . . . . . . . S50S5.6 Quantifying crosstalk for cognate and non-cognate cgRNA/trigger pairs . . . . . . . . . . . . . . . . . . . . . S51

S6 Additional Studies S52S6.1 Constitutively active terminator switch using alternative plasmid layout and constitutive trigger expression in

E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S52S6.1.1 Plasmids used for alternative terminator switch plasmid layout (cf. Section S3.1) . . . . . . . . . . . . S54S6.1.2 Flow cytometry replicates for ON state, OFF state, and conditional response (cf. Figure 2c and Sec-

tion S5.1.1) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S57S6.1.3 Flow cytometry replicates for orthogonal library studies (cf. Figure 2d and Section S5.1.2) . . . . . . . S57

S6.2 Single and double sequence inserts for construction of allosteric cgRNAs in E. coli . . . . . . . . . . . . . . . S58S6.2.1 Quantifying performance of candidate cgRNAs using 71 E. coli strains . . . . . . . . . . . . . . . . . S58S6.2.2 Candidate cgRNA and trigger sequences used for single and double insert studies . . . . . . . . . . . . S61

S6.3 Characterization of splinted switch conditional response to lacI-regulated trigger expression at different timepoints in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S64

List of FiguresS1 Target test tubes for sequence design of orthogonal terminator switch cgRNAs . . . . . . . . . . . . . . . . . . S5S2 Nucleotide defect weights for sequence design of terminator switch cgRNAs . . . . . . . . . . . . . . . . . . . S6S3 Target test tubes for sequence design of orthogonal splinted switch cgRNAs . . . . . . . . . . . . . . . . . . . S7S4 Nucleotide defect weights for sequence design of splinted switch cgRNAs . . . . . . . . . . . . . . . . . . . . S8S5 Target test tubes for sequence design of orthogonal toehold switch cgRNAs . . . . . . . . . . . . . . . . . . . S10S6 Nucleotide defect weights for sequence design of toehold switch cgRNAs . . . . . . . . . . . . . . . . . . . . S10S7 Illustration of gates used for flow cytometry analysis of E. coli. . . . . . . . . . . . . . . . . . . . . . . . . . . S12S8 Illustration of gates used for flow cytometry analysis of HEK 293T cells. . . . . . . . . . . . . . . . . . . . . . S15S9 Example plasmid map for terminator switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S26S10 Example annotated plasmid sequence for terminator switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . S27S11 Example plasmid map for splinted switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S29S12 Example annotated plasmid sequence for splinted switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . S30S13 Example plasmid map for toehold switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S32S14 Example annotated plasmid sequence for toehold switch in E. coli . . . . . . . . . . . . . . . . . . . . . . . . S33S15 Plasmid map for pdCas9+lacI in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S34S16 Annotated plasmid sequence for expression of pdCas9+lacI in E. coli . . . . . . . . . . . . . . . . . . . . . . S35S17 Example plasmid map for terminator switch cgRNA in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . S37S18 Example annotated plasmid sequence for terminator switch cgRNA in HEK 293T cells . . . . . . . . . . . . . S38S19 Example plasmid map for terminator switch trigger in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . S39S20 Example annotated plasmid sequence for terminator switch trigger in HEK 293T cells . . . . . . . . . . . . . . S40S21 Plasmid map for induction assay reporter in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . S41

S2

S22 Annotated plasmid sequence for induction assay reporter in HEK 293T cells . . . . . . . . . . . . . . . . . . . S42S23 Schematics of putative ON and OFF states for terminator switch mechanism . . . . . . . . . . . . . . . . . . . S43S24 Schematics of putative ON and OFF states for splinted switch mechanism . . . . . . . . . . . . . . . . . . . . S43S25 Schematics of putative OFF and ON states for toehold switch mechanism . . . . . . . . . . . . . . . . . . . . S44S26 Flow cytometry replicates for terminator switch ON state, OFF state, and conditional response in E. coli (cf.

Figure 2c) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S45S27 Flow cytometry replicates for terminator switch orthogonal response in E. coli (cf. Figure 2d) . . . . . . . . . . S45S28 Flow cytometry replicates for splinted switch ON state, OFF state, and conditional response in E. coli (cf.

Figure 3c) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S46S29 Flow cytometry replicates for splinted switch orthogonal response in E. coli (cf. Figure 3d) . . . . . . . . . . . S46S30 Flow cytometry replicates for toehold switch ON state, OFF state, and conditional response in E. coli (cf. Figure

4c) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S47S31 Flow cytometry replicates for toehold switch orthogonal response in E. coli (cf. Figure 4d) . . . . . . . . . . . S47S32 Flow cytometry replicates for terminator switch ON state, OFF state, and conditional response in HEK 293T

cells (cf. Figure 5b) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S48S33 Flow cytometry replicates for terminator switch orthogonal response in HEK 293T cells (cf. Figure 5c) . . . . S49S34 Constitutively active terminator switch cgRNAs (ON!OFF logic) using alternative plasmid layout and consti-

tutive trigger expression with silencing dCas9 in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S53S35 Example plasmid map for alternative terminator switch plasmid layout with constitutive trigger expression in

E. coli (cf. Figure S9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S55S36 Example annotated plasmid sequence for alternative terminator switch plasmid layout with constitutive trigger

expression in E. coli (cf. Figure S10) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S56S37 Flow cytometry replicates for terminator switch ON state, OFF state, and conditional response in E. coli with

constitutive trigger expression (cf. Figure S26) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S57S38 Flow cytometry replicates for terminator switch orthogonal response in E. coli with constitutive trigger expres-

sion (cf. Figure S27) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S57S39 Performance of single and double sequence inserts for construction of allosteric cgRNAs in E. coli . . . . . . . S59S40 Characterization of splinted switch response to lacI-regulated trigger expression at different time points in E. coliS64

List of TablesS1 Example duplex fragments for Golden Gate assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S13S2 Mammalian induction assay components . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S14S3 Terminator switch sequences for studies in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S21S4 Splinted switch sequences for studies in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S21S5 Toehold switch sequences for studies in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S22S6 Terminator switch sequences for studies in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . . . . . . . S22S7 Control gRNA sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S22S8 Transcriptional promoter and terminator sequences . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S23S9 Plasmids used with terminator switch cgRNAs in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S25S10 Plasmids used with splinted switch cgRNAs in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S28S11 Plasmids used with toehold switch cgRNAs in E. coli . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S31S12 Plasmids used with terminator switch cgRNAs in HEK 293T cells . . . . . . . . . . . . . . . . . . . . . . . . S36S13 Quantifying ON state, OFF state, fold change, and dynamic range (cf. Figures 2d, 3d, 4d, 5c) . . . . . . . . . . S50S14 Quantifying crosstalk for cognate and non-cognate cgRNA/trigger pairs (cf. Figures 2d, 3d, 4d, 5c) . . . . . . . S51S15 Plasmids used for alternative terminator switch plasmid layout with constitutive trigger expression in E. coli

(cf. Table S9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S54S16 Quantifying ON state, OFF state, fold change, and dynamic range for candidate cgRNAs with designed single

and double inserts into the standard gRNA structure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . S60S17 Candidate cgRNA sequences used for single and double insert studies in E. coli . . . . . . . . . . . . . . . . . S62S18 Trigger sequences used for single and double insert studies in E. coli . . . . . . . . . . . . . . . . . . . . . . . S63

S3

S1 Methods

S1.1 Rational design of libraries of orthogonal cgRNAs using NUPACKFor each mechanism, orthogonal cgRNA/trigger pairs were designed using the reaction pathway engineering tools withinNUPACK (nupack.org; see the NUPACK 3.2 User Guide).1, 2 Target test tubes were specified using the general formulationof Section S2.2.1 in the Supplementary Information of Wolfe et al.2 using the definitions provided below (Section S1.1.1 forthe terminator switch, Section S1.1.2 for the splinted switch, and Section S1.1.3 for the toehold switch). Sequence designswere performed for libraries of 4 orthogonal cgRNA/trigger pairs. For a given design trial, the sequences were optimized bymutating the sequence set to reduce the multi-tube ensemble defect2 subject to the diverse sequence constraints detailed below.Within the ensemble defect, defect weights (see Section S1.6 in the Supplementary Information of Wolfe et al.2) were appliedto prioritize design effort as described below. Designs were performed using RNA parameters for 37 ◦C in 1M Na+.3 Afterperforming several independent design trials for a given mechanism, a final sequence set was selected for experimental testingbased on inspection of the predicted structural defects (fraction of nucleotides in the incorrect base-pairing state within theensemble of an on-target complex) and concentration defects (fraction of nucleotides in the incorrect base-pairing state becausethere is a deficiency in the concentration of an on-target complex) for species in the context of the target test tubes,2, 4 as wellas for each cgRNA in the presence of each non-cognate trigger (e.g., computational orthogonality study of Figure 6b (right)).After preliminary experimental studies, 3 cgRNA/trigger pairs (termed A, B, C for bacterial studies and Q, R, S for mammalianstudies) were selected for full experimental characterization.

S4

S1.1.1 Target test tube specification for terminator switch mechanism

To design N orthogonal systems, the total number of target test tubes is |⌦| =P

n=1,...,N {Step 0, Step 1}n + Crosstalk =2N + 1; the target test tubes in the multi-tube ensemble, ⌦, are indexed by h = 1, . . . , |⌦|. Lmax = 2 for all tubes (i.e., eachtarget test tube contains all off-target complexes of up to 2 strands). Final sequence designs for orthogonal cgRNA/trigger pairsA, B, C are shown in Table S3 for bacterial studies and Q, R, S are shown in Table S6 for mammalian studies.

Reactants for system n

• cgRNAs: Gn

• Triggers: Xn

Elementary step tubes for system n

• Step 0n tube: products

0n⌘ {G, X}n; reactants

0n⌘ ;; exclude

0n⌘ {G·X}n


1n⌘ {G·X}n; reactants

1n⌘ {G, X}n; exclude

1n⌘ ;

Global crosstalk tube

• Crosstalk tube: reactive

global⌘ [n=1,...,N{reactive

n }; crosstalk

global⌘ LLmax

global− [n=1,...,N{cognate

n }

The reactive species and cognate products for system n are:

• simplen ⌘ {G, X}n

• ss-outn ⌘ Xn

• ss-inn ⌘ Gss

n , the 30nt single stranded terminator loop insert domain• reactive

n ⌘ {G, X, Gss}n• cognate

n ⌘ {G·X, Gss·X}n

Step 0n Step 1n

Gn

10nM

Xn

10nM

10nM

Gn·Xn

Gn

Xn

10nM

10nM

10nM

Global Crosstalk Tube

Constrained by target geneConstrained by dCas9Constrained by synthetic terminator

Elementary Step Tubes

n=1,...,N

Sequence Constraints

Gssn

Complementarity between Gn and Xn

Tube On-targets ( onh ) Off-targets ( o↵

h )

Step 0n {G, X}n LLmax

0n− {G·X}n

Step 1n {G·X}n {G, X}n [ LLmax

1n

Crosstalk [n=1,...,N{reactiven } LLmax

global − [n=1,...,N{cognaten }

Figure S1: Target test tubes for sequence design of orthogonal terminator switch cgRNAs. Top: Target test tube schematics. Bottom:Target test tube details. Each target test tube contains the depicted on-target complexes (each with the depicted target structure and a targetconcentration of 10 nM) and the off-target complexes listed in the table (each with vanishing target concentration). The on-target structuresdepicted above are used in the mechanism schematic of Figure 2b. To simultaneously design N orthogonal systems, the total number of targettest tubes is |⌦| = 2N + 1. Lmax = 2 for all tubes. Domain shading reflects sequence constraints. Design conditions: RNA in 1 M Na+ at37 ◦C.

S5

Sequence constraints

• Assignment constraints: portions of the cgRNA are constrained to match standard gRNA sequences for use with dCas9(shaded gray in Figure 2b, Figure S1, and Tables S3 and S6), the synthetic terminators for the cgRNA and trigger are fullyconstrained (shaded tan in Figure 2b, Figure S1, and Tables S3 and S6).

• Watson–Crick constraints: cgRNA sequence domains “d-e-f” are constrained to be complementary to the trigger sequencedomains “f*-e*-d*” (shaded blue in Figure 2b, Figure S1, and Tables S3 and S6).

• Assignment constraint: cgRNA domain “u” is constrained to be complementary to a subsequence of the target gene mRFP(full template sequence in Section S2.3, constrained sequence shaded orange in Figure 2b, Figure S1, and Tables S3 andS6).

• Pattern prevention constraints: for the bacterial cgRNA mechanisms (cgRNA A, cgRNA B, cgRNA C) the following pat-terns are prevented for cgRNA sequence domain “f”: AAAA, CCCC, GGGG, UUUU. For the mammalian cgRNA mech-anisms (cgRNA Q, cgRNA R, cgRNA S) the following patterns are prevented for cgRNA sequence domains “d”, “e”, and“f”: AAAA, CCCC, GGGG, UUUU, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY.

Defect weights

• Test tube weight for each elementary step tube: 1• Test tube weight for global crosstalk tube: 4• Nucleotide weights are depicted for each complex in Figure S2

Gn Xn Gss Gn·Xn

w = 1

w = 1.5

w = 3

w = 1

w = 1 w = 3

w = 1

w = 3

w = 0

w = 0

w = 3

w = 0

w = 0

w = 0 Constrained by target geneConstrained by dCas9Constrained by synthetic terminatorComplementarity between Gn and Xn

n

Figure S2: Nucleotide defect weights for sequence design of terminator switch cgRNAs. Within the target test tubes of Figure S1, thenucleotides in a given sequence domain within a given complex are assigned a defect weight w as depicted.

S6

S1.1.2 Target test tube specification for splinted switch mechanism

To design N orthogonal systems, the total number of target test tubes is |⌦| =P

n=1,...,N {Step 0, Step 1}n + Crosstalk =2N + 1; the target test tubes in the multi-tube ensemble, ⌦, are indexed by h = 1, . . . , |⌦|. Lmax = 2 for all tubes (i.e., eachtarget test tube contains all off-target complexes of up to 2 strands). Final sequence designs for orthogonal cgRNAs/triggerpairs A, B, C are shown in Table S4.


• cgRNAs: Gn

• Triggers: Xn



0n⌘ {G, X}n; reactants

0n⌘ ;; exclude

0n⌘ {G·X}n



1n⌘ {G, X}n; exclude

1n⌘ ;

Global crosstalk tube

• Crosstalk tube: reactive

global⌘ [n=1,...,N{reactive

n }; crosstalk

global⌘ LLmax

global− [n=1,...,N{cognate

n }

The reactive species and cognate products for system n are:

• simplen ⌘ {G, X}n

• ss-outn ⌘ Xn

• ss-inn ⌘ Gss

n , the 35nt single stranded handle and terminator loop insert domains with intervening gRNA sequence• reactive

n ⌘ {G, X, Gss}n• cognate

n ⌘ {G·X, Gss·X}n

Step 0n

Global Crosstalk Tube

Step 1n

Gn

10nM

Xn

10nM

10nM

Gn·XnGn

Xn10nM

10nM

10nM

Constrained by target geneConstrained by dCas9Constrained by synthetic terminatorComplementarity between Gn and Xn

Gssn

Elementary Step Tubes

n=1,...,N

Sequence Constraints


h )

Step 0n {G, X}n LLmax

0n− {G·X}n


1n

Crosstalk [n=1,...,N{reactiven } LLmax

global − [n=1,...,N{cognaten }

Figure S3: Target test tubes for sequence design of orthogonal splinted switch cgRNAs. Top: Target test tube schematics. Bottom:Target test tube details. Each target test tube contains the depicted on-target complexes (each with the depicted target structure and a targetconcentration of 10 nM) and the off-target complexes listed in the table (each with vanishing target concentration). The on-target structuresdepicted above are used in the mechanism schematic of Figure 3b. To simultaneously design N orthogonal systems, the total number of targettest tubes is |⌦| = 2N + 1. Lmax = 2 for all tubes. Domain shading reflects sequence constraints. Design conditions: RNA in 1 M Na+ at37 ◦C.

S7


• Assignment constraints: portions of the cgRNA are constrained to match standard gRNA sequences for use with dCas9(shaded gray in Figure 3b, Figure S3, and Table S4), the synthetic terminator for the trigger is fully constrained (shaded tanin Figure 3b, Figure S3, and Table S4).

• Watson–Crick constraints: cgRNA sequence domains “d” and “e” are constrained to be complementary to the triggersequence domains “d*” and “e*” (shaded blue in Figure 3b, Figure S3, and Table S4).

• Assignment constraint: cgRNA domain “u” is constrained to be complementary to a subsequence of the target gene sfGFP(full template sequence in Section S2.3, constrained sequence shaded orange in Figure 3b, Figure S3, and Table S4).

• Pattern prevention constraints: the following patterns are prevented for cgRNA sequence domains “d” and “e”: AAAA,CCCC, GGGG, UUUU.

Defect weights

• Test tube weight for each elementary step tube: 1• Test tube weight for global crosstalk tube: 4• Nucleotide weights are depicted for each complex in Figure S4

w = 1

Gn Xn Gss Gn·Xn

w = 1.5w = 1.5

w = 3

w = 3w = 1

w = 1

w = 1

w = 3

w = 3

w = 3

w = 0

w = 0

w = 0

w = 3

w = 3 w = 3 w = 3

w = 0

w = 0

w = 0

n


Figure S4: Nucleotide defect weights for sequence design of splinted switch cgRNAs. Within the target test tubes of Figure S3, thenucleotides in a given sequence domain within a given complex are assigned a defect weight w as depicted.

S8

S1.1.3 Target test tube specification for toehold switch mechanism

Computational sequence design of the toehold switch mechanism was performed using a previous version of NUPACK that didnot yet support exclusion of a set of complexes from a target test tube ensemble. For example, to design N orthogonal systems,the test tube specifications detailed in the bottom tables of Figures S1 and S3 use the “minus” operator to enable compactspecification of one Reactants tube for each system n = 1, . . . , N and a single Global Crosstalk tube. Lacking the minusoperator at the time the toehold switch library was designed, we used a more verbose target test tube specification with separateReactants tubes for cgRNA and trigger for each system n = 1, . . . , N , as well as one Crosstalk tube for each non-cognatecgRNA/trigger pair. To design N orthogonal systems, the total number of target test tubes is:

|⌦| =X

n=1,...,N

{Step 0G, Step 0X, Step 1}n +X

n = 1, . . . , Np = 1, . . . , N

p 6= n

Crosstalkp,n = 3N +N(N − 1) = N2 + 2N

The target test tubes in the multi-tube ensemble, ⌦, are indexed by h = 1, . . . , |⌦|. Lmax = 2 for all tubes (i.e., each target testtube contains all off-target complexes of up to 2 strands). Final sequence designs for orthogonal cgRNAs/trigger pairs A, B, Care shown in Table S5.


• cgRNAs: Gn

• Triggers: Xn


• Step 0Gn tube: products

0n⌘ Gn; reactants

0n⌘ ;

• Step 0Xn tube: products

0n⌘ Xn; reactants

0n⌘ ;



1n⌘ {G, X}n

Crosstalk tubes for system n

• Crosstalk tubes: products

crosstalkp, n⌘ {Gn, Xp}; reactants

crosstalkp, n⌘ ;, for each non-cognate cgRNA/trigger pair (p 6= n)


• Assignment constraints: portions of the cgRNA are constrained to match standard gRNA sequences for use with dCas9(shaded gray in Figure 4b, Figure S5 and Table S5), the synthetic terminator for the trigger is fully constrained (shaded tanin Figure 4b, Figure S5 and Table S5).

• Watson–Crick constraints: cgRNA sequence domain “d” is constrained to be complementary to the trigger sequence domain“d*” (shaded blue in Figure 4b, Figure S5 and Table S5).

• Assignment constraint: cgRNA domain “u” is constrained to be complementary to a subsequence of the target gene mRFP(full template sequence in Section S2.3, constrained sequence shaded orange in Figure 4b, Figure S5 and Table S5).

• Pattern prevention constraints: the following patterns are prevented for cgRNA sequence domain “d”: AAAA, CCCC,GGGG, UUUU, KKKKKK, MMMMMM, RRRRRR, SSSSSS, WWWWWW, YYYYYY.

Defect weights

• Test tube weight for each elementary step tube: 1• Test tube weight for crosstalk tubes: 1• Nucleotide weights are depicted for each complex in Figure S6

S9

Step 0Xn Crosstalkp,nStep 1n

Xn

10nM

10nM

Gn·Xn

Step 0Gn

Gn

10nMGn

20nM

Xp

200nM

Crosstalk Tubes

Constrained by target geneConstrained by dCas9Constrained by synthetic terminator

Sequence ConstraintsElementary Step Tubes

Complementarity between Gn and Xn


h )

Step 0Gn Gn LLmax

0Gn

Step 0Xn Xn LLmax

0Xn


1n

Crosstalkp,n {Gn, Xp} {Gn·Xp} [ LLmax

crosstalkp,n

Figure S5: Target test tubes for sequence design of orthogonal toehold switch cgRNAs. Top: Target test tube schematics. Bottom:Target test tube details. Each target test tube contains the depicted on-target complexes (each with the depicted target structure and targetconcentration) and the off-target complexes listed in the table (each with vanishing target concentration). The on-target structures depictedabove are used in the mechanism schematic of Figure 4b. To simultaneously design N orthogonal systems, the total number of target testtubes is |⌦| = N2 + 2N . Lmax = 2 for all tubes. Domain shading reflects sequence constraints. Design conditions: RNA in 1 M Na+ at 37◦C.

Gn Xn Gn·Xn

w = 200 w = 200

w = 200 w = 200

w = 200

w = 200

w = 1

w = 50 (4nt loop)

w = 100

w = 100

w = 300

w = 25

w = 200

w = 200

w = 1

w = 50 w = 300

w = 1


Figure S6: Nucleotide defect weights for sequence design of toehold switch cgRNAs. Within the target test tubes of Figure S5, thenucleotides in a given sequence domain within a given complex are assigned a defect weight w as depicted.

S10

S1.2 Methods for bacterial studies in E. coliS1.2.1 Plasmid construction and molecular cloning for bacterial cgRNA studies

Sequences for parts used in bacterial studies are provided in Section S2. Plasmid layouts for each construct as well as exampleplasmid maps and corresponding full sequences are provided in Sections S3.1-S3.4.

Control gRNA and cgRNA constructs were generated by inverse PCR, inserting sequence modifications into the previouslydescribed pgRNA-bacteria vector5 (Addgene plasmid #44251; gift from S. Qi). All PCR steps for the generation of exper-imental constructs were performed using Q5 Hot Start High-Fidelity polymerase (NEB #M0494) according to manufacturerinstructions using primers designed using standard molecular cloning techniques and synthesized by Integrated DNA Technolo-gies. Introduced sequences were verified by Sanger sequencing for single colony picks via colony PCR using GoTaq Greenpolymerase (Promega #M7122).

For terminator switch cgRNAs (Figure 2), trigger-expressing constructs were generated by cloning lacI-regulated promoter(BioBrick part number BBa R0011), trigger template, and synthetic terminator (BBa B1002) directly into the cgRNA vectorvia inverse PCR. For splinted switch cgRNAs (Figure 3), trigger-expressing constructs were generated by first cloning syn-thetic promoter, trigger template, and synthetic terminator (BBa B1006) into a trigger-only cassette via inverse PCR, and thencgRNA+trigger expressing constructs were cloned by inserting trigger cassette into the cgRNA vector using BioBrick assem-bly.6, 7 For toehold switch cgRNAs (Figure 4), trigger-expressing constructs were generated by first cloning synthetic promoter,trigger template, and synthetic terminator (BBa B0050) into a trigger-only cassette via inverse PCR, and then cgRNA+triggerexpressing constructs were cloned by inserting trigger cassette into the cgRNA vector using DNA assembly according to man-ufacturer instructions (NEBuilder HiFi DNA Assembly, NEB #E2621).

A lacI+dCas9 expression construct was generated by inserting a lacI template sequence with J23108 constitutive promoter8

into the previously described pdCas9-bateria vector5 (Addgene plasmid #44249; gift from S. Qi) between the dCas9 gene andthe p15A origin with a synthetic terminator (BBa B0010) added upstream of lacI as a transcriptional terminator for dCas9 (seeSection S3.4), using DNA assembly according to manufacturer instructions (NEBuilder HiFi DNA Assembly, NEB #E2621).

S1.2.2 Bacterial culture and silencing assay for cgRNA studies

A previously described E. coli MG1655 strain with constitutively expressed mRFP and sfGFP inserted into the nfsA locus5

(Ec001; gift from S. Qi) was used for all fluorescence assays. For experiments with constitutive expression of trigger, thepreviously described pdCas9-bacteria vector5 (Addgene plasmid #44251) was used for tetR-regulated dCas9 expression. Forexperiments with lacI-regulated expression of trigger, the lacI+dCas9 vector was used for tetR-regulated dCas9 expression andconstitutive expression of lacI. Chemically competent chloramphenicol-resistant cells carrying either the dCas9 or lacI+dCas9construct were transformed with gRNA, cgRNA, or cgRNA+trigger expression vectors and cultivated in EZ-RDM (Teknova#M2105) containing 100 µg/mL carbenicillin and 34 µg/mL chloramphenicol (EZ-RDM+Carb+Cam).

Sequence-verified strains were grown overnight in EZ-RDM+Carb+Cam, then seeded at 100⇥ dilution in 100 µL freshmedium and grown at 37 ◦C with shaking in the Neo2 microplate reader (Biotek) to monitor absorbance at 600 nm. When cellshad reached mid-log phase (⇡4 h), cells were again diluted ⇡100-fold in fresh medium with cell density normalized by A600and, if applicable, dCas9 expression and trigger expression were induced with aTc and IPTG, respectively, in N = 3 replicatewells at 400 µL final volume in a 96-well high-volume glass bottom plate (Matriplate, Brooks #MGB096-1-2-LG-L). A finalworking concentration of 200 nM aTc and 5 mM IPTG was used for terminator switch experiments (Figure 2). A final workingconcentration of 2 nM aTc was used for splinted switch experiments (Figure 3). A final working concentration of 200 nM aTcwas used for toehold switch experiments (Figure 4). Induced cells were grown at 37 ◦C with continuous shaking for 12 h.

S1.2.3 Flow cytometry for bacterial cgRNA studies

Protein fluorescence was measured using the MACSQuant VYB flow cytometer (Miltenyi Biotec) using FSC/SSC to gate for20,000 live cells per well (see example of Figure S7) at a flow rate of 25 µL/min. sfGFP fluorescence was measured using theB1 channel (488 nm laser, 525/50 nm filter) and mRFP fluorescence was measured using the Y2 channel (561 nm laser, 615/20nm filter).

S11

ba

FSC-A

SSC

-A

10-1 101 103 10510-1

101

103

105

FSC-A

SSC

-A

10-1 101 103 10510-1

101

103

105

101 103 105

Fluorescence intensity (au)

0

1000

2000

Cou

nts

No-target gRNA, ungated

101 103 105


0

1000

2000

Cou

nts

No-target gRNA, gated

Figure S7: Illustration of gates used for flow cytometry analysis of E. coli. (a) Scatter plots for ungated sample (top) and gated sample(bottom): side scatter area (SSC-A) vs. forward scatter area (FSC-A). (b) Fluorescence intensity histogram for ungated sample (top) andgated sample (bottom). mRFP fluorescence for no-target gRNA control used for terminator switch characterization in Figure 2c.

S12

S1.3 Methods for mammalian studies in HEK 293T cellsS1.3.1 Plasmid construction and molecular cloning for mammalian cgRNA studies

Sequences for parts used in mammalian studies are provided in Section S2. Plasmid layouts for each construct as well asexample plasmid maps and corresponding full sequences are provided in Section S3.5.

gRNA, cgRNA and trigger constructs were generated via a modified Golden Gate assembly protocol. For cgRNA-expressingplasmids, the pU6 gRNA handle U6t plasmid (gift from T. Lu, Addgene plasmid #49016)9 was mutated to remove the BsaIsite in the ampicillin resistance gene, EBFP2 was replaced with miRFP670 cloned from the pmiRFP670-N1 plasmid (gift fromV. Verkhusha, Addgene plasmid #79987),10 and the gRNA scaffold was replaced with two BsaI sites and two BbsI sites for sub-sequent Golden Gate assembly. For trigger-expressing plasmids, the pU6 gRNA handle U6t plasmid was mutated to removethe BsaI site in the ampicillin resistance gene, and the gRNA scaffold was replaced with two BsaI sites for subsequent GoldenGate assembly.

To avoid synthesis difficulties introduced by the gRNA scaffold secondary structure, gRNAs and cgRNAs were decomposedinto four duplexes with 4-nt sticky ends (see example: Table S1). Triggers were designed to have minimal secondary structureand were synthesized as a single duplex with 4-nt sticky ends. Duplex fragments were ordered as RxnReady duplexes from IDT.Duplexes were phosphorylated with T4 Polynucleotide Kinase (NEB, #M0201) and annealed (90 ◦C for 90 s, cool 1 ◦C permin to 23 ◦C). For gRNA and cgRNAs, the four duplexes were ligated at 150 nM each using T4 DNA ligase (NEB, #M0202).The ligated gRNA/cgRNA or trigger was then cloned into the appropriately modified pU6 gRNA handle U6t plasmid usingthe standard Golden Gate assembly guidelines provided by NEB (https://www.neb.com/protocols/2018/06/05/golden-gate-24-fragment-assembly-protocol).

Duplex Sequence

P1 gRNA (P1 g) duplexesP1-d1 50-CGAAACACCGAGTCGCGTGTAGCGAAGCAGTTTTAGAGCTA-30

|||||||||||||||||||||||||||||||||30- TGTGGCTCAGCGCACATCGCTTCGTCAAAATCT -50

P1-d2 50- GAAATAGCAAGTTAAAATAAGGCTAGTC-30

||||||||||||||||||||||||30-CGATCTTTATCGTTCAATTTTATTCCGA -50

P1-d3 50- CGTTATCAACTTGAAAAAGTGGCA-30

||||||||||||||||||||30-TCAGGCAATAGTTGAACTTTTTCA -50

P1-d4 50- CCGAGTCGGTG -30

|||||||||||30-CCGTGGCTCAGCCACGAAA-50

Trigger duplexTrmS tQ-d1 30-CGAAACACCGAAACATGACAGAAATACGAACGAAACTAACGCGCAAAGAT -50

||||||||||||||||||||||||||||||||||||||||||||||30- TGTGGCTTTGTACTGTCTTTATGCTTGCTTTGATTGCGCGTTTCTAGAAA-50

Table S1: Example duplex fragments for Golden Gate assembly.

S13

S1.3.2 Mammalian cell culture and induction assay for cgRNA studies

Mammalian cgRNA performance was assayed using a modified version of previously described fluorescent protein gene induc-tion assays.11–13 Briefly, four plasmids (Table S2) were co-transfected into HEK 293T cells and CRISPR/dCas9-mediated fluo-rescent protein gene induction was assayed by flow cytometry after 24 h. The SP-dCas9-VPR plasmid was a gift from G. Church(Addgene plasmid #63798).14 The reporter-gT1 plasmid was also a gift from G. Church (Addgene plasmid #47320)11 and wasmodified to replace the T1 gRNA target-binding region with the P1 gRNA target-binding region.12

Plasmid Function Fluorescent protein Parent plasmid ng/well Relative copytransfected number

SP-dCas9-VPR S. pyogenes dCas9 fused to VPR inductiondomains (CMV promoter)

none Addgene 63798 16.4 0.2

reporter-gP1 P1 gRNA binding site upstream of a min-imal CMV promoter expressing dTomatoreporter

dTomato Addgene 47320 233.6 6.8

cgRNA hU6 expression of a single cgRNA miRFP670 (pPGK) Addgene 49016 32.0 1.0trigger hU6 expression of a single trigger EBFP2 (pPGK) Addgene 49016 218.0 7.4

Table S2: Mammalian induction assay components.

HEK 293T (ATCC, #CRL-3216) cells were cultured in high-glucose DMEM (Gibco, #11995073) supplemented with 10%FBS (Gibco, #16140071) and grown at 37 ◦C with 5% CO2. Cells were subcultivated every 2 to 3 days at a ratio of 1:5 to 1:10for a maximum of two months. Plasmids (Table S12) were transiently transfected using Lipofectamine 3000 (Thermo FisherScientific, #L3000) according to manufacturer’s instructions. One day prior to transfection, 24-well plates were seeded with125,000 cells in 0.5 mL medium per well. For each well, a total of 500 ng plasmid was transfected simultaneously (16.4 ngSP-dCas9-VPR plasmid, 233.6 ng reporter-gP1 plasmid, 32 ng gRNA/cgRNA plasmid, and 218 ng trigger plasmid) with 1.5 µLLipofectamine 3000 and 1 µL P3000 reagent. For cgRNA-only studies, the equivalent amount of no-trigger control plasmidwas used in place of the trigger plasmid. 24 hours post transfection, the cells were detached from the plate with 50 µL EDTA-free trypsin (Gibco, #15090046) incubated at 37 ◦C for 5 min. Trypsin was inhibited with 50 µL 1 mg/mL soybean trypsininhibitor (Sigma-Aldrich, #T6522), and 150 uL flow cytometry buffer [2.5 mg/mL fraction V BSA (VWR, #0332), 10 mMpH 7.5 HEPES buffer (Teknova, #H1035), 50 µg/mL DNase1 (Sigma-Aldrich, #D4513), 1 mM MgCl2 (Ambion, #AM9530G)in 1X HBSS (Gibco, #14175)] was added to each well and the entire solution was triturated. From each well, 200 µL of cellsolution was filtered through a 96-well 30–40 µM PP/PE non-woven media filter plate (Pall, AcroPrep Advance Filter Plate,#8027) into a round bottom 96-well plate.

S14

S1.3.3 Flow cytometry for mammalian cgRNA studies

Protein fluorescence was measured with the CytoFLEX S Flow Cytometer (Beckman Coulter) in the Caltech Flow CytometryFacility. SSC-H vs. FSC-H was used to gate for live cells, followed by FSC-H vs. FSC-W to gate for single cells (seeexample in Figure S8). 50,000 live, single cells (or a maximum of 90 µL sample) were collected for each well at a flow rate of60 µL/min. EBFP2 fluorescence (trigger plasmid transfection control) was measured using the 405 nm laser with the 450/45 nmfilter. miRFP670 fluorescence (cgRNA plasmid transfection control) was measured using the 640 nm laser with the 660/20 nmfilter. dTomato fluorescence (target gene Y) was measured using the 561 nm laser with the 585/42 nm filter. Using the standardgRNA, high dTomato expression resulted in saturation of the detector for some cells (see histogram, Figure 5b) despite usingthe lowest gain setting.

Flow cytometry fluorescence values were compensated manually using cells expressing a single fluorescent protein. Todetermine the amount of compensation required, the median channel value of cells within the live, single gates with high fluo-rescent protein signal was matched to the median channel value of cells within the live, single gates with no or low fluorescentprotein signal. Live, single cells were then gated to include only cells with high levels of both fluorescent protein transfectioncontrols (>100,000 AU for EBFP2 and miRFP670 expression), which varied from 1.56% to 11.7% of the sample. Cell countsin this population varied from 426–7714 cells. Bar graphs (Figure 5c) display fluorescence measurements based on all cells inthe highly-transfected gate. Flow cytometry replicates for mammalian cgRNA studies are shown in Section S5.4. To facilitatecomparisons, fluorescence distributions display the same number of cells for all experiment types and replicates for a givencgRNA (Figure 5b, Figure S32 and Figure S33): 3500 for Q, 3000 for R, 2400 for S (with the exception that one experimentfor S had only 415 counts; see Figure S33). Distributions display the last-measured cells (e.g., 3500 for Q) from the highlytransfected gate (after first removing any cells with non-positive fluorescence readings to enable display in a log plot).

Live59.8%

0 200K 400K 600KFSC-H

0

200K

400K

600K

800K

1.0M

SSC

-H

Ungated

Single93.9%

0 1.0K 2.0K 3.0KFSC-W

0

200K

400K

600K

FSC

-H

Live Q17.48%

Q29.17%

Q31.96%

Q481.4%

101 102 103 104 105 106miRFP670 Expression

101

102

103

104

105

106

EBFP

2 Ex

pres

sion

Single

101 103 105 107dTomato Expression

0

500

1.0K

1.5K

Cou

nt

Subset CountUngated 112,408 Live 67,245Single 63,145Q2: miRFP670+, EBFP2+ 5,789

a

b

Figure S8: Illustration of gates used for flow cytometry analysis of HEK 293T cells. (a) Example of progressive gating for one replicate ofthe mammalian terminator switch cgRNA Q (without trigger). First, live cells are gated using side scatter height (SSC-H) vs. forward scatterheight (FSC-H). Live cells are then gated for single cells using forward scatter height (FSC-H) vs. forward scatter width (FSC-W). Finally,only highly transfected cells are included in the analysis (Q2: miRFP670+ and EBFP2+). (b) dTomato fluorescence intensity histogram forungated sample, live-cell gated sample, single-cell gated sample, and [miRFP670+ and EBFP2+] gated sample.

S15

S1.4 Quantitative fluorescence analysis for E. coliS1.4.1 Measuring signal in E. coli

For a bacterial strain containing a fluorescent reporter protein, the total fluorescence (SIG+AF) in the relevant fluorescentchannel (mRFP for the terminator switch of Figure 2, sfGFP for the splinted switch of Figure 3, mRFP for the toehold switch ofFigure 4) is a combination of signal (SIG) from the reporter and autofluorescence (AF) inherent to the cells. Autofluorescenceis characterized for a given fluorescent channel in strain MG1655 containing no fluorescent reporters. For cell j of replicatewell i of a given bacterial strain, we denote the autofluorescence:

XAF

i,j

the signalXSIG

i,j

and the total fluorescence (SIG + AF):XSIG+AF

i,j .

For replicate well i of a given strain, we measure the mean fluorescence (X̄SIG+AF

i for a strain containing the reporter, X̄AF

i forstrain MG1655 lacking reporters) over N = 20, 000 cells. Performance across N = 3 replicate wells is characterized by thesample means (X̄SIG+AF and X̄AF) and estimated standard errors (sX̄SIG+AF and sX̄AF ). Let (n, p) denote a strain containingcgRNA n and trigger p. The mean signal is estimated as

X̄(n, p)SIG = X̄(n, p)SIG+AF − X̄AF

with the standard error estimated via uncertainty propagation as

sX̄(n,p)SIG q(sX̄(n,p)SIG+AF)2 + (sX̄AF)2.

The upper bound on estimated standard error holds under the assumption that the correlation between SIG and AF is non-negative.

S1.4.2 Fold change for constitutively active cgRNAs (ON!OFF logic) with silencing dCas9 in E. coli

For a constitutively active cgRNA with silencing dCas9 (Figures 2 and 3), the ON state for cgRNA n corresponds to lowfluorescence using no trigger (p = 0) and the OFF state corresponds to high fluorescence using cognate trigger (p = n). Thefold change is estimated as

X̄(n)OFF:ON = X̄(n, n)SIG/X̄(n, 0)SIG

with standard error estimated via uncertainty propagation as

sX̄(n)OFF:ON X̄(n)OFF:ON

s✓sX̄(n,n)SIG

X̄(n, n)SIG

◆2

+

✓sX̄(n,0)SIG

X̄(n, 0)SIG

◆2

.

The upper bound on estimated standard error holds under the assumption that the correlation between SIG in the two strains isnon-negative.

S1.4.3 Dynamic range for constitutively active cgRNAs (ON!OFF logic) with silencing dCas9 in E. coli

For a constitutively active cgRNA with silencing dCas9 (Figures 2 and 3), the ON state for cgRNA n corresponds to lowfluorescence using no trigger (p = 0) and the OFF state corresponds to high fluorescence using cognate trigger (p = n). Thedynamic range is estimated as

X̄(n)DR = X̄(n, n)SIG+AF − X̄(n, 0)SIG+AF


sX̄(n)DR q(sX̄(n,n)SIG+AF)2 + (sX̄(n,0)SIG+AF)2.

The upper bound on estimated standard error holds under the assumption that the correlation between SIG+AF in the two strainsis non-negative.

S16

S1.4.4 Fold change for constitutively inactive cgRNAs (OFF!ON logic) with silencing dCas9 in E. coli

For a constitutively inactive cgRNA with silencing dCas9 (Figure 4), the OFF state for cgRNA n corresponds to high fluo-rescence using no trigger (p = 0) and the ON state corresponds to low fluorescence using cognate trigger (p = n). The foldchange is estimated as

X̄(n)OFF:ON = X̄(n, 0)SIG/X̄(n, n)SIG


sX̄(n)OFF:ON X̄(n)OFF:ON

s✓sX̄(n,0)SIG

X̄(n, 0)SIG

◆2

+

✓sX̄(n,n)SIG

X̄(n, n)SIG

◆2

.

The upper bound on estimated standard error holds under the assumption that the correlation between SIG in the two strains isnon-negative.

S1.4.5 Dynamic range for constitutively inactive cgRNAs (OFF!ON logic) with silencing dCas9 in E. coli

For a constitutively inactive cgRNA with silencing dCas9 (Figure 4), the OFF state for cgRNA n corresponds to high fluores-cence using no trigger (p = 0) and the ON state corresponds to low fluorescence using cognate trigger (p = n). The dynamicrange is estimated as

X̄(n)DR = X̄(n, 0)SIG+AF − X̄(n, n)SIG+AF


sX̄(n)DR q(sX̄(n,0)SIG+AF)2 + (sX̄(n,n)SIG+AF)2.

The upper bound on estimated standard error holds under the assumption that the correlation between SIG+AF in the two strainsis non-negative.

S1.4.6 Fractional dynamic range for cgRNAs with silencing dCas9 in E. coli

For a cgRNA with silencing dCas9 (Figures 2, 3, and 4), the ideal OFF state corresponds to high fluorescence with a no-targetgRNA lacking the target-binding region and the ideal ON state corresponds to low fluorescence with a standard gRNA with atarget-binding region for the target Y. The ideal dynamic range is estimated as

X̄DR

ideal= X̄SIG+AF

no−target− X̄SIG+AF

standard


sX̄DR

ideal

q(sX̄SIG+AF

notarget

)2 + (sX̄SIG+AF

standard

)2.

The upper bound on estimated standard error holds under the assumption that the correlation between SIG+AF in the two strainsis non-negative. The fractional dynamic range is estimated as

X̄(n)FDR = X̄(n)DR/X̄DR

ideal


sX̄(n)FDR X̄(n)FDR

s✓sX̄(n)DR

X̄(n)DR

◆2

+

✓sX̄DR

ideal

X̄DR

ideal

◆2

.

The upper bound on estimated standard error holds under the assumption that the correlation between the ideal dynamic rangeand the cgRNA dynamic range is non-negative.

S1.4.7 Crosstalk for orthogonal cgRNAs in E. coli

Crosstalk (CT) is estimated for cgRNA n with trigger p as

X̄(n, p)CT = [X̄(n, p)SIG+AF − X̄(n, 0)SIG+AF]/[X̄(n, n)SIG+AF − X̄(n, 0)SIG+AF]


S17

sX̄(n,p)CT X̄(n, p)CT

vuut✓q

(sX̄(n,p)SIG+AF)2 + (sX̄(n,0)SIG+AF)2

X̄(n, p)SIG+AF − X̄(n, 0)SIG+AF

◆2

+

✓q

(sX̄(n,n)SIG+AF)2 + (sX̄(n,0)SIG+AF)2

X̄(n, n)SIG+AF − X̄(n, 0)SIG+AF

◆2

.

The upper bound on estimated standard error holds under the assumption that the correlation between strains is non-negative.Note that crosstalk values can be positive or negative. Table S14abc reports X̄(n, p)CT ± sX̄(n,p)CT based on the mean single-cell fluorescence over 20,000 cells for N = 3 replicate wells. The bar graphs of Figures 2d (right), 3d (right), 4d (right) areannotated with X̄(n, p)CT except that in instances where |X̄(n, p)CT| < sX̄(n,p)CT , we instead report X̄(n, p)CT + sX̄(n,p)CT

as an estimated upper bound.The bar graphs for the bacterial studies of Figures 2d (right), 3d (right), 4d (right) plot normalized fluorescence. For replicate

well i of cgRNA n and trigger p, the normalized mean fluorescence over 20,000 cells is

X̄(n, p)Norm

i = [X̄(n, p)SIG+AF

i − X̄(n, 0)SIG+AF]/[X̄(n, n)SIG+AF − X̄(n, 0)SIG+AF].

The bar graphs display the mean normalized fluorescence over N = 3 replicate wells (X̄(n, p)Norm) ± the standard error ofthe mean (sX̄(n,p)Norm) calculated treating the normalizing values X̄(n, 0)SIG+AF and X̄(n, n)SIG+AF as exact (i.e. sX̄Norm

n,pis

calculated without error propagation) so as to display the uncertainty corresponding to the particular trigger p with cgRNA n.By contrast, sX̄(n,p)CT is calculated with error propagation so as to characterize the uncertainty arising from cognate trigger(p = n), non-cognate trigger (p 6= n), and no trigger (p = 0) with cgRNA n.

S1.5 Quantitative fluorescence analysis for HEK 293T cellsS1.5.1 Measuring signal in HEK 293T cells

For HEK 293T cells utilizing the CRIPSR/Cas9 gene induction assay, the total fluorescence (SIG + BACK) in the relevantfluorescent channel (dTomato) is a combination of signal (SIG) from gRNA/cgRNA-mediated induction of dTomato geneexpression and background (BACK) consisting of the autofluorescence inherent to the cells plus basal expression of dTomatofrom the minimal CMV promoter. Background fluorescence is characterized using the no-target control. For cell j of replicatewell i, we denote the background:

XBACK

i,j

the signalXSIG

i,j

and the total fluorescence (SIG + BACK):XSIG+BACK

i,j .

For replicate well i, we measure the mean fluorescence (X̄SIG+BACK

i for cells expressing a gRNA/cgRNA, X̄BACK

i for cellsexpression a no-target control) over all the cells in the [miRFP670+ and EBFP2+] gate (corresponding to cells highly ex-pressing cgRNA and trigger plasmid transfection controls). Performance across N = 3 replicate wells is characterized by thesample means (X̄SIG+BACK and X̄BACK) and estimated standard errors (sX̄SIG+BACK and sX̄BACK). Let (n, p) denote wellstransfected with cgRNA n and trigger p. The mean signal is estimated as

X̄(n, p)SIG = X̄(n, p)SIG+BACK − X̄BACK


sX̄(n,p)SIG q(sX̄(n,p)SIG+BACK)2 + (sX̄BACK)2.

The upper bound on estimated standard error holds under the assumption that the correlation between SIG and BACK is non-negative.

S1.5.2 Fold change for constitutively active cgRNAs (ON!OFF logic) with inducing dCas9 in HEK 293T cells

For a constitutively active cgRNA with inducing dCas9 (Figure 5), the ON state for cgRNA n corresponds to high fluorescenceusing no trigger (p = 0) and the OFF state corresponds to low fluorescence using cognate trigger (p = n). The fold change isestimated as

X̄(n)ON:OFF = X̄(n, 0)SIG/X̄(n, n)SIG

S18


sX̄(n)ON:OFF X̄(n)ON:OFF

s✓sX̄(n,0)SIG

X̄(n, 0)SIG

◆2

+

✓sX̄(n,n)SIG

X̄(n, n)SIG

◆2

The upper bound on estimated standard error holds under the assumption that the correlation between SIG in the two transfectionconditions is non-negative.

S1.5.3 Dynamic range for constitutively active cgRNAs (ON!OFF logic) with inducing dCas9 in HEK 293T cells

For a constitutively active cgRNA with inducing dCas9 (Figure 5), the ON state for cgRNA n corresponds to high fluorescenceusing no trigger (p = 0) and the OFF state corresponds to low fluorescence using cognate trigger (p = n). The dynamic rangeis estimated as

X̄(n)DR = X̄(n, 0)SIG+BACK − X̄(n, n)SIG+BACK


sX̄(n)DR q(sX̄(n,0)SIG+BACK)2 + (sX̄(n,n)SIG+BACK)2.

The upper bound on estimated standard error holds under the assumption that the correlation between SIG+BACK in the twotransfection conditions is non-negative.

S1.5.4 Fractional dynamic range for cgRNAs with inducing dCas9 in HEK 293T cells

For a cgRNA with inducing dCas9 (Figure 5), the ideal OFF state corresponds to low fluorescence with a no-target gRNAlacking the target-binding region and the ideal ON state corresponds to high fluorescence with a standard gRNA with a target-binding region for the target Y. The ideal dynamic range is estimated as

X̄DR

ideal= X̄SIG+BACK

standard− X̄SIG+BACK

no−target


sX̄DR

ideal

q(sX̄SIG+BACK

standard

)2 + (sX̄SIG+BACK

notarget

)2.

The upper bound on estimated standard error holds under the assumption that the correlation between SIG+BACK in the twotransfection conditions is non-negative. The fractional dynamic range is estimated as

X̄(n)FDR = X̄(n)DR/X̄DR

ideal


sX̄(n)FDR X̄(n)FDR

s✓sX̄(n)DR

X̄(n)DR

◆2

+

✓sX̄DR

ideal

X̄DR

ideal

◆2

.

The upper bound on estimated standard error holds under the assumption that the correlation between the ideal dynamic rangeand the cgRNA dynamic range is non-negative.

S1.5.5 Crosstalk for orthogonal cgRNAs in HEK 293T cells

Crosstalk (CT) is estimated for cgRNA n with trigger p as

X̄(n, p)CT = [X̄(n, p)SIG+BACK − X̄(n, 0)SIG+BACK]/[X̄(n, n)SIG+BACK − X̄(n, 0)SIG+BACK]


s(n, p)CT X̄(n, p)CT

vuut✓q

(sX̄(n,p)SIG+BACK)2 + (sX̄(n,0)SIG+BACK)2

X̄(n, p)SIG+BACK − X̄(n, 0)SIG+BACK

◆2

+

✓q

(sX̄(n,n)SIG+BACK)2 + (sX̄(n,0)SIG+BACK)2

X̄(n, n)SIG+BACK − X̄(n, 0)SIG+BACK

◆2

.

S19

The upper bound on estimated standard error holds under the assumption that the correlation between transfection conditionsis non-negative. Note that crosstalk values can be positive or negative. Table S14d reports X̄(n, p)CT ± sX̄(n,p)CT based onthe mean single-cell fluorescence over 426–7714 transfected cells for N = 3 replicate wells (corresponding to cells with highexpression of the two fluorescent reporter proteins, miRFP670 and EBFP2, that serve as transfection controls for the cgRNAplasmid and the trigger plasmid respectively). The bar graph of Figure 5c (right) is annotated with X̄(n, p)CT except that ininstances where |X̄(n, p)CT| < sX̄(n,p)CT , we instead report X̄(n, p)CT + sX̄(n,p)CT as an estimated upper bound.

The bar graph for the mammalian study of Figure 5c (right) plots normalized fluorescence. For replicate well i of cgRNAn and trigger p, the normalized mean fluorescence over 426–7714 transfected cells is

X̄(n, p)Norm

i = [X̄(n, p)SIG+AF

i − X̄(n, 0)SIG+AF]/[X̄(n, n)SIG+AF − X̄(n, 0)SIG+AF].

The bar graphs display the mean normalized fluorescence over N = 3 replicate wells (X̄(n, p)Norm) ± the standard error ofthe mean (sX̄(n,p)Norm) calculated treating the normalizing values X̄(n, 0)SIG+AF and X̄(n, n)SIG+AF as exact (i.e. sX̄Norm

n,pis

calculated without error propagation) so as to display the uncertainty corresponding to the particular trigger p with cgRNA n.By contrast, sX̄(n,p)CT is calculated with error propagation so as to characterize the uncertainty arising from cognate trigger(p = n), non-cognate trigger (p 6= n), and no trigger (p = 0) with cgRNA n.

S20

S2 Sequences

S2.1 Sequences for cgRNAs, triggers, and control gRNAs

Terminator switch mechanism in E. coliName Sequence Figure Legend

TrmS cgA 50-AACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGATCAAACGGGTAAACAAACAGGATAATTAAGGAGGCAGTACCCGGGCACCGAGTCGGTGCTTTTTTT-30

2c2d

cgRNAcgRNA A

TrmS cgB 50-AACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTATCATGGGGTTGTGTGTTGTTGTAAGTGTGTGTGTGTTGCCCCGGCACCGAGTCGGTGCTTTTTTT-30

2d cgRNA B

TrmS cgC 50-AACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGAATATAGGGGAAGAGAAAGAAGAAGAGAAGAGAAAGATGTCCCCGGCACCGAGTCGGTGCTTTTTTT-30

2d cgRNA C

TrmS tA 50-TACTGCCTCCTTAATTATCCTGTTTGTTTACCCGTTTGAT-30 2c2d

TriggerTrigger XA

TrmS tB 50-CAACACACACACACTTACAACAACACACAACCCCATGATA-30 2d Trigger XB

TrmS tC 50-ACATCTTTCTCTTCTCTTCTTCTTTCTCTTCCCCTATATT-30 2d Trigger XC

Table S3: Terminator switch sequences for studies in E. coli. Nucleotides shaded orange are constrained by the target gene. Nucleotidesshaded gray are constrained by dCas9. Nucleotides shaded blue are designed as described in Section S1.1.

Splinted switch mechanism in E. coliName Sequence Figure Legend

SplS cgA 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTACACCTTACGCCGGTTCAATTCCAAGTCCCTTCCAGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAACACCCTTTACAAACCTTCCTCTTCCTTTACCCTAAGTGGCACCGAGTCGGTGCTTTTTTT-30

3c3d

cgRNAcgRNA A

SplS cgB 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGTAATCGAATCATAGTAAATTTCCCATCGTCATAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTCATACGGGTCTGAAGTAGTTCATTCTTATACAGTCAAGTGGCACCGAGTCGGTGCTTTTTTT-30

3d cgRNA B

SplS cgC 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGTCGTTACCTTATCAATATCAACCTCCGCATACACTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGCACATAGGACCCAACATGCCAACAGAGAAGAGTTAAGTGGCACCGAGTCGGTGCTTTTTTT-30

3d cgRNA C

SplS tA 50-AGGGTAAAGGAAGAGGAAGGTTTGTAAAGGGTGTTCTGGAAGGGACTTGGAATTGAACCGGCGTAAGGTG-30

3c3d

TriggerTrigger XA

SplS tB 50-GACTGTATAAGAATGAACTACTTCAGACCCGTATGTTATGACGATGGGAAATTTACTATGATTCGATTAC-30

3d Trigger XB

SplS tC 50-AACTCTTCTCTGTTGGCATGTTGGGTCCTATGTGCGTGTATGCGGAGGTTGATATTGATAAGGTAACGAC-30

3d Trigger XC

Table S4: Splinted switch sequences for studies in E. coli. Nucleotides shaded orange are constrained by the target gene. Nucleotidesshaded gray are constrained by dCas9. Nucleotides shaded blue are designed as described in Section S1.1.

S21

Toehold switch mechanism in E. coliName Sequence Figure Legend

ToeS cgA 50-ATGTTCGTTGTATTAAGACCGCTAAACTGAAAGTTACACGCCCAACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

4c4d

cgRNAcgRNA A

ToeS cgB 50-GTATATGAAATTGAAAGACCGCTAAACTGAAAGTTACACGCCCAACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

4d cgRNA B

ToeS cgC 50-AAGGTGATAGTAAAGAGACCGCTAAACTGAAAGTTACACGCCCAACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

4d cgRNA C

ToeS tA 50-AACTTTCAGTTTAGCGGTCTTAATACAACGAACAT-30 4c4d

TriggerTrigger XA

ToeS tB 50-AACTTTCAGTTTAGCGGTCTTTCAATTTCATATAC-30 4d Trigger XB

ToeS tC 50-AACTTTCAGTTTAGCGGTCTCTTTACTATCACCTT-30 4d Trigger XC

Table S5: Toehold switch sequences for studies in E. coli. Nucleotides shaded orange are constrained by the target gene. Nucleotidesshaded gray are constrained by dCas9. Nucleotides shaded blue are designed as described in Section S1.1.

Terminator switch mechanism in HEK 293TName Sequence Figure Legend

TrmS cgQ 50-GAGTCGCGTGTAGCGAAGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGATCTTTGCGCGTTAGTTTCGTTCGTATTTCTGTCATGTTTGCGCGGCACCGAGTCGGTGCTTTTTTT-30

5b5c

cgRNAcgRNA Q

TrmS cgR 50-GAGTCGCGTGTAGCGAAGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTATCGCCGGGTTCAAGCAGATGTGGCATTTCAGTGTAGTTCCCGGGCACCGAGTCGGTGCTTTTTTT-30

5c cgRNA R

TrmS cgS 50-GAGTCGCGTGTAGCGAAGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTCCATTCGGGTTTACTATTACAATCTTACGTGTTCTCATTCCCGGGCACCGAGTCGGTGCTTTTTTT-30

5c cgRNA S

TrmS tQ 50-AAACATGACAGAAATACGAACGAAACTAACGCGCAAAGATCTTTTTTT-30 5b5c

TriggerTrigger XQ

TrmS tR 50-AACTACACTGAAATGCCACATCTGCTTGAACCCGGCGATACTTTTTTT-30 5c Trigger XR

TrmS tS 50-AATGAGAACACGTAAGATTGTAATAGTAAACCCGAATGGACTTTTTTT-30 5c Trigger XS

Table S6: Terminator switch sequences for studies in HEK 293T cells. Nucleotides shaded orange are constrained by the target plasmid.Nucleotides shaded gray are constrained by dCas9. Nucleotides shaded blue are designed as described in Section S1.1. Nucleotides shadedtan are constrained by mammalian transcriptional termination.

Control gRNA sequencesName Sequence Figure Legend

mRFP g 50-AACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

2c4c

Standard gRNAStandard gRNA

sfGFP g 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

3c Standard gRNA

P1 g 50-GAGTCGCGTGTAGCGAAGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

5b Standard gRNA

NT g 50-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

2c3c4c5bc

No-target gRNA, AutofluorescenceNo-target gRNA, AutofluorescenceNo-target gRNA, AutofluorescenceNo-target gRNA

S22

Table S7: Control gRNA sequences. For the standard gRNAs, nucleotides shaded orange are constrained by the target gene (mRFP orsfGFP for bacterial studies) or target plasmid (P1 gRNA binding site for mammalian studies). The no-target gRNA contains no target-bindingregion. For bacterial studies, the autofluorescence control strain was transformed with the no-target gRNA control plasmid (sequence NT g).

S2.2 Transcriptional promoter and terminator sequences

Name Type Sequence

BBa J23100 Constitutive promoter 50-TTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGC-30

BBa J23108 Constitutive promoter 50-CTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGC-30

BBa J23114 Constitutive promoter 50-TTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGC-30

BBa R0011 lacI promoter 50-AATTGTGAGCGGATAACAATTGACATTGTGAGCGGATAACAAGATACTGAGCACA-30

BBa B0015 Synthetic terminator 50-CCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATA-30

BBa B0050 Synthetic terminator 50-AAAAAAAGGATCTCAAGAAGATCCTTTGATTTT-30

BBa B1002 Synthetic terminator 50-CGCAAAAAACCCCGCTTCGGCGGGGTTTTTTCGC-30

BBa B1006 Synthetic terminator 50-AAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTT-30

BBa B1010 Synthetic terminator 50-CGCCGCAAACCCCGCCCCTGACAGGGCGGGGTTTCGCCGC-30

hU6 H. sapiens U6 promoter(Pol III)

50-AAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGCTTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACG-30

Table S8: Transcriptional promoter and terminator sequences.

S2.3 Genomically incorporated gene sequences (E. coli Ec001)mRFP template sequence

Nucleotides highlighted in orange indicate the gRNA/cgRNA target sequence.

1 ATGGCGAGTA GCGAAGACGT TATCAAAGAG TTCATGCGTT TCAAAGTTCG TATGGAAGGT TCCGTTAACG71 GTCACGAGTT CGAAATCGAA GGTGAAGGTG AAGGTCGTCC GTACGAAGGT ACCCAGACCG CTAAACTGAA

141 AGTTACCAAA GGTGGTCCGC TGCCGTTCGC TTGGGACATC CTGTCCCCGC AGTTCCAGTA CGGTTCCAAA211 GCTTACGTTA AACACCCGGC TGACATCCCG GACTACCTGA AACTGTCCTT CCCGGAAGGT TTCAAATGGG281 AACGTGTTAT GAACTTCGAA GACGGTGGTG TTGTTACCGT TACCCAGGAC TCCTCCCTGC AAGACGGTGA351 GTTCATCTAC AAAGTTAAAC TGCGTGGTAC CAACTTCCCG TCCGACGGTC CGGTTATGCA GAAAAAAACC421 ATGGGTTGGG AAGCTTCCAC CGAACGTATG TACCCGGAAG ACGGTGCTCT GAAAGGTGAA ATCAAAATGC491 GTCTGAAACT GAAAGACGGT GGTCACTACG ACGCTGAAGT TAAAACCACC TACATGGCTA AAAAACCGGT561 TCAGCTGCCG GGTGCTTACA AAACCGACAT CAAACTGGAC ATCACCTCCC ACAACGAAGA CTACACCATC631 GTTGAACAGT ACGAACGTGC TGAAGGTCGT CACTCCACCG GTGCTTAA

sfGFP template sequence

Nucleotides highlighted in orange indicate the gRNA/cgRNA target sequence.

1 ATGAGCAAAG GAGAAGAACT TTTCACTGGA GTTGTCCCAA TTCTTGTTGA ATTAGATGGT GATGTTAATG71 GGCACAAATT TTCTGTCCGT GGAGAGGGTG AAGGTGATGC TACAAACGGA AAACTCACCC TTAAATTTAT

141 TTGCACTACT GGAAAACTAC CTGTTCCGTG GCCAACACTT GTCACTACTC TGACCTATGG TGTTCAATGC211 TTTTCCCGTT ATCCGGATCA CATGAAACGG CATGACTTTT TCAAGAGTGC CATGCCCGAA GGTTATGTAC281 AGGAACGCAC TATATCTTTC AAAGATGACG GGACCTACAA GACGCGTGCT GAAGTCAAGT TTGAAGGTGA351 TACCCTTGTT AATCGTATCG AGTTAAAGGG TATTGATTTT AAAGAAGATG GAAACATTCT TGGACACAAA421 CTCGAGTACA ACTTTAACTC ACACAATGTA TACATCACGG CAGACAAACA AAAGAATGGA ATCAAAGCTA491 ACTTCAAAAT TCGCCACAAC GTTGAAGATG GTTCCGTTCA ACTAGCAGAC CATTATCAAC AAAATACTCC

S23

561 AATTGGCGAT GGCCCTGTCC TTTTACCAGA CAACCATTAC CTGTCGACAC AATCTGTCCT TTCGAAAGAT631 CCCAACGAAA AGCGTGACCA CATGGTCCTT CTTGAGTTTG TAACTGCTGC TGGGATTACA CATGGCATGG701 ATGAGCTCTA CAAA

S24

S3Pl

asm

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Con

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resc

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pg-m

RFP

Col

E1

ori;

amp-

R;B

Ba

J231

14-m

RFP

g-B

Ba

B10

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Stan

dard

gRN

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cgA

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Col

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ori;

amp-

R;B

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A-t

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-Trm

Scg

A-B

Ba

B10

06;B

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R00

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tC-B

Ba

B10

022d

cgR

NA

A,t

rigg

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C

pTrm

S-cg

B-n

TC

olE

1or

i;am

p-R

;BB

aJ2

3114

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Scg

B-B

Ba

B10

06;B

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Ba

B10

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BpT

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Col

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amp-

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Ba

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14-T

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1006

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2dcg

RN

AB

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Figu

reS9

:Exa

mpl

epl

asm

idm

apfo

rte

rmin

ator

switc

hin

E.c

oli.

Plas

mid

:pTr

mS-

cgA

-tA

forc

gRN

AA

+tr

igge

rA.

S26

1 AATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTG

GAATTC

TAAAGATCTTTTATGGCTAGCTCAGTCCTAGGTACAATGCTAGC

AACTTTCAGTTTAGCGGTCT

GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG

ATCAAACGGGTAAACAAACA

200

>>>>>>

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

>>>>>>>>>>>>>>>>>>>>

>>>>>>

EcoRI BglII BBa_J23114 mRFP target-specific spacer de novo sequence d

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

>>>>

Standard gRNA sequence de novo sequence e

>>>>>>>>>>

de novo sequence f

201

GGATAATTAAGGAGGCAGTACCCG

GGCACCGAGTCGGTGCTTTTTTTA

AAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTT

GAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGA 400

>>>>>>>>>>>>>>>>>>>>>>>>

>

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

de novo sequence f de novo sequence e* BBa_B1006

>>>>>>>>>>>>>>>>>>>>>>>

Standard gRNA sequence

401 TGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAA 600

601 AGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACTGGATCA

ATTGTGAGCGGATAACAATTGACATTGTGAGCGGATAACAAGATACTGAGCACA

TACTGCCTCCTTAATTATCCTGTTTGTTTACCCGTTTGAT

CGCAAAAAACCCCGCTTCGGCGGGGTTTTTTCGC

TCGAGTCTAGAC 800

>

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

BBa_R0011 de novo sequence f* de novo sequence e*

>>>>>>

de novo sequence d*

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

BBa_B1002

801 TGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAG

GCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCC

1000

>

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1001

CCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAG

1200

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1201

TTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGC

1400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1401

CTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCT

1600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1601

GACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGA

GATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAG

TTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCAT

1800

287

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

ColE1 AmpR

1801

AGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGG

2000

267

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2001

AAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAG

2200

200

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2201

TTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCT

2400

133

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2401

CATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCA

2600

67

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2601

TACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTT

ATTGTCTCATGAGCGGATACATATTTGAAT

GTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA 2773

287

<

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR AmpR_promoter

Figu

reS1

0:E

xam

ple

anno

tate

dpl

asm

idse

quen

cefo

rte

rmin

ator

switc

hin

E.c

oli.

Plas

mid

:pTr

mS-

cgA

-tA

forc

gRN

AA

+tr

igge

rA.

S27

S3.2

Con

stitu

tivel

yac

tive

splin

ted

switc

hin

E.c

oli

Nam

ePa

rts

Figu

reL

egen

d

pdC

as9-

bact

eria

Add

gene

plas

mid

#442

493c

dpa

g-N

TC

olE

1or

i;am

p-R

;BB

aJ2

3108

-NT

g-B

Ba

B10

063c 3d

No-

targ

etgR

NA

;Aut

ofluo

resc

ence

Aut

ofluo

resc

ence

pg-s

fGFP

Col

E1

ori;

amp-

R;B

Ba

J231

08-s

fGFP

g-B

Ba

B10

063c

Stan

dard

gRN

ApS

plS-

cgA

-nT

Col

E1

ori;

amp-

R;B

Ba

J231

08-S

plS

cgA

-BB

aB

1006

3c 3dcg

RN

Acg

RN

AA

pSpl

S-cg

A-t

AC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Spl

StA

-BB

aB

1006

-BB

aB

0015

;BB

aJ2

3108

-Spl

Scg

A-B

Ba

B10

06Se

eFi

gure

S11,

Figu

reS1

23c 3d

cgR

NA

+tr

igge

rcg

RN

AA

,tri

gger

XA

pSpl

S-cg

A-t

BC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Spl

StB

-BB

aB

1006

-BB

aB

0015

;BB

aJ2

3108

-Spl

Scg

A-B

Ba

B10

063d

cgR

NA

A,t

rigg

erX

B

pSpl

S-cg

A-t

CC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Spl

StC

-BB

aB

1006

-BB

aB

0015

;BB

aJ2

3108

-Spl

Scg

A-B

Ba

B10

063d

cgR

NA

A,t

rigg

erX

C

pSpl

S-cg

B-n

TC

olE

1or

i;am

p-R

;BB

aJ2

3108

-Spl

Scg

B-B

Ba

B10

063d

cgR

NA

BpS

plS-

cgB

-tA

Col

E1

ori;

amp-

R;B

Ba

J231

00-S

plS

tA-B

Ba

B10

06-B

Ba

B00

15;B

Ba

J231

08-S

plS

cgB

-BB

aB

1006

3dcg

RN

AB

,tri

gger

XA

pSpl

S-cg

B-t

BC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Spl

StB

-BB

aB

1006

-BB

aB

0015

;BB

aJ2

3108

-Spl

Scg

B-B

Ba

B10

063d

cgR

NA

B,t

rigg

erX

B

pSpl

S-cg

B-t

CC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Spl

StC

-BB

aB

1006

-BB

aB

0015

;BB

aJ2

3108

-Spl

Scg

B-B

Ba

B10

063d

cgR

NA

B,t

rigg

erX

C

pSpl

S-cg

C-n

TC

olE

1or

i;am

p-R

;BB

aJ2

3108

-Spl

Scg

C-B

Ba

B10

063d

cgR

NA

CpS

plS-

cgC

-tA

Col

E1

ori;

amp-

R;B

Ba

J231

00-S

plS

tA-B

Ba

B10

06-B

Ba

B00

15;B

Ba

J231

08-S

plS

cgC

-BB

aB

1006

3dcg

RN

AC

,tri

gger

XA

pSpl

S-cg

C-t

BC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Spl

StB

-BB

aB

1006

-BB

aB

0015

;BB

aJ2

3108

-Spl

Scg

C-B

Ba

B10

063d

cgR

NA

C,t

rigg

erX

B

pSpl

S-cg

C-t

CC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Spl

StC

-BB

aB

1006

-BB

aB

0015

;BB

aJ2

3108

-Spl

Scg

C-B

Ba

B10

063d

cgR

NA

C,t

rigg

erX

C

Tabl

eS1

0:Pl

asm

idsu

sed

with

splin

ted

switc

hcg

RN

Asi

nE

.col

i.

S28

BBa_

B100

6

de n

ovo

sequ

ence

e

de n

ovo

sequ

ence

dsf

GFP

targ

et-s

peci

fic s

pace

rBB

a_J2

3108

Scar

BBa_

B001

5BB

a_B1

006

de n

ovo

sequ

ence

d*

de n

ovo

sequ

ence

e*

BBa_

J231

00Bg

lIIEc

oRI

Bam

HI

Col

E1

AmpR

pSpl

S-cg

A-tA

3049

bp

Stan

dard

gR

NA

sequ

ence

AmpR

_pro

mot

er

Figu

reS1

1:E

xam

ple

plas

mid

map

for

splin

ted

switc

hin

E.c

oli.

Plas

mid

:pSp

lS-c

gA-t

Afo

rcgR

NA

A+

trig

gerA

.

S29

1 AATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCGTGGTAGGAGATCTGCATAAGGAGTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAGGGTAAAGGAAGAGGAAGGTTTGTAAAGGGTGTTCTGGAAGGGACTTGGAATTGAACCGGCGTAAG 200

>>>>>> >>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

EcoRI BglII BBa_J23100 de novo sequence e* de novo sequence d*

201 GTGAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTCTACACCCTTCTAACAACTCCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGATCTGAA 400

>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

de novo sequence d* BBa_B0015 Scar

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

BBa_B1006

401 ACATGACGAGCTGGTGAGCATAAGGAGCTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCCATCTAATTCAACAAGAATTGTTTTAGAGCTACACCTTACGCCGGTTCAATTCCAAGTCCCTTCCAGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAACACCCTTTACAAACCTTCCTCTTCCTTTACCCT 600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

BBa_J23108 sfGFP target-specific spacer de novo sequence d Standard gRNA sequence de novo sequence e

>>>>>>>>>>>>

Standard gRNA sequence

601 AAGTGGCACCGAGTCGGTGCTTTTTTTAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTTGCGCTATCAGTTGACACAGTGAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGA 800

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

Standard gRNA sequence BBa_B1006

801 TGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAA 1000

1001 AGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACTGGATCCTTACTCGAGTCTAGACTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACAT 1200

>>>>>>

BamHI

1201 GTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGC 1400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1401 TTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCA 1600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1601 CTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTT 1800

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1801 TTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGT 2000

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

2001 AAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAG 2200

287

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2201 CCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAG 2400

225

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2401 GCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTA 2600

159

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2601 TGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTT 2800

92

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2801 TCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGT 3000

25

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR AmpR_promoter

3001 GCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA 3049

Figu

reS1

2:E

xam

ple

anno

tate

dpl

asm

idse

quen

cefo

rsp

linte

dsw

itch

inE

.col

i.Pl

asm

id:p

SplS

-cgA

-tA

forc

gRN

AA

+tr

igge

rA.

S30

S3.3

Con

stitu

tivel

yin

activ

eto

ehol

dsw

itch

inE

.col

i

Nam

ePa

rts

Figu

reL

egen

d

pdC

as9-

bact

eria

Add

gene

plas

mid

#442

494c

dps

g-N

TC

olE

1or

i;am

p-R

;BB

aJ2

3108

-NT

g4c 4d

No-

targ

etgR

NA

;Aut

ofluo

resc

ence

Aut

ofluo

resc

ence

psg-

mR

FPC

olE

1or

i;am

p-R

;BB

aJ2

3108

-mR

FPg

4cSt

anda

rdgR

NA

pToe

S-cg

A-n

TC

olE

1or

i;am

p-R

;BB

aJ2

3100

-BB

aB

0050

;BB

aJ2

3108

-Toe

Scg

A4c 4d

cgR

NA

cgR

NA

ApT

oeS-

cgA

-tA

Col

E1

ori;

amp-

R;B

Ba

J231

00-T

oeS

tA-B

Ba

B00

50;B

Ba

J231

08-T

oeS

cgA

See

Figu

reS1

3,Fi

gure

S14

4c 4dcg

RN

A+

trig

ger

cgR

NA

A,t

rigg

erX

A

pToe

S-cg

A-t

BC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Toe

StB

-BB

aB

0050

;BB

aJ2

3108

-Toe

Scg

A4d

cgR

NA

A,t

rigg

erX

B

pToe

S-cg

A-t

CC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Toe

StC

-BB

aB

0050

;BB

aJ2

3108

-Toe

Scg

A4d

cgR

NA

A,t

rigg

erX

C

pToe

S-cg

B-n

TC

olE

1or

i;am

p-R

;BB

aJ2

3100

-BB

aB

0050

;BB

aJ2

3108

-Toe

Scg

B4d

cgR

NA

BpT

oeS-

cgB

-tA

Col

E1

ori;

amp-

R;B

Ba

J231

00-T

oeS

tA-B

Ba

B00

50;B

Ba

J231

08-T

oeS

cgB

4dcg

RN

AB

,tri

gger

XA

pToe

S-cg

B-t

BC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Toe

StB

-BB

aB

0050

;BB

aJ2

3108

-Toe

Scg

B4d

cgR

NA

B,t

rigg

erX

B

pToe

S-cg

B-t

CC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Toe

StC

-BB

aB

0050

;BB

aJ2

3108

-Toe

Scg

B4d

cgR

NA

B,t

rigg

erX

C

pToe

S-cg

C-n

TC

olE

1or

i;am

p-R

;BB

aJ2

3100

-BB

aB

0050

;BB

aJ2

3108

-Toe

Scg

C4d

cgR

NA

CpT

oeS-

cgC

-tA

Col

E1

ori;

amp-

R;B

Ba

J231

00-T

oeS

tA-B

Ba

B00

50;B

Ba

J231

08-T

oeS

cgC

4dcg

RN

AC

,tri

gger

XA

pToe

S-cg

C-t

BC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Toe

StB

-BB

aB

0050

;BB

aJ2

3108

-Toe

Scg

C4d

cgR

NA

C,t

rigg

erX

B

pToe

S-cg

C-t

CC

olE

1or

i;am

p-R

;BB

aJ2

3100

-Toe

StC

-BB

aB

0050

;BB

aJ2

3108

-Toe

Scg

C4d

cgR

NA

C,t

rigg

erX

C

Tabl

eS1

1:Pl

asm

idsu

sed

with

toeh

old

switc

hcg

RN

Asi

nE

.col

i.

S31

Stan

dard

gR

NA

sequ

ence

mR

FP ta

rget

-spe

cific

spa

cer

mR

FP ta

rget

-spe

cific

spa

cer a

ntis

ense

de n

ovo

sequ

ence

d

BBa_

J231

08Sc

ar

BBa_

B005

0de

nov

o se

quen

ce d

*m

RFP

targ

et-s

peci

fic s

pace

rBB

a_J2

3100

BglII

EcoR

I

Bam

HI

AmpR

pToe

S-cg

A-tA

3105

bp

Col

E1

AmpR

_pro

mot

er

Figu

reS1

3:E

xam

ple

plas

mid

map

for

toeh

old

switc

hin

E.c

oli.

Plas

mid

:pTo

eS-c

gA-t

Afo

rcgR

NA

A+

trig

gerA

.

S32

1 AATAGGCGTATCACGAGGCAGAATTTCAGATAAAAAAAATCCTTAGCTTTCGCTAAGGATGATTTCTGGAATTCTAAAGATCTTTGACGGCTAGCTCAGTCCTAGGTACAGTGCTAGCAACTTTCAGTTTAGCGGTCTTAATACAACGAACATAAAAAAAGGATCTCAAGAAGATCCTTTGATTTTTGAAGCTTGGGCCC 200

>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

EcoRI BglII BBa_J23100 mRFP target-specific spacer BBa_B0050

>>>>>>>>>>>>>>>

de novo sequence d*

201 GAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCACCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCT 400

401 GACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACTGGATCTCTGACAGCTAGCTCAGTCCTAGGTATAATGCTAGCATGT 600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

Scar BBa_J23108 de novo sequence d

601 TCGTTGTATTAAGACCGCTAAACTGAAAGTTACACGCCCAACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTGAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGAC 800

>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>

de novo sequence d mRFP target-specific spacer

>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

mRFP target-specific spacer antisense Standard gRNA sequence

801 CATCATCATCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAATTTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGT 1000

1001 AGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACTGGATCCTTACTCGAGTCTAGACTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCAC 1200

>>>>>>

BamHI

1201 TCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATA 1400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1401 CCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCG 1600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1601 CCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGA 1800

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1801 AAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCAC 2000

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

2001 CTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTG 2200

287

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2201 CAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTG 2400

244

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2401 GTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGT 2600

177

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2601 AAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGA 2800

111

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2801 TGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTT 3000

44

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR AmpR_promoter

3001 GAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA 3105

<<<

AmpR_promoter

Figu

reS1

4:E

xam

ple

anno

tate

dpl

asm

idse

quen

cefo

rto

ehol

dsw

itch

inE

.col

i.Pl

asm

id:p

ToeS

-cgA

-tA

forc

gRN

AA

+tr

igge

rA.

S33

S3.4

pdC

as9+

lacI

inE

.col

i

TetR

Cam

R

dCas

9

BBa_

B003

4J2

3108

BBa_

B101

0

lacI

(C00

12m

-LVA

)

p15A

lacI

+dC

as9

7924

bp

Figu

reS1

5:Pl

asm

idm

apfo

rpd

Cas

9+la

cIin

E.c

oli.

S34

1 TT

ACGC

CCCG

CCCT

GCCA

CTCA

TCGC

AGTA

CTGT

TGTA

ATTC

ATTA

AGCA

TTCT

GCCG

ACAT

GGAA

GCCA

TCAC

AAAC

GGCA

TGAT

GAAC

CTGA

ATCG

CCAG

CGGC

ATCA

GCAC

CTTG

TCGC

CTTG

CGTA

TAAT

ATTT

GCCC

ATGG

TGAA

AACG

GGGG

CGAA

GAAG

TTGT

CCAT

ATTG

GCCA

CGTT

TAAA

TCAA

AACT

GGTG

AAAC

TCAC

CCAG

GGAT

TGGC

TGAG

ACGA

AAAA

CATA

TTCT

CAAT

AAAC

CCTTTA

GGGA

AATA

GGCC

AGGT

TTTC

ACCG

TAAC

ACGC

CACA

TCTT

GCGA

ATAT

ATGT

GTAG

AAAC

TGCC

GGAA

ATCG

TCGT

GGTA

TTCA

CTCC

AGAG

CG 3

50

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

CamR

35

1 AT

GAAA

ACGT

TTCA

GTTT

GCTC

ATGG

AAAA

CGGT

GTAA

CAAG

GGTG

AACA

CTAT

CCCA

TATC

ACCA

GCTC

ACCG

TCTT

TCAT

TGCC

ATAC

GAAA

TTCC

GGAT

GAGC

ATTC

ATCA

GGCG

GGCA

AGAA

TGTG

AATA

AAGG

CCGG

ATAA

AACT

TGTG

CTTA

TTTT

TCTT

TACG

GTCT

TTAA

AAAG

GCCG

TAAT

ATCC

AGCT

GAAC

GGTC

TGGT

TATA

GGTA

CATT

GAGC

AACT

GACT

GAAA

TGCC

TCAA

AATG

TTCTTT

ACGA

TGCC

ATTG

GGAT

ATAT

CAAC

GGTG

GTAT

ATCC

AGTG

ATTT

TTTT

CTCC

ATTT

TAGC

TTCC

TTAG

CTCC

TGAA

AATC

TCGA

TAAC

TCAA

AA 7

00

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

CamR

70

1 AA

TACG

CCCG

GTAG

TGAT

CTTA

TTTC

ATTA

TGGT

GAAA

GTTG

GAAC

CTCT

TACG

TGCC

GATC

AACG

TCTC

ATTT

TCGC

CAGA

TATC

GACG

TCTT

AAGA

CCCA

CTTT

CACA

TTTA

AGTT

GTTT

TTCT

AATC

CGCA

TATG

ATCA

ATTC

AAGG

CCGA

ATAA

GAAG

GCTG

GCTC

TGCA

CCTT

GGTG

ATCA

AATA

ATTC

GATA

GCTT

GTCG

TAAT

AATG

GCGG

CATA

CTAT

CAGT

AGTA

GGTG

TTTC

CCTT

TCTT

CTTT

AGCG

ACTT

GATG

CTCT

TGAT

CTTC

CAAT

ACGC

AACC

TAAA

GTAA

AATG

CCCC

ACAG

CGCT

GAGT

GCAT

ATAA

TGCA

TTCT

CTAG

TGAA

AAAC

CTTG

105

0

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<

TetR

105

1 TT

GGCA

TAAA

AAGG

CTAA

TTGA

TTTT

CGAG

AGTT

TCAT

ACTG

TTTT

TCTG

TAGG

CCGT

GTAC

CTAA

ATGT

ACTT

TTGC

TCCA

TCGC

GATG

ACTT

AGTA

AAGC

ACAT

CTAA

AACT

TTTA

GCGT

TATT

ACGT

AAAA

AATC

TTGC

CAGC

TTTC

CCCT

TCTA

AAGG

GCAA

AAGT

GAGT

ATGG

TGCC

TATC

TAAC

ATCT

CAAT

GGCT

AAGG

CGTC

GAGC

AAAG

CCCG

CTTA

TTTT

TTAC

ATGC

CAAT

ACAA

TGTA

GGCTGC

TCTA

CACC

TAGC

TTCT

GGGC

GAGT

TTAC

GGGT

TGTT

AAAC

CTTC

GATT

CCGA

CCTC

ATTA

AGCA

GCTC

TAAT

GCGC

TGTT

AATC

ACTT

TACT

TT 1

400

<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

<<<<

Te

tR

140

1 TA

TCTA

ATCT

AGAC

ATCA

TTAA

TTCC

TAAT

TTTT

GTTG

ACAC

TCTA

TCGT

TGAT

AGAG

TTAT

TTTA

CCAC

TCCC

TATC

AGTG

ATAG

AGAA

AAGA

ATTC

AAAA

GATC

TAAA

GAGG

AGAA

AGGA

TCTA

TGGA

TAAG

AAAT

ACTC

AATA

GGCT

TAGC

TATC

GGCA

CAAA

TAGC

GTCG

GATG

GGCG

GTGA

TCAC

TGAT

GAAT

ATAA

GGTT

CCGT

CTAA

AAAG

TTCA

AGGT

TCTG

GGAA

ATAC

AGAC

CGCC

ACAG

TATC

AAAA

AAAA

TCTT

ATAG

GGGC

TCTT

TTAT

TTGA

CAGT

GGAG

AGAC

AGCG

GAAG

CGAC

TCGT

CTCA

AACG

GACA

GCTC

GTAG

AAGG

TATA

CACG

TCGG

175

0

<<<<

<<<<

<<<<

<<<<

>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

Te

tR

dCa

s9

175

1 AA

GAAT

CGTA

TTTG

TTAT

CTAC

AGGA

GATT

TTTT

CAAA

TGAG

ATGG

CGAA

AGTA

GATG

ATAG

TTTC

TTTC

ATCG

ACTT

GAAG

AGTC

TTTT

TTGG

TGGA

AGAA

GACA

AGAA

GCAT

GAAC

GTCA

TCCT

ATTT

TTGG

AAAT

ATAG

TAGA

TGAA

GTTG

CTTA

TCAT

GAGA

AATA

TCCA

ACTA

TCTA

TCAT

CTGC

GAAA

AAAA

TTGG

TAGA

TTCT

ACTG

ATAA

AGCG

GATT

TGCG

CTTA

ATCT

ATTT

GGCC

TTAG

CGCATA

TGAT

TAAG

TTTC

GTGG

TCAT

TTTT

TGAT

TGAG

GGAG

ATTT

AAAT

CCTG

ATAA

TAGT

GATG

TGGA

CAAA

CTAT

TTAT

CCAG

TTGG

TACA

AACC

TA 2

100

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

210

1 CA

ATCA

ATTA

TTTG

AAGA

AAAC

CCTA

TTAA

CGCA

AGTG

GAGT

AGAT

GCTA

AAGC

GATT

CTTT

CTGC

ACGA

TTGA

GTAA

ATCA

AGAC

GATT

AGAA

AATC

TCAT

TGCT

CAGC

TCCC

CGGT

GAGA

AGAA

AAAT

GGCT

TATT

TGGG

AATC

TCAT

TGCT

TTGT

CATT

GGGT

TTGA

CCCC

TAAT

TTTA

AATC

AAAT

TTTG

ATTT

GGCA

GAAG

ATGC

TAAA

TTAC

AGCT

TTCA

AAAG

ATAC

TTAC

GATG

ATGA

TTTA

GATAAT

TTAT

TGGC

GCAA

ATTG

GAGA

TCAA

TATG

CTGA

TTTG

TTTT

TGGC

AGCT

AAGA

ATTT

ATCA

GATG

CTAT

TTTA

CTTT

CAGA

TATC

CTAA

GAGT

AA 2

450

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

245

1 AT

ACTG

AAAT

AACT

AAGG

CTCC

CCTA

TCAG

CTTC

AATG

ATTA

AACG

CTAC

GATG

AACA

TCAT

CAAG

ACTT

GACT

CTTT

TAAA

AGCT

TTAG

TTCG

ACAA

CAAC

TTCC

AGAA

AAGT

ATAA

AGAA

ATCT

TTTT

TGAT

CAAT

CAAA

AAAC

GGAT

ATGC

AGGT

TATA

TTGA

TGGG

GGAG

CTAG

CCAA

GAAG

AATT

TTAT

AAAT

TTAT

CAAA

CCAA

TTTT

AGAA

AAAA

TGGA

TGGT

ACTG

AGGA

ATTA

TTGG

TGAA

ACTAAA

TCGT

GAAG

ATTT

GCTG

CGCA

AGCA

ACGG

ACCT

TTGA

CAAC

GGCT

CTAT

TCCC

CATC

AAAT

TCAC

TTGG

GTGA

GCTG

CATG

CTAT

TTTG

AGAA

GA 2

800

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

280

1 CA

AGAA

GACT

TTTA

TCCA

TTTT

TAAA

AGAC

AATC

GTGA

GAAG

ATTG

AAAA

AATC

TTGA

CTTT

TCGA

ATTC

CTTA

TTAT

GTTG

GTCC

ATTG

GCGC

GTGG

CAAT

AGTC

GTTT

TGCA

TGGA

TGAC

TCGG

AAGT

CTGA

AGAA

ACAA

TTAC

CCCA

TGGA

ATTT

TGAA

GAAG

TTGT

CGAT

AAAG

GTGC

TTCA

GCTC

AATC

ATTT

ATTG

AACG

CATG

ACAA

ACTT

TGAT

AAAA

ATCT

TCCA

AATG

AAAA

AGTA

CTAC

CAAAAC

ATAG

TTTG

CTTT

ATGA

GTAT

TTTA

CGGT

TTAT

AACG

AATT

GACA

AAGG

TCAA

ATAT

GTTA

CTGA

AGGA

ATGC

GAAA

ACCA

GCAT

TTCT

TTCA

GG 3

150

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

315

1 TG

AACA

GAAG

AAAG

CCAT

TGTT

GATT

TACT

CTTC

AAAA

CAAA

TCGA

AAAG

TAAC

CGTT

AAGC

AATT

AAAA

GAAG

ATTA

TTTC

AAAA

AAAT

AGAA

TGTT

TTGA

TAGT

GTTG

AAAT

TTCA

GGAG

TTGA

AGAT

AGAT

TTAA

TGCT

TCAT

TAGG

TACC

TACC

ATGA

TTTG

CTAA

AAAT

TATT

AAAG

ATAA

AGAT

TTTT

TGGA

TAAT

GAAG

AAAA

TGAA

GATA

TCTT

AGAG

GATA

TTGT

TTTA

ACAT

TGAC

CTTA

TTTGAA

GATA

GGGA

GATG

ATTG

AGGA

AAGA

CTTA

AAAC

ATAT

GCTC

ACCT

CTTT

GATG

ATAA

GGTG

ATGA

AACA

GCTT

AAAC

GTCG

CCGT

TATA

CTGG

TT 3

500

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

350

1 GG

GGAC

GTTT

GTCT

CGAA

AATT

GATT

AATG

GTAT

TAGG

GATA

AGCA

ATCT

GGCA

AAAC

AATA

TTAG

ATTT

TTTG

AAAT

CAGA

TGGT

TTTG

CCAA

TCGC

AATT

TTAT

GCAG

CTGA

TCCA

TGAT

GATA

GTTT

GACA

TTTA

AAGA

AGAC

ATTC

AAAA

AGCA

CAAG

TGTC

TGGA

CAAG

GCGA

TAGT

TTAC

ATGA

ACAT

ATTG

CAAA

TTTA

GCTG

GTAG

CCCT

GCTA

TTAA

AAAA

GGTA

TTTT

ACAG

ACTG

TAAA

AGTTGT

TGAT

GAAT

TGGT

CAAA

GTAA

TGGG

GCGG

CATA

AGCC

AGAA

AATA

TCGT

TATT

GAAA

TGGC

ACGT

GAAA

ATCA

GACA

ACTC

AAAA

GGGC

CAGA

AA 3

850

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

385

1 AA

TTCG

CGAG

AGCG

TATG

AAAC

GAAT

CGAA

GAAG

GTAT

CAAA

GAAT

TAGG

AAGT

CAGA

TTCT

TAAA

GAGC

ATCC

TGTT

GAAA

ATAC

TCAA

TTGC

AAAA

TGAA

AAGC

TCTA

TCTC

TATT

ATCT

CCAA

AATG

GAAG

AGAC

ATGT

ATGT

GGAC

CAAG

AATT

AGAT

ATTA

ATCG

TTTA

AGTG

ATTA

TGAT

GTCG

ATGC

CATT

GTTC

CACA

AAGT

TTCC

TTAA

AGAC

GATT

CAAT

AGAC

AATA

AGGT

CTTA

ACGC

GTTCTG

ATAA

AAAT

CGTG

GTAA

ATCG

GATA

ACGT

TCCA

AGTG

AAGA

AGTA

GTCA

AAAA

GATG

AAAA

ACTA

TTGG

AGAC

AACT

TCTA

AACG

CCAA

GTTA

AT 4

200

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

420

1 CA

CTCA

ACGT

AAGT

TTGA

TAAT

TTAA

CGAA

AGCT

GAAC

GTGG

AGGT

TTGA

GTGA

ACTT

GATA

AAGC

TGGT

TTTA

TCAA

ACGC

CAAT

TGGT

TGAA

ACTC

GCCA

AATC

ACTA

AGCA

TGTG

GCAC

AAAT

TTTG

GATA

GTCG

CATG

AATA

CTAA

ATAC

GATG

AAAA

TGAT

AAAC

TTAT

TCGA

GAGG

TTAA

AGTG

ATTA

CCTT

AAAA

TCTA

AATT

AGTT

TCTG

ACTT

CCGA

AAAG

ATTT

CCAA

TTCT

ATAA

AGTA

CGTGAG

ATTA

ACAA

TTAC

CATC

ATGC

CCAT

GATG

CGTA

TCTA

AATG

CCGT

CGTT

GGAA

CTGC

TTTG

ATTA

AGAA

ATAT

CCAA

AACT

TGAA

TCGG

AGTT

TG 4

550

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

455

1 TC

TATG

GTGA

TTAT

AAAG

TTTA

TGAT

GTTC

GTAA

AATG

ATTG

CTAA

GTCT

GAGC

AAGA

AATA

GGCA

AAGC

AACC

GCAA

AATA

TTTC

TTTT

ACTC

TAAT

ATCA

TGAA

CTTC

TTCA

AAAC

AGAA

ATTA

CACT

TGCA

AATG

GAGA

GATT

CGCA

AACG

CCCT

CTAA

TCGA

AACT

AATG

GGGA

AACT

GGAG

AAAT

TGTC

TGGG

ATAA

AGGG

CGAG

ATTT

TGCC

ACAG

TGCG

CAAA

GTAT

TGTC

CATG

CCCC

AAGT

CAATAT

TGTC

AAGA

AAAC

AGAA

GTAC

AGAC

AGGC

GGAT

TCTC

CAAG

GAGT

CAAT

TTTA

CCAA

AAAG

AAAT

TCGG

ACAA

GCTT

ATTG

CTCG

TAAA

AAAG

AC 4

900

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

490

1 TG

GGAT

CCAA

AAAA

ATAT

GGTG

GTTT

TGAT

AGTC

CAAC

GGTA

GCTT

ATTC

AGTC

CTAG

TGGT

TGCT

AAGG

TGGA

AAAA

GGGA

AATC

GAAG

AAGT

TAAA

ATCC

GTTA

AAGA

GTTA

CTAG

GGAT

CACA

ATTA

TGGA

AAGA

AGTT

CCTT

TGAA

AAAA

ATCC

GATT

GACT

TTTT

AGAA

GCTA

AAGG

ATAT

AAGG

AAGT

TAAA

AAAG

ACTT

AATC

ATTA

AACT

ACCT

AAAT

ATAG

TCTT

TTTG

AGTT

AGAA

AACG

GTCGTA

AACG

GATG

CTGG

CTAG

TGCC

GGAG

AATT

ACAA

AAAG

GAAA

TGAG

CTGG

CTCT

GCCA

AGCA

AATA

TGTG

AATT

TTTT

ATAT

TTAG

CTAG

TCAT

TA 5

250

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

525

1 TG

AAAA

GTTG

AAGG

GTAG

TCCA

GAAG

ATAA

CGAA

CAAA

AACA

ATTG

TTTG

TGGA

GCAG

CATA

AGCA

TTAT

TTAG

ATGA

GATT

ATTG

AGCA

AATC

AGTG

AATT

TTCT

AAGC

GTGT

TATT

TTAG

CAGA

TGCC

AATT

TAGA

TAAA

GTTC

TTAG

TGCA

TATA

ACAA

ACAT

AGAG

ACAA

ACCA

ATAC

GTGA

ACAA

GCAG

AAAA

TATT

ATTC

ATTT

ATTT

ACGT

TGAC

GAAT

CTTG

GAGC

TCCC

GCTG

CTTT

TAAA

TATTTT

GATA

CAAC

AATT

GATC

GTAA

ACGA

TATA

CGTC

TACA

AAAG

AAGT

TTTA

GATG

CCAC

TCTT

ATCC

ATCA

ATCC

ATCA

CTGG

TCTT

TATG

AAAC

AC 5

600

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

560

1 GC

ATTG

ATTT

GAGT

CAGC

TAGG

AGGT

GACT

AACT

CGAG

TAAG

GATC

TCGC

CGCA

AACC

CCGC

CCCT

GACA

GGGC

GGGG

TTTC

GCCG

CCTG

ACAG

CTAG

CTCA

GTCC

TAGG

TATA

ATGC

TAGC

AAAA

GAAT

TCAA

AAGA

TCTA

AAGA

GGAG

AAAG

GATC

TATG

GTGA

ATGT

GAAA

CCAG

TAAC

GTTA

TACG

ATGT

CGCA

GAGT

ATGC

CGGT

GTCT

CTTA

TCAG

ACCG

TTTC

CCGC

GTGG

TGAA

CCAG

GCCA

GCCA

CGTT

TCTG

CGAA

AACG

CGGG

AAAA

AGTG

GAAGCG

GCGA

TGGC

GGAG

CTGA

ATTA

CATT

CCCA

ACCG

CGTG

GCAC

AACA

ACTG

GCGG

GCAA

ACAG

TC 5

950

1

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>

>>>>

>>>>

>>>

>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

dCas

9

B

Ba_B

1010

J

2310

8

BBa

_B00

34

l

acI

(C00

12m

-LVA

)

595

1 GT

TGCT

GATT

GGCG

TTGC

CACC

TCCA

GTCT

GGCC

CTGC

ACGC

GCCG

TCGC

AAAT

TGTC

GCGG

CGAT

TAAA

TCTC

GCGC

CGAT

CAAC

TGGG

TGCC

AGCG

TGGT

GGTG

TCGA

TGGT

AGAA

CGAA

GCGG

CGTC

GAAG

CCTG

TAAA

GCGG

CGGT

GCAC

AATC

TTCT

CGCG

CAAC

GCGT

CAGT

GGGC

TGAT

CATT

AACT

ATCC

GCTG

GATG

ACCA

GGAT

GCCA

TTGC

TGTG

GAAG

CTGC

CTGC

ACTA

ATGT

TCCG

GCGTTA

TTTC

TTGA

TGTC

TCTG

ACCA

GACA

CCCA

TCAA

CAGT

ATTA

TTTT

CTCC

CATG

AGGA

CGGT

ACGC

GACT

GGGC

GTGG

AGCA

TCTG

GTCG

CATT

GG 6

300

65

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

lacI

(C0

012m

-LV

A)

630

1 GT

CACC

AGCA

AATC

GCGC

TGTT

AGCG

GGCC

CATT

AAGT

TCTG

TCTC

GGCG

CGTC

TGCG

TCTG

GCTG

GCTG

GCAT

AAAT

ATCT

CACT

CGCA

ATCA

AATT

CAGC

CGAT

AGCG

GAAC

GGGA

AGGC

GACT

GGAG

TGCC

ATGT

CCGG

TTTT

CAAC

AAAC

CATG

CAAA

TGCT

GAAT

GAGG

GCAT

CGTT

CCCA

CTGC

GATG

CTGG

TTGC

CAAC

GATC

AGAT

GGCG

CTGG

GCGC

AATG

CGCG

CCAT

TACC

GAGT

CCGG

GCTGCG

CGTT

GGTG

CGGA

TATC

TCGG

TAGT

GGGA

TACG

ACGA

TACC

GAAG

ATAG

CTCA

TGTT

ATAT

CCCG

CCGT

TAAC

CACC

ATCA

AACA

GGAT

TTTC

GC 6

650

18

2

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

lacI

(C0

012m

-LV

A)

665

1 CT

GCTG

GGGC

AAAC

CAGC

GTGG

ACCG

CTTG

CTGC

AACT

CTCT

CAGG

GCCA

GGCG

GTGA

AGGG

CAAT

CAGC

TGTT

GCCC

GTCT

CACT

GGTG

AAAA

GAAA

AACC

ACCC

TGGC

GCCC

AATA

CGCA

AACC

GCCT

CTCC

CCGC

GCGT

TGGC

CGAT

TCAT

TAAT

GCAG

CTGG

CACG

ACAG

GTTT

CCCG

ACTG

GAAA

GCGG

GCAG

TAAC

TCGA

ATAA

GGAT

CTCC

AGGC

ATCA

AATA

AAAC

GAAA

GGCT

CAGT

CGAA

AGAC

TGGG

CCTT

TCGT

TTTA

TCTG

TTGT

TTGT

CGGT

GAAC

GCTC

TCTA

CTAG

AGTC

ACAC

TGGC

TCAC

CTTC

GGGT

GGGC

CTTT

CTGC

GTTT

ATAC

CTAG

700

0

298

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>

la

cI (

C001

2m -

LVA)

700

1 GG

ATAT

ATTC

CGCT

TCCT

CGCT

CACT

GACT

CGCT

ACGC

TCGG

TCGT

TCGA

CTGC

GGCG

AGCG

GAAA

TGGC

TTAC

GAAC

GGGG

CGGA

GATT

TCCT

GGAA

GATG

CCAG

GAAG

ATAC

TTAA

CAGGGA

AGTG

AGAG

GGCC

GCGG

CAAA

GCCG

TTTT

TCCA

TAGG

CTCC

GCCC

CCCT

GACA

AGCA

TCAC

GAAA

TCTG

ACGC

TCAA

ATCA

GTGG

TGGC

GAAA

CCCG

ACAG

GACT

ATAA

AGAT

ACCA

GGCG

TTTC

CCCC

TGGC

GGCT

CCCT

CGTG

CGCT

CTCC

TGTT

CCTG

CCTT

TCGG

TTTA

CCGG

TGTC

ATTC

CGCT

GTTA

TGGC

CGCG

TTTG

TCTC

ATTC

CACG

CCTG

ACAC

TC 7

350

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

p1

5A

735

1 AG

TTCC

GGGT

AGGC

AGTT

CGCT

CCAA

GCTG

GACT

GTAT

GCAC

GAAC

CCCC

CGTT

CAGT

CCGA

CCGC

TGCG

CCTT

ATCC

GGTA

ACTA

TCGT

CTTG

AGTC

CAAC

CCGG

AAAG

ACAT

GCAA

AAGC

ACCA

CTGG

CAGC

AGCC

ACTG

GTAA

TTGA

TTTA

GAGG

AGTT

AGTC

TTGA

AGTC

ATGC

GCCG

GTTA

AGGC

TAAA

CTGA

AAGG

ACAA

GTTT

TGGT

GACT

GCGC

TCCT

CCAA

GCCA

GTTA

CCTC

GGTT

CAAA

GAGTTG

GTAG

CTCA

GAGA

ACCT

TCGA

AAAA

CCGC

CCTG

CAAG

GCGG

TTTT

TTCG

TTTT

CAGA

GCAA

GAGA

TTAC

GCGC

AGAC

CAAA

ACGA

TCTC

AAGA

AG 7

700

>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>>>

>>

p15A

770

1 AT

CATC

TTAT

TAAT

CAGA

TAAA

ATAT

TTCT

AGAT

TTCA

GTGC

AATT

TATC

TCTT

CAAA

TGTA

GCAC

CTGA

AGTC

AGCC

CCAT

ACGA

TATA

AGTT

GTTA

CTAG

TGCT

TGGA

TTCT

CACC

AATAAA

AAAC

GCCC

GGCG

GCAA

CCGA

GCGT

TCTG

AACA

AATC

CAGA

TGGA

GTTC

TGAG

GTCA

TTAC

TGGA

TCTA

TCAA

CAGG

AGTC

CAAG

CGAG

CTCG

ATAT

CAAA

792

4

Figu

reS1

6:A

nnot

ated

plas

mid

sequ

ence

for

expr

essi

onof

pdC

as9+

lacI

inE

.col

i.Pl

asm

id:p

dCas

9+la

cI.

S35

S3.5

Con

stitu

tivel

yac

tive

term

inat

orsw

itch

inH

EK

293T

cells

Nam

ePa

rts

Figu

reL

egen

d

SP-d

Cas

9-V

PRA

ddge

nepl

asm

id#6

3798

5bc

repo

rter

-gP1

pUC

ori;

kan-

R;P

1gR

NA

targ

etsi

te–P

AM

–min

imal

CM

Vpr

omot

er–d

Tom

ato–

SV40

poly

(A)s

igna

l5b

cpj

g-N

TpU

Cor

i;am

p-R

;hU

6pr

omot

er–N

Tg;

PGK

prom

oter

–miR

FP67

0–SV

40po

ly(A

)sig

nal

5bc

No-

targ

etgR

NA

pg-P

1pU

Cor

i;am

p-R

;hU

6pr

omot

er–P

1g;

PGK

prom

oter

–miR

FP67

0–SV

40po

ly(A

)sig

nal

5bSt

anda

rdgR

NA

pTrm

S-cg

QpU

Cor

i;am

p-R

;hU

6pr

omot

er–T

rmS

cgQ

;PG

Kpr

omot

er–m

iRFP

670–

SV40

poly

(A)s

igna

l5b 5c

cgR

NA

cgR

NA

QpT

rmS-

cgR

pUC

ori;

amp-

R;h

U6

prom

oter

–Trm

Scg

R;P

GK

prom

oter

–miR

FP67

0–SV

40po

ly(A

)sig

nal

5ccg

RN

AR

pTrm

S-cg

SpU

Cor

i;am

p-R

;hU

6pr

omot

er–T

rmS

cgS;

PGK

prom

oter

–miR

FP67

0–SV

40po

ly(A

)sig

nal

5ccg

RN

AS

pTrm

S-nT

pUC

ori;

amp-

R;P

GK

prom

oter

–EB

FP2–

SV40

poly

(A)s

igna

l5b

cTr

igge

r(-)

pTrm

S-tQ

pUC

ori;

amp-

R;h

U6

prom

oter

–Trm

StQ

;PG

Kpr

omot

er–E

BFP

2–SV

40po

ly(A

)sig

nal

5b 5cTr

igge

rTr

igge

rXQ

pTrm

S-tR

pUC

ori;

amp-

R;h

U6

prom

oter

–Trm

StR

;PG

Kpr

omot

er–E

BFP

2–SV

40po

ly(A

)sig

nal

5cTr

igge

rXR

pTrm

S-tS

pUC

ori;

amp-

R;h

U6

prom

oter

–Trm

StS

;PG

Kpr

omot

er–E

BFP

2–SV

40po

ly(A

)sig

nal

5cTr

igge

rXS

Tabl

eS1

2:Pl

asm

idsu

sed

with

term

inat

orsw

itch

cgR

NA

sin

HE

K29

3Tce

lls.

S36

pUC

Orig

in

hU6

prom

oter

P1 ta

rget

-spe

cific

spa

cer

Stan

dard

gR

NA

sequ

ence

de n

ovo

sequ

ence

dde

nov

o se

quen

ce e

de n

ovo

sequ

ence

fde

non

o se

quen

ce e

*St

anda

rd g

RN

A se

quen

ce

miR

FP67

0

PGK

prom

oter

SV40

sig

nal

pTrm

S-cg

Q

4444

bp

AmpR

Figu

reS1

7:E

xam

ple

plas

mid

map

for

term

inat

orsw

itch

cgR

NA

inH

EK

293T

cells

.Pla

smid

:pTr

mS-

cgQ

forc

gRN

AQ

.

S37

1 TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCT 200

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

pUC Origin

201 CACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA 400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

pUC Origin

401 GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT 600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

pUC Origin

601 CTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTC 800

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

801 TATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGCGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT 1000

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

1001 ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT 1200

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

1201 CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCA 1400

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

1401 GAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGG 1600

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

1601 AAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTTGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTAC 1800

<<<<<<<<<<<<<<<<<

AmpR

1801 CAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGC 2000

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

hU6 promoter

2001 TTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAGTCGCGTGTAGCGAAGCAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGATCTTTGCGCGTTAGTTTCGTTCGTATTTCTGTCATGTTTGCGCGGCACCGAGTCGGTGCTTTTTTTCTAGAA 2200

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>> >>>>>> >>>>

hU6 promoter P1 target-specific spacer de novo sequence d de nono sequence e*

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>> >>>>>>>>>>>>>>>>>>>>>>>

Standard gRNA sequence de novo sequence e Standard gRNA sequence

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

de novo sequence f

2201 AGCTTGTCTTCGAGCTCCTCGAGGAAGACCTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTAGACGTCGAGGAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTGGAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGT 2400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

PGK promoter

2401 AGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAAGTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATA 2600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

PGK promoter

2601 GCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGCATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCATCCCAAGCT 2800

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

PGK promoter

2801 TGCCACCATGGTAGCAGGTCATGCCTCTGGCAGCCCCGCATTCGGGACCGCCTCTCATTCGAATTGCGAACATGAAGAGATCCACCTCGCCGGCTCGATCCAGCCGCATGGCGCGCTTCTGGTCGTCAGCGAACATGATCATCGCGTCATCCAGGCCAGCGCCAACGCCGCGGAATTTCTGAATCTCGGAAGCGTACTCG 3000

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

miRFP670

3001 GCGTTCCGCTCGCCGAGATCGACGGCGATCTGTTGATCAAGATCCTGCCGCATCTCGATCCCACCGCCGAAGGCATGCCGGTCGCGGTGCGCTGCCGGATCGGCAATCCCTCTACGGAGTACTGCGGTCTGATGCATCGGCCTCCGGAAGGCGGGCTGATCATCGAACTCGAACGTGCCGGCCCGTCGATCGATCTGTCA 3200

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

miRFP670

3201 GGCACGCTGGCGCCGGCGCTGGAGCGGATCCGCACGGCGGGTTCACTGCGCGCGCTGTGCGATGACACCGTGCTGCTGTTTCAGCAGTGCACCGGCTACGACCGGGTGATGGTGTATCGTTTCGATGAGCAAGGCCACGGCCTGGTATTCTCCGAGTGCCATGTGCCTGGGCTCGAATCCTATTTCGGCAACCGCTATCC 3400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

miRFP670

3401 GTCGTCGACTGTCCCGCAGATGGCGCGGCAGCTGTACGTGCGGCAGCGCGTCCGCGTGCTGGTCGACGTCACCTATCAGCCGGTGCCGCTGGAGCCGCGGCTGTCGCCGCTGACCGGGCGCGATCTCGACATGTCGGGCTGCTTCCTGCGCTCGATGTCGCCGTGCCATCTGCAGTTCCTGAAGGACATGGGCGTGCGCG 3600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

miRFP670

3601 CCACCCTGGCGGTGTCGCTGGTGGTCGGCGGCAAGCTGTGGGGCCTGGTTGTCTGTCACCATTATCTGCCGCGCTTCATCCGTTTCGAGCTGCGGGCGATCTGCAAACGGCTCGCCGAAAGGATCGCGACGCGGATCACCGCGCTTGAGAGCTAAGTACCACTAGTACCGGTAGATATCAGCCATGGCTTCCCGCCGGCG 3800

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

miRFP670

3801 GTGGCGGCGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAGGCTAGTCGAGCAGACATGATAAGATACATTGATGAGTTTGGACAAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGC 4000

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

SV40 signal

4001 TATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCCGATAAGGATCGATCCGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCA 4200

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

SV40 signal

4201 ACAGTTGCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGC 4400

4401 CAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT 4444

Figu

reS1

8:E

xam

ple

anno

tate

dpl

asm

idse

quen

cefo

rte

rmin

ator

switc

hcg

RN

Ain

HE

K29

3Tce

lls.P

lasm

id:p

Trm

S-cg

Qfo

rcgR

NA

Q.

S38

AmpR

hU6

prom

oter

EBFP

2

PGK

prom

oter

Poly

-Tde

nov

o se

quen

ce d

*de

nov

o se

quen

ce e

*de

nov

o se

quen

ce f*

SV40

sig

nal

pTrm

S-tQ

4099

bp

pUC

Orig

in

Figu

reS1

9:E

xam

ple

plas

mid

map

for

term

inat

orsw

itch

trig

ger

inH

EK

293T

cells

.Pla

smid

:pTr

mS-

tQfo

rtri

gger

Q.

S39

1 TTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCT 200

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

pUC Origin

201 CACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACA 400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

pUC Origin

401 GAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGAT 600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

pUC Origin

601 CTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTC 800

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

801 TATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGCGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCT 1000

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

1001 ATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGAT 1200

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

1201 CGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCA 1400

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

1401 GAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGG 1600

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

1601 AAATGTTGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTTGTACAAAAAAGCAGGCTTTAAAGGAACCAATTCAGTCGACTGGATCCGGTAC 1800

<<<<<<<<<<<<<<<<<

AmpR

1801 CAAGGTCGGGCAGGAAGAGGGCCTATTTCCCATGATTCCTTCATATTTGCATATACGATACAAGGCTGTTAGAGAGATAATTAGAATTAATTTGACTGTAAACACAAAGATATTAGTACAAAATACGTGACGTAGAAAGTAATAATTTCTTGGGTAGTTTGCAGTTTTAAAATTATGTTTTAAAATGGACTATCATATGC 2000

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

hU6 promoter

2001 TTACCGTAACTTGAAAGTATTTCGATTTCTTGGCTTTATATATCTTGTGGAAAGGACGAAACACCGAAACATGACAGAAATACGAACGAAACTAACGCGCAAAGATCTTTTTTTCTAGACCCAGCTTTCTTGTACAAAGTTGGCATTAGACGTCGAGGAATTCTACCGGGTAGGGGAGGCGCTTTTCCCAAGGCAGTCTG 2200

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

hU6 promoter de novo sequence f* de novo sequence e* PGK promoter

>>>>>>

de novo sequence d*

>>>>>>>

Poly-T

2201 GAGCATGCGCTTTAGCAGCCCCGCTGGGCACTTGGCGCTACACAAGTGGCCTCTGGCCTCGCACACATTCCACATCCACCGGTAGGCGCCAACCGGCTCCGTTCTTTGGTGGCCCCTTCGCGCCACCTTCTACTCCTCCCCTAGTCAGGAAGTTCCCCCCCGCCCCGCAGCTCGCGTCGTGCAGGACGTGACAAATGGAA 2400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

PGK promoter

2401 GTAGCACGTCTCACTAGTCTCGTGCAGATGGACAGCACCGCTGAGCAATGGAAGCGGGTAGGCCTTTGGGGCAGCGGCCAATAGCAGCTTTGCTCCTTCGCTTTCTGGGCTCAGAGGCTGGGAAGGGGTGGGTCCGGGGGCGGGCTCAGGGGCGGGCTCAGGGGCGGGGCGGGCGCCCGAAGGTCCTCCGGAGGCCCGGC 2600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

PGK promoter

2601 ATTCTGCACGCTTCAAAAGCGCACGTCTGCCGCGCTGTTCTCCTCTTCCTCATCTCCGGGCCTTTCGACCTGCATCCCAAGCTTGCCACCATGGTGAGCAAGGGCGAGGAGCTGTTCACCGGGGTGGTGCCCATCCTGGTCGAGCTGGACGGCGACGTAAACGGCCACAAGTTCAGCGTGAGGGGCGAGGGCGAGGGCGA 2800

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

PGK promoter EBFP2

2801 TGCCACCAACGGCAAGCTGACCCTGAAGTTCATCTGCACCACCGGCAAGCTGCCCGTGCCCTGGCCCACCCTCGTGACCACCCTGAGCCACGGCGTGCAGTGCTTCGCCCGCTACCCCGACCACATGAAGCAGCACGACTTCTTCAAGTCCGCCATGCCCGAAGGCTACGTCCAGGAGCGCACCATCTTCTTCAAGGACG 3000

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

EBFP2

3001 ACGGCACCTACAAGACCCGCGCCGAGGTGAAGTTCGAGGGCGACACCCTAGTGAACCGCATCGAGCTGAAGGGCGTCGACTTCAAGGAGGACGGCAACATCCTGGGGCACAAGCTGGAGTACAACTTCAACAGCCACAACATCTATATCATGGCCGTCAAGCAGAAGAACGGCATCAAGGTGAACTTCAAGATCCGCCAC 3200

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

EBFP2

3201 AACGTGGAGGACGGCAGCGTGCAGCTCGCCGACCACTACCAGCAGAACACCCCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAGCCACTACCTGAGCACCCAGTCCGTGCTGAGCAAAGACCCCAACGAGAAGCGCGATCACATGGTCCTGCTGGAGTTCCGCACCGCCGCCGGGATCACTCTCGGCATGGACGAGCT 3400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

EBFP2

3401 GTACAAGTAAGTACCACTAGTACCGGTAGATATCAGCCATGGCTTCCCGCCGGCGGTGGCGGCGCAGGATGATGGCACGCTGCCCATGTCTTGTGCCCAGGAGAGCGGGATGGACCGTCACCCTGCAGCCTGTGCTTCTGCTAGGATCAATGTGTAGGCTAGTCGAGCAGACATGATAAGATACATTGATGAGTTTGGAC 3600

>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>

EBFP2 SV40 signal

3601 AAACCACAACTAGAATGCAGTGAAAAAAATGCTTTATTTGTGAAATTTGTGATGCTATTGCTTTATTTGTAACCATTATAAGCTGCAATAAACAAGTTAACAACAACAATTGCATTCATTTTATGTTTCAGGTTCAGGGGGAGATGTGGGAGGTTTTTTAAAGCAAGTAAAACCTCTACAAATGTGGTAAAATCCGATAA 3800

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

SV40 signal

3801 GGATCGATCCGGGCTGGCGTAATAGCGAAGAGGCCCGCACCGATCGCCCTTCCCAACAGTTGCCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGCGCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAAT 4000

4001 ACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTT 4099

Figu

reS2

0:E

xam

ple

anno

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dpl

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quen

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rte

rmin

ator

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S-tQ

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.

S40

pUC

Orig

in

KanR

dTom

ato

min

imal

CM

V pr

omot

erP1

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nal

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4766

bp

Figu

reS2

1:Pl

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duct

ion

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Are

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S41

1 GGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCG 200

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

pUC Origin

201 TGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGG 400

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

pUC Origin

401 TATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGCCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGG 600

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

pUC Origin

601 ATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAGCACGTGTCAGTCCTGCTCCTCGGCCACGAAGTGCACGCAGTTGCCGGCCGGGTCGCGCAGGG 800

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

pUC Origin

801 CGAACTCCCGCCCCCACGGCTGCTCGCCGATCTCGGTCATGGCCGGCCCGGAGGCGTCCCGGAAGTTCGTGGACACGACCTCCGACCACTCGGCGTACAGCTCGTCCAGGCCGCGCACCCACACCCAGGCCAGGGTGTTGTCCGGCACCACCTGGTCCTGGACCGCGCTGATGAACAGGGTCACGTCGTCCCGGACCACA 1000

1001 CCGGCGAAGTCGTCCTCCACGAAGTCCCGGGAGAACCCGAGCCGGTCGGTCCAGAACTCGACCGCTCCGGCGACGTCGCGCGCGGTGAGCACCGGAACGGCACTGGTCAACTTGGCCATGGTGGCCCTCCTCACGTGCTATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTA 1200

1201 GAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGATGCGGTGTGAAATACCGCACAGATGCGTAAGGAGAAAATACCGCATCAGGAAATTGTAAGCGTTAATAATTCAGAAGAACTCGTCAAGAAGGCGATAGAAGGCGATGCGCTGCGAATCGGGAGCGGCGATACCGTAAAGCACGAGG 1400

265

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

KanR

1401 AAGCGGTCAGCCCATTCGCCGCCAAGCTCTTCAGCAATATCACGGGTAGCCAACGCTATGTCCTGATAGCGGTCCGCCACACCCAGCCGGCCACAGTCGATGAATCCAGAAAAGCGGCCATTTTCCACCATGATATTCGGCAAGCAGGCATCGCCATGGGTCACGACGAGATCCTCGCCGTCGGGCATGCTCGCCTTGAG 1600

239

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

KanR

1601 CCTGGCGAACAGTTCGGCTGGCGCGAGCCCCTGATGCTCTTCGTCCAGATCATCCTGATCGACAAGACCGGCTTCCATCCGAGTACGTGCTCGCTCGATGCGATGTTTCGCTTGGTGGTCGAATGGGCAGGTAGCCGGATCAAGCGTATGCAGCCGCCGCATTGCATCAGCCATGATGGATACTTTCTCGGCAGGAGCAA 1800

173

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

KanR

1801 GGTGAGATGACAGGAGATCCTGCCCCGGCACTTCGCCCAATAGCAGCCAGTCCCTTCCCGCTTCAGTGACAACGTCGAGCACAGCTGCGCAAGGAACGCCCGTCGTGGCCAGCCACGATAGCCGCGCTGCCTCGTCTTGCAGTTCATTCAGGGCACCGGACAGGTCGGTCTTGACAAAAAGAACCGGGCGCCCCTGCGCT 2000

106

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

KanR

2001 GACAGCCGGAACACGGCGGCATCAGAGCAGCCGATTGTCTGTTGTGCCCAGTCATAGCCGAATAGCCTCTCCACCCAAGCGGCCGGAGAACCTGCGTGCAATCCATCTTGTTCAATCATGCGAAACGATCCTCATCCTGTCTCTTGATCAGAGCTTGATCCCCTGCGCCATCAGATCCTTGGCGGCAAGAAAGCCATCCA 2200

39

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

KanR

2201 GTTTACTTTGCAGGGCTTCCCAACCTTACCAGAGGGCGCCCCAGCTGGCAATTCCGGTTCGCTTGCTGTCCATAAAACCGCCCAGTCTAGCTATCGCCATGTAAGCCCACTGCAAGCTACCTGCTTTCTCTTTGCGCTTGCGTTTTCCCTTGTCCAGATAGCCCAGTAGCTGACATTCATCCGGGGTCAGCACCGTTTCT 2400

2401 GCGGACTGGCTTTCTACGTGAAAAGGATCTAGGTGAAGATCCTTTTTGATAATCTCATGCCTGACATTTATATTCCCCAGAACATCAGGTTAATGGCGTTTTTGATGTCATTTTCGCGGTGGCTGAGATCAGCCACTTCTTCCCCGATAACGGAGACCGGCACACTGGCCATATCGGTGGTCATCATGCGCCAGCTTTCA 2600

2601 TCCCCGATATGCACCACCGGGTAAAGTTCACGGGAGACTTTATCTGACAGCAGACGTGCACTGGCCAGGGGGATCACCATCCGTCGCCCCGGCGTGTCAATAATATCACTCTGTACATCCACAAACAGACGATAACGGCTCTCTCTTTTATAGGTGTAAACCTTAAACTGCCGTACGTATAGGCTGCGCAACTGTTGGGA 2800

2801 AGGGCGATCGGTGCGGGCCTCTTCGCTATTACGCCAGCTGGCGAAAGGGGGATGTGCTGCAAGGCGATTAAGTTGGGTAACGCCAGGGTTTTCCCAGTCACGACGTTGTAAAACGACGGCCAGTGAATTGTAATACGACTCACTATAGGGCGAATTGGGCCCTCTAGATGCATGCTCGAGCGGCCGCCAGTGTGATGGAT 3000

3001 ATCTGCAGAATTCGCCCTTGCGACGTAGGGATAACAGGGTAATAGTAGTCGCGTGTAGCGAAGCAGGGCGAGGTAGGCGTGTACGGTGGGAGGCCTATATAAGCAGAGCTCGTTTAGTGAACCGTCAGATCGCCTGGAGAATTCGCCACCATGGACTACAAGGATGACGACGATAAAACTTCCGGTGGCGGACTGGGTTC 3200

<<<<<<<<<<<<<<<<<<< >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

P1 target minimal CMV promoter

3201 CACCGTGAGCAAGGGCGAGGAGGTCATCAAAGAGTTCATGCGCTTCAAGGTGCGCATGGAGGGCTCCATGAACGGCCACGAGTTCGAGATCGAGGGCGAGGGCGAGGGCCGCCCCTACGAGGGCACCCAGACCGCCAAGCTGAAGGTGACCAAGGGCGGCCCCCTGCCCTTCGCCTGGGACATCCTGTCCCCCCAGTTCA 3400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

dTomato

3401 TGTACGGCTCCAAGGCGTACGTGAAGCACCCCGCCGACATCCCCGATTACAAGAAGCTGTCCTTCCCCGAGGGCTTCAAGTGGGAGCGCGTGATGAACTTCGAGGACGGCGGTCTGGTGACCGTGACCCAGGACTCCTCCCTGCAGGACGGCACGCTGATCTACAAGGTGAAGATGCGCGGCACCAACTTCCCCCCCGAC 3600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

dTomato

3601 GGCCCCGTAATGCAGAAGAAGACCATGGGCTGGGAGGCCTCCACCGAGCGCCTGTACCCCCGCGACGGCGTGCTGAAGGGCGAGATCCACCAGGCCCTGAAGCTGAAGGACGGCGGCCACTACCTGGTGGAGTTCAAGACCATCTACATGGCCAAGAAGCCCGTGCAACTGCCCGGCTACTACTACGTGGACACCAAGCT 3800

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

dTomato

3801 GGACATCACCTCCCACAACGAGGACTACACCATCGTGGAACAGTACGAGCGCTCCGAGGGCCGCCACCACCTGTTCCTGTACGGCATGGACGAGCTGTACAAGTAAGAATTCGAGCTCGGTACCCGGGGATCCTCTAGTCAGCTGACGCGTGCTAGCGCGGCCGCATCGATAAGCTTGTCGACGATATCTCTAGAGGATC 4000

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

dTomato

4001 ATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTTTCACTGCCTCGAGCTTCCTCGCT 4200

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

SV40 signal

4201 CACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATAAAGGGCGAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCCGAGCTCGGTACCAAGCTTGATGCATAGCTTGAGTATTCTATAGTGTCACCTAAATAGCTTGGCGTAATCATGGTCATAGCTGTTTCCTGT 4400

4401 GTGAAATTGTTATCCGCTCACAATTCCACACAACATACGAGCCGGAAGCATAAAGTGTAAAGCCTGGGGTGCCTAATGAGTGAGCTAACTCACATTAATTGCGTTGCGCTCACTGCCCGCTTTCCAGTCGGGAAACCTGTCGTGCCAGCTGCATTAATGAATCGGCCAACGCGCGGGGAGAGGCGGTTTGCGTATTGGGC 4600

4601 GCTCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAA 4766

Figu

reS2

2:A

nnot

ated

plas

mid

sequ

ence

for

indu

ctio

nas

say

repo

rter

inH

EK

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cells

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smid

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orte

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S42

S4 Schematics of putative ON and OFF states

S4.1 Constitutively active terminator switch cgRNA

cgRNA:triggercomplex

......

Target gene Y

......

Terminator switch mechanismOFF State

(presence of trigger X)ON State

(absence of trigger X)

cgRNA dCas9

Low target expression using silencing dCas9 High target expression using silencing dCas9

cgRNA:dCas9:targetcomplex

+ +

Figure S23: Schematics of putative ON and OFF states for the terminator switch mechanism. ON state: the terminator switch cgRNA isconstitutively active, directing the function of protein effector dCas9 to a target gene Y in the absence of trigger; the extended loop andmodified sequence domains in the terminator region (blue) are intended not to interfere with the activity of the cgRNA:dCas9 complex. OFFstate: in the presence of RNA trigger X, hybridization of the trigger is intended to form a structure incompatible with cgRNA mediation ofdCas9 function.

S4.2 Constitutively active splinted switch cgRNA


Target gene Y

......

Splinted switch mechanismOFF State

(presence of trigger X)ON State

(absence of trigger X)

cgRNA dCas9

Low target expression using silencing dCas9 High target expression using silencing dCas9

cgRNA:dCas9:targetcomplex

+ + ......

Figure S24: Schematics of putative ON and OFF states for the splinted switch mechanism. ON state: the splinted switch cgRNA isconstitutively active, directing the function of protein effector dCas9 to a target gene Y in the absence of trigger; the extended loops inthe Cas9 handle and terminator region (blue) are intended not to interfere with the activity of the cgRNA:dCas9 complex. OFF state: in thepresence of RNA trigger X, hybridization of the trigger is intended to form a splint that is structurally incompatible with cgRNA mediationof dCas9 function.

S43

S4.3 Constitutively inactive toehold switch cgRNA

cgRNATarget gene Y

......

Toehold switch mechanismOFF State

(absence of trigger X)ON State

(presence of trigger X)


dCas9

Low target expression using silencing dCas9High target expression using silencing dCas9

cgRNA:trigger:dCas9:targetcomplex

+ +

......

uu*

uuu*

Figure S25: Schematics of putative OFF and ON states for the toehold switch mechanism. OFF state: The toehold switch cgRNA isconstitutively inactive; the target-binding region (domain “u”; orange) is initially sequestered by a 50 extension to inhibit recognition of targetgene Y. ON state: in the presence of RNA trigger X, hybridization of the trigger to this extension via the toehold region (blue) is intended tode-sequester the target-binding region and enable cgRNA direction of dCas9 function to target gene Y.

S44

S5 Flow cytometry replicatesFlow cytometry replicates are shown for conditional response and crosstalk studies with all cgRNA mechanisms in Sec-tions S5.1–S5.4. These replicates are used to quantify the ON state, OFF state, fold change, and dynamic range of each cgRNAin Section S5.5 and the crosstalk between non-cognate cgRNA/trigger pairs in Section S5.6 using uncertainty propagation asdetailed in Sections S1.4 and S1.5.

S5.1 Constitutively active terminator switch in E. coliS5.1.1 ON state, OFF state, and conditional response (cf. Figure 2c)

101 103 105


0

1000

2000

Cou

nts

AutofluorescenceStandard gRNA (ideal ON)cgRNA (ON state)cgRNA + trigger (OFF state)No-target gRNA (ideal OFF)

ON OFF

Terminator switch cgRNA in bacteria (ON OFF logic)

ConstitutivelyActive

cgRNAXY

silencing dCas9

Figure S26: Flow cytometry replicates for terminator switch ON state, OFF state, and conditional response in E. coli (cf. Figure 2c).Single-cell fluorescence intensities. Expression of RNA trigger X toggles the cgRNA from ON!OFF, leading to an increase in fluorescence.Induced expression (aTc) of silencing dCas9 and constitutive expression of mRFP target gene Y and either: standard gRNA (ideal ON state),cgRNA (ON state), cgRNA + RNA trigger X (OFF state; trigger expression is IPTG-induced), no-target gRNA that lacks target-bindingregion (ideal OFF state). Autofluorescence: cells with no mRFP. Traces of the same color correspond to N = 3 replicate wells assayed onthe same day (20,000 cells per well).

S5.1.2 Orthogonal library studies (cf. Figure 2d)

101 103 105


0

1000

2000

Cou

nts

AutofluorescenceStandard gRNAcgRNA AcgRNA A + trigger AcgRNA A + trigger BcgRNA A + trigger C

a b c

101 103 105


0

1000

2000

Cou

nts

AutofluorescenceStandard gRNAcgRNA BcgRNA B + trigger AcgRNA B + trigger BcgRNA B + trigger C

101 103 105


0

1000

2000

Cou

nts

AutofluorescenceStandard gRNAcgRNA CcgRNA C + trigger AcgRNA C + trigger BcgRNA C + trigger C

Figure S27: Flow cytometry replicates for terminator switch orthogonal response in E. coli (cf. Figure 2d). (a) cgRNA A. (b) cgRNAB. (c) cgRNA C. Single-cell fluorescence intensities. Induced expression (aTc) of silencing dCas9 and constitutive expression of mRFP targetgene Y and either: standard gRNA, cgRNA without trigger, cgRNA + cognate trigger, or cgRNA + a non-cognate trigger (trigger expressionis IPTG-induced). Autofluorescence: cells with no mRFP. Expression of the cognate RNA trigger (XA for cgRNA A, XB for cgRNA B, XC

for cgRNA C) toggles the cgRNA from ON!OFF, leading to an increase in fluorescence. Traces of the same color correspond to N = 3replicate wells assayed on the same day (20,000 cells per well).

S45

S5.2 Constitutively active splinted switch in E. coliS5.2.1 ON state, OFF state, and conditional response (cf. Figure 3c)

101 103 105


0

1000

2000

Cou

nts


ON OFF

Splinted switch cgRNA in bacteria (ON OFF logic)


cgRNAXY

silencing dCas9

Figure S28: Flow cytometry replicates for splinted switch ON state, OFF state, and conditional response in E. coli (cf. Figure 3c).Single-cell fluorescence intensities. Expression of RNA trigger X toggles the cgRNA from ON!OFF, leading to an increase in fluorescence.Induced expression (aTc) of silencing dCas9 and constitutive expression of sfGFP target gene Y and either: standard gRNA (ideal ON state),cgRNA (ON state), cgRNA + RNA trigger X (OFF state), no-target gRNA that lacks target-binding region (ideal OFF state). Autofluo-rescence: cells with no sfGFP. Traces of the same color correspond to N = 3 replicate wells assayed on the same day (20,000 cells perwell).


101 103 105


0

1000

2000

Cou

nts

101 103 105


0

1000

2000

Cou

nts

101 103 105


0

1000

2000C

ount

sa b c




Figure S29: Flow cytometry replicates for splinted switch orthogonal response in E. coli (cf. Figure 3d). (a) cgRNA A. (b) cgRNA B.(c) cgRNA C. Single-cell fluorescence intensities. Induced expression (aTc) of silencing dCas9 and constitutive expression of sfGFP targetgene Y and either: standard gRNA, cgRNA without trigger, cgRNA + cognate trigger, or cgRNA + a non-cognate trigger. Autofluorescence:cells with no sfGFP. Expression of the cognate RNA trigger (XA for cgRNA A, XB for cgRNA B, XC for cgRNA C) toggles the cgRNA fromON!OFF, leading to an increase in fluorescence. Traces of the same color correspond to N = 3 replicate wells assayed on the same day(20,000 cells per well).

S46

S5.3 Constitutively inactive toehold switch in E. coliS5.3.1 ON state, OFF state, and conditional response (cf. Figure 4c)

cgRNA + trigger (ON state)cgRNA (OFF state)No-target gRNA (ideal OFF)

101 103 105


0

1000

2000

Cou

nts

AutofluorescenceStandard gRNA (ideal ON)

ON OFF

Toehold switch cgRNA in bacteria (OFF ON logic)

ConstitutivelyInactive

cgRNAXY

silencing dCas9

Figure S30: Flow cytometry replicates for toehold switch ON state, OFF state, and conditional response in E. coli (cf. Figure 4c).Single-cell fluorescence intensities. Expression of RNA trigger X toggles the cgRNA from OFF!ON, leading to a decrease in fluores-cence. Induced expression (aTc) of silencing dCas9 and constitutive expression of mRFP target gene Y and either: no-target gRNA thatlacks target-binding region (ideal OFF state), cgRNA (OFF state), cgRNA + RNA trigger X (ON state), standard gRNA (ideal ON state).Autofluorescence: cells with no mRFP. Traces of the same color correspond to N = 3 replicate wells assayed on the same day (20,000 cellsper well).


101 103 105


0

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nts

a b c

101 103 105


0

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nts

101 103 105


0

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ount

s




Figure S31: Flow cytometry replicates for toehold switch orthogonal response in E. coli (cf. Figure 4d). Flow cytometry fluorescenceassay shows selective activation of Toehold Switch cgRNA with expression of cognate RNA trigger. (a) cgRNA A. (b) cgRNA B. (c) cgRNAC. Single-cell fluorescence intensities. Induced expression (aTc) of silencing dCas9 and constitutive expression of mRFP target gene Y andeither: standard gRNA, cgRNA without trigger, cgRNA + cognate trigger, or cgRNA + a non-cognate trigger. Autofluorescence: cells with nomRFP. Expression of the cognate RNA trigger (XA for cgRNA A, XB for cgRNA B, XC for cgRNA C) toggles the cgRNA from OFF!ON,leading to decrease in fluorescence. Traces of the same color correspond to N = 3 replicate wells assayed on the same day (20,000 cells perwell).

S47

S5.4 Constitutively active terminator switch in HEK 293T cellsS5.4.1 ON state, OFF state, and conditional response (cf. Figure 5b)

0

100

200

10 1 10 3 10 5 10 7


Cou

nts

Terminator switch cgRNA in HEK 293T cells (ON OFF logic)


cgRNAXY

inducing dCas9

0

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0.8

1

10 1 10 3 10 5 10 7


ECD

F

a b

No-target gRNA (ideal OFF)

Standard gRNA (ideal ON)cgRNA (ON state)cgRNA + trigger (OFF state)

Figure S32: Flow cytometry replicates for terminator switch ON state, OFF state, and conditional response in HEK 293T cells (cf.Figure 5b). (a) Single-cell fluorescence intensities. (b) Empirical cumulative distribution function (ECDF) with bootstrapped 95% confidenceintervals (gray) obtained by sampling 10,000 bootstrap replicates of the ECDF (sampling M cells with replacement for each replicate) andthen calculating the 2.5th and 97.5th percentiles from the replicates for each plotted fluorescence value.15, 16 The mean for each replicate isdisplayed as a vertical line. The confidence intervals are tight around the ECDFs, and the OFF state replicates (cgRNA + cognate trigger)exhibit a consistent shift to the left relative to the ON state replicates (cgRNA-only), contributing to a measurable mean conditional ON!OFFresponse. Expression of RNA trigger X toggles the cgRNA from ON!OFF, leading to a decrease in fluorescence. Transient expression ofinducing dCas9 and dTomato target gene Y and either: standard gRNA (ideal ON state), cgRNA (ON state), cgRNA + RNA trigger X (OFFstate), no-target gRNA that lacks target-binding region (ideal OFF state). Traces of the same color correspond to N = 3 replicate wellstransfected on the same day and assayed via flow cytometry after 24 h (M = 3500 cells from the high-transfection gate per well).

S48

S5.4.2 Orthogonal library studies (cf. Figure 5c)


Standard gRNA (ideal ON)cgRNA Q (ON state)

cgRNA Q + trigger Q (OFF state)cgRNA Q + trigger R (ON state)cgRNA Q + trigger S (ON state)

aC

ount

s

10 1 10 3 10 5 10 70

100

200


b


Standard gRNA (ideal ON)cgRNA R (ON state)

cgRNA R + trigger Q (ON state)cgRNA R + trigger R (OFF state)cgRNA R + trigger S (ON state)


Cou

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10 1 10 3 10 5 10 70

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Fluorescence intensity (au)10 1 10 3 10 5 10 70

0.2

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Standard gRNA (ideal ON)cgRNA S (ON state)

cgRNA S + trigger Q (ON state)cgRNA S + trigger R (ON state)cgRNA S + trigger S (OFF state)


Cou

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10 1 10 3 10 5 10 70

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cgR

NA

Qcg

RN

A R

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NA

S

shift

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Figure S33: Flow cytometry replicates for terminator switch orthogonal response in HEK 293T cells (cf. Figure 5c). (a) Single-cellfluorescence intensities. (b) Empirical cumulative distribution function (ECDF) with bootstrapped 95% confidence intervals (gray) obtainedby sampling 10,000 bootstrap replicates of the ECDF (sampling M cells with replacement for each replicate) and then calculating the 2.5thand 97.5th percentiles from the replicates for each plotted fluorescence value.15, 16 The mean for each replicate is displayed as a vertical line.The confidence intervals are tight around the ECDFs, and the OFF state replicates (cgRNA + cognate trigger) exhibit a consistent shift tothe left at the top right corner of the ECDF relative to the ON state replicates (cgRNA-only or cgRNA + non-cognate trigger), contributingto a measurable mean conditional ON!OFF response. Top: cgRNA Q. Middle: cgRNA R. Bottom: cgRNA S. Transient expression ofinducing dCas9 and dTomato target gene Y and either: standard gRNA, cgRNA without trigger, cgRNA + cognate trigger, cgRNA + a non-cognate trigger, or no-target gRNA that lacks target-binding region. Expression of the cognate RNA trigger (XQ for cgRNA Q, XR for cgRNAR, XS for cgRNA S) toggles the cgRNA from ON!OFF, leading to a decrease in fluorescence. Traces of the same color correspond toN = 3 replicate wells transfected on the same day and assayed via flow cytometry after 24 h. To enable comparison of distributions betweenreplicates, the same number of cells are displayed for all replicates of a given cgRNA: M = 3500 cells for Q (top), M = 3000 cells for R(middle), M = 2400 cells for S (bottom), with the exception that one replicate for S yielded only M = 415 cells (displayed with dottedlines; note that the confidence intervals for this replicate are not as tight due to the smaller number of cells).

S49

S5.5 Quantifying ON state, OFF state, fold change, and dynamic range

cgRNA ON OFF Fold Change Dynamic Range Fractional Dynamic Range

a Terminator switch mechanism in E. coliIdeal 1200 ± 20 108 900± 1800 91 ± 2 108 000± 2000 1cgRNA A 4020 ± 60 23 700± 900 5.9 ± 0.2 19 700± 900 0.182 ± 0.009cgRNA B 5200 ± 300 20 700± 800 4.0 ± 0.3 15 500± 900 0.144 ± 0.009cgRNA C 5200 ± 500 13 100± 500 2.5 ± 0.3 7900± 700 0.074 ± 0.006

b Splinted switch mechanism in E. coliIdeal 100 ± 40 35 450± 80 300 ± 100 35 350± 70 1cgRNA A 150 ± 40 2270± 50 15 ± 4 2120± 30 0.0599± 0.0008cgRNA B 140 ± 40 2450± 50 18 ± 5 2310± 30 0.0654± 0.0009cgRNA C 160 ± 40 1320± 70 8 ± 2 1160± 60 0.033 ± 0.002

c Toehold switch mechanism in E. coliIdeal 340 ± 30 102 000± 4000 300 ± 30 102 000± 4000 1cgRNA A 4400 ± 200 18 400± 400 4.2 ± 0.2 14 000± 500 0.138 ± 0.007cgRNA B 10 200 ± 400 16 000± 300 1.57± 0.07 5800± 500 0.057 ± 0.005cgRNA C 4890 ± 110 15 300± 500 3.13± 0.12 10 400± 500 0.102 ± 0.006

d Terminator switch mechanism in HEK 293T cellsIdeal 5 400 000 ± 500 000 0± 3000 5 300 000± 500 000 1cgRNA Q 1 780 000 ± 30 000 450 000± 40 000 4.0 ± 0.4 1 330 000± 50 000 0.25 ± 0.03cgRNA R 1 500 000 ± 120 000 690 000± 50 000 2.2 ± 0.2 810 000± 130 000 0.15 ± 0.03cgRNA S 785 000 ± 97 000 150 000± 30 000 5.2 ± 1.2 630 000± 100 000 0.12 ± 0.02

Table S13: Quantifying ON state, OFF state, fold change, and dynamic range. (a) Terminator switch in E. coli (cf. Figure 2d). (b)Splinted switch in E. coli (cf. Figure 3d). (c) Toehold switch in E. coli (cf. Figure 4d). (d) Terminator switch in HEK 293T cells (cf.Figure 5c). See Sections S1.4 and S1.5 for quantification details (ON and OFF values are background-subtracted mean fluorescent signal;for background subtraction, autofluorescence (AF) values were 34 ± 5 (terminator switch), 210 ± 40 (splinted switch), and 33 ± 2 (toeholdswitch) in E. coli and no-target gRNA fluorescence was 21000 ± 2000 for the terminator switch in HEK 293T cells). Ideal values correspondto a standard gRNA for ideal ON and a no-target gRNA for ideal OFF. Using silencing dCas9 in E. coli (a,b,c): fold change = OFF/ON,dynamic range = OFF−ON, fractional dynamic range = [OFF−ON]/[ideal OFF − ideal ON], M = 20, 000 cells per well; using inducingdCas9 in HEK 293T cells (d): fold change = ON/OFF, dynamic range = ON−OFF, fractional dynamic range = [ON−OFF]/[ideal ON −ideal OFF], M = 426–7714 cells per well. Mean ± estimated standard error (with uncertainty propagation) based on the mean single-cellfluorescence over M cells for each of N = 3 replicate wells.

S50

S5.6 Quantifying crosstalk for cognate and non-cognate cgRNA/trigger pairs

cgRNA Trigger Signal Crosstalk

a Terminator switch mechanism in E. coliA no trigger 4020 ± 60A A 23 700 ± 900 1.00 ± 0.06A B 3690 ± 80 −0.017± 0.005A C 1660 ± 50 −0.120± 0.007B no trigger 5200 ± 300B A 6000 ± 300 0.05 ± 0.03B B 20 700 ± 800 1.00 ± 0.08B C 5500 ± 400 0.02 ± 0.04C no trigger 5200 ± 500C A 4800 ± 300 −0.05 ± 0.07C B 4200 ± 100 −0.13 ± 0.06C C 13 100 ± 500 1.00 ± 0.12

b Splinted switch mechanism in E. coliA no trigger 150 ± 40A A 2270 ± 50 1.00 ± 0.02A B 110 ± 40 −0.019± 0.003A C 110 ± 40 −0.021± 0.005B no trigger 140 ± 40B A 190 ± 40 0.021± 0.006B B 2450 ± 50 1.00 ± 0.02B C 420 ± 40 0.122± 0.007C no trigger 160 ± 40C A 380 ± 40 0.19 ± 0.02C B 150 ± 40 −0.007± 0.011C C 1320 ± 70 1.00 ± 0.07

c Toehold switch mechanism in E. coliA no trigger 18 400 ± 400A A 4400 ± 200 1.00 ± 0.05A B 14 100 ± 300 0.31 ± 0.04A C 12 800 ± 400 0.40 ± 0.05B no trigger 16 000 ± 300B A 17 500 ± 900 −0.3 ± 0.2B B 10 200 ± 400 1.00 ± 0.12B C 18 500 ± 600 −0.43 ± 0.12C no trigger 15 300 ± 500C A 14 000 ± 500 0.12 ± 0.07C B 13 000 ± 60 0.22 ± 0.05C C 4890 ± 110 1.00 ± 0.06

d Terminator switch mechanism in HEK 293T cellsQ no trigger 1 780 000 ± 30 000Q Q 450 000 ± 40 000 1.00 ± 0.05Q R 1 160 000 ± 110 000 0.46 ± 0.09Q S 1 350 000 ± 60 000 0.32 ± 0.05R no trigger 1 500 000 ± 120 000R Q 1 560 000 ± 40 000 −0.1 ± 0.2R R 690 000 ± 50 000 1.0 ± 0.2R S 1 430 000 ± 80 000 0.1 ± 0.2S no trigger 785 000 ± 97 000S Q 690 000 ± 50 000 0.1 ± 0.2S R 480 000 ± 30 000 0.5 ± 0.2S S 150 000 ± 30 000 1.0 ± 0.2

Table S14: Quantifying crosstalk for cognate and non-cognate cgRNA/trigger pairs. (a) Terminator switch in E. coli (cf. Figure 2d). (b)Splinted switch in E. coli (cf. Figure 3d). (c) Toehold switch in E. coli (cf. Figure 4d). (d) Terminator switch in HEK 293T cells (cf. Figure5c). See Sections S1.4 and S1.5 for quantification details (for background subtraction, autofluorescence (AF) values were 34 ± 5 (terminatorswitch), 210 ± 40 (splinted switch), and 33 ± 2 (toehold switch) in E. coli and no-target gRNA fluorescence was 21000 ± 2000 for theterminator switch in HEK 293T cells). Using silencing dCas9 in E. coli (a,b,c): M = 20, 000 cells per well; using inducing dCas9 in HEK293T cells (d): M = 426–7714 cells per well. Mean ± estimated standard error (with uncertainty propagation) based on the mean single-cellfluorescence over M cells for each of N = 3 replicate wells.

S51

S6 Additional Studies

S6.1 Constitutively active terminator switch using alternative plasmid layout and constitutive trig-ger expression in E. coli

For the terminator switch mechanism, many of the E. coli strains generate bimodal fluorescence histograms (see Figure 2c andthe replicates of Figures S26 and S27). The fact that the second hump is observed for the standard gRNA control strain as wellas for the cgRNA-only strains suggests that it is a property of the assay and not related to the terminator switch mechanism.Preliminary studies demonstrated variation of the relative size of the two humps between biological replicates, consistent withescape mutation. To test whether the second hump could be eliminated by using the same gRNA/cgRNA/trigger sequences(Table S3) in a different plasmid context, we generated 14 new terminator switch strains (3 cgRNA ⇥ [3 triggers + 1 no-trigger] + standard gRNA + no-target gRNA [autofluorescence strain same as previous]) using plasmids (Section S6.1.1) similarin construction to those of the splinted switch mechanism (Section S3.2), including constitutive trigger expression (dCas9-only plasmid) rather than the lacI-regulated trigger expression (dCas9+lacI plasmid) previously used for the terminator switch(Figure 2). Constructs were generated by inserting cgRNA and trigger gene fragments (ordered as gBlocks from IDT) intothe pg-gRNA derived backbone5 (Addgene plasmid #44251; gift from S. Qi), and subsequently inserting trigger cassette intothe cgRNA vector using BioBrick assembly.6, 7 The previously described pdCas9-bacteria vector5 (Addgene plasmid #44251)was used for tetR-regulated dCas9 expression, and cells were transformed, cultivated and prepared for the fluorescence assayas described in Section S1.2. A final working concentration of 2 nM aTc was used for induction of dCas9 expression. NoIPTG was added to experimental wells, as trigger was constitutively expressed. Protein fluorescence was measured using theMACSQuant VYB flow cytometer (Miltenyi Biotec) as described in Section S1.2.3.

As with the terminator switch cgRNA mechanism with trigger expression regulated by lacI (Figure 2), an E. coli strainexpressing the cgRNA exhibits low fluorescence (ON state) while a strain expressing both the cgRNA and the cognate RNAtrigger exhibit high fluorescence (OFF state), achieving a conditional ON!OFF response (Figure S34a). To test programma-bility, we again tested three orthogonal cgRNA/trigger pairs (Figure S34b), achieving a median ⇡6-fold conditional ON!OFFresponse to expression of the cognate trigger (left) and negative median crosstalk between non-cognate cgRNA/trigger com-binations (right). Compared with the characterization of the terminator switch mechanism in the setting with lacI-regulatedtrigger expression (Figure 2c), the ON state is significantly closer to the ideal ON state (standard gRNA), with significant roomfor improvement in the OFF state relative to the ideal OFF state (no-target gRNA) (Figure S34a). Although typical crosstalkbetween non-cognate cgRNA/trigger pairs remains low, we observed a significant increase in crosstalk between cgRNA A andtrigger XC. Notably, examining the standard gRNA and cgRNA-only histograms that previously exhibited bimodal fluorescence(Figures S26 and S27), the new strains exhibit unimodal fluorescence (Figures S37 and S38), confirming that the existence of asecond hump is not a property of the terminator switch.

S52

A

B

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XC

XAXB

b

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Foldchange

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Trigger

XA

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-

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-

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B

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101 103 1050

1000

2000ON OFF


Cou

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6.2 ± 0.4

2.87 ± 0.09

Crosstalk

AutofluorescenceStandard gRNAcgRNAcgRNA + triggerNo-target gRNA

0 0.5 1.00 1000 2000

a

0.06-0.1

-0.04 0.4

Figure S34: Constitutively active terminator switch cgRNAs (ON!OFF logic) using alternative plasmid layout and constitutive trig-ger expression with silencing dCas9 in E. coli. (a) Expression of RNA trigger X (40 nt + synthetic terminator) toggles the cgRNA fromON!OFF, leading to an increase in fluorescence. Single-cell fluorescence intensities via flow cytometry. Induced expression (aTc) of silenc-ing dCas9 and constitutive expression of mRFP target gene Y and either: standard gRNA (ideal ON state), cgRNA (ON state), cgRNA + RNAtrigger X (OFF state; constitutive trigger expression), no-target gRNA that lacks target-binding region (ideal OFF state). Autofluorescence(AF): cells with no mRFP. (d) Programmable conditional regulation using 3 orthogonal cgRNAs (A, B, C). Left: Raw fluorescence depictingON!OFF conditional response to cognate trigger (fold change = OFF/ON = [cognate trigger−AF]/[no trigger−AF]). Right: normalized flu-orescence depicting orthogonality between non-cognate cgRNA/trigger pairs (crosstalk = [non-cognate trigger − no trigger]/[cognate trigger− no trigger]). Bar graphs depict mean ± estimated standard error calculated based on the mean single-cell fluorescence over 20,000 cellsfor each of N = 3 replicate wells (OFF:ON ratio and crosstalk calculated with uncertainty propagation).

S53

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>>>>>> >>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

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201 GGGGTTTTTTTTCTACACCCTTCTAACAACTCCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACGCTCTCTACTAGAGTCACACTGGCTCACCTTCGGGTGGGCCTTTCTGCGTTTATAGGATCTGAAACATGACGAGCTGGTGAGCATAAGGAGCTG 400

>>>>>>>>>>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>

BBa_B1006 BBa_B0015 Scar BBa_J23108

401 ACAGCTAGCTCAGTCCTAGGTATAATGCTAGCAACTTTCAGTTTAGCGGTCTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGATCAAACGGGTAAACAAACAGGATAATTAAGGAGGCAGTACCCGGGCACCGAGTCGGTGCTTTTTTTAAAAAAAAACCCCGCCCCTGACAGGGCGGGGTTTTTTTT 600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> >>>>>> >>>> >>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

BBa_J23108 mRFP target-specific spacer de novo sequence d de novo sequence e* BBa_B1006

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Standard gRNA sequence de novo sequence e Standard gRNA sequence

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

de novo sequence f

601 GCGCTATCAGTTGACACAGTGAAGCTTGGGCCCGAACAAAAACTCATCTCAGAAGAGGATCTGAATAGCGCCGTCGACCATCATCATCATCATCATTGAGTTTAAACGGTCTCCAGCTTGGCTGTTTTGGCGGATGAGAGAAGATTTTCAGCCTGATACAGATTAAATCAGAACGCAGAAGCGGTCTGATAAAACAGAAT 800

801 TTGCCTGGCGGCAGTAGCGCGGTGGTCCCACCTGACCCCATGCCGAACTCAGAAGTGAAACGCCGTAGCGCCGATGGTAGTGTGGGGTCTCCCCATGCGAGAGTAGGGAACTGCCAGGCATCAAATAAAACGAAAGGCTCAGTCGAAAGACTGGGCCTTTCGTTTTATCTGTTGTTTGTCGGTGAACTGGATCCTTACTC 1000

>>>>>>

BamHI

1001 GAGTCTAGACTGCAGGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGGGGATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAG 1200

>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1201 GCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTA 1400

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1401 GGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTG 1600

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1601 AAGTGGTGGCCTAACTACGGCTACACTAGAAGGACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTC 1800

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>

ColE1

1801 TACGGGGTCTGACGCTCAGTGGAACGAAAACTCACGTTAAGGGATTTTGGTCATGAGATTATCAAAAAGGATCTTCACCTAGATCCTTTTAAATTAAAAATGAAGTTTTAAATCAATCTAAAGTATATATGAGTAAACTTGGTCTGACAGTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTC 2000

>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>>> <<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

ColE1 AmpR

2001 GTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGTGCTGCAATGATACCGCGAGACCCACGCTCACCGGCTCCAGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAAT 2200

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2201 TGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTTGCGCAACGTTGTTGCCATTGCTACAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGT 2400

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2401 CAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTCAATACGGGATAATACCGCGCCACATAGCAGAACTT 2600

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2601 TAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGT 2800

<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR

2801 TGAATACTCATACTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTTCCGCGCACATTTCCCCGAAAAGTGCCACCTGACGTCTAAGAAACCATTATTATCATGACATTAACCTATAAA 2983

<<<<<<<<<<< <<<<<<<<<<<<<<<<<<<<<<<<<<<<<

AmpR AmpR_promoter

Figu

reS3

6:E

xam

ple

anno

tate

dpl

asm

idse

quen

cefo

ral

tern

ativ

ete

rmin

ator

switc

hpl

asm

idla

yout

with

cons

titut

ive

trig

ger

expr

essi

onin

E.c

oli(

cf.F

igur

eS1

0).P

lasm

id:

paTr

mS-

cgA

-tA

forc

gRN

AA

+tr

igge

rA.

S56

S6.1.2 Flow cytometry replicates for ON state, OFF state, and conditional response (cf. Figure 2c and Section S5.1.1)

101 103 105


0

1000

2000

Cou

nts


ON OFF

Terminator switch cgRNA in bacteria (ON OFF logic)


cgRNAXY

silencing dCas9

Figure S37: Flow cytometry replicates for terminator switch ON state, OFF state, and conditional response in E. coli with constitutivetrigger expression (cf. Figure S26). Single-cell fluorescence intensities. Expression of RNA trigger X toggles the cgRNA from ON!OFF,leading to an increase in fluorescence. Induced expression (aTc) of silencing dCas9 and constitutive expression of mRFP target gene Y andeither: standard gRNA (ideal ON state), cgRNA (ON state), cgRNA + RNA trigger X (OFF state; constitutive trigger expression), no-targetgRNA that lacks target-binding region (ideal OFF state). Autofluorescence: cells with no mRFP. Traces of the same color correspond toN = 3 replicate wells assayed on the same day (20,000 cells per well).

S6.1.3 Flow cytometry replicates for orthogonal library studies (cf. Figure 2d and Section S5.1.2)

101 103 105


0

1000

2000

Cou

nts


a b c

101 103 105


0

1000

2000

Cou

nts


101 103 105


0

1000

2000

Cou

nts


Figure S38: Flow cytometry replicates for terminator switch orthogonal response in E. coli with constitutive trigger expression (cf.Figure S27). (a) cgRNA A. (b) cgRNA B. (c) cgRNA C. Single-cell fluorescence intensities. Induced expression (aTc) of silencing dCas9and constitutive expression of mRFP target gene Y and either: standard gRNA, cgRNA without trigger, cgRNA + cognate trigger, or cgRNA+ a non-cognate trigger (constitutive trigger expression). Autofluorescence: cells with no mRFP. Expression of the cognate RNA trigger (XA

for cgRNA A, XB for cgRNA B, XC for cgRNA C) toggles the cgRNA from ON!OFF, leading to an increase in fluorescence. Traces of thesame color correspond to N = 3 replicate wells assayed on the same day (20,000 cells per well).

S57

S6.2 Single and double sequence inserts for construction of allosteric cgRNAs in E. coliS6.2.1 Quantifying performance of candidate cgRNAs using 71 E. coli strains

To establish a basis for engineering allosteric cgRNAs with trigger sequence X independent of the target gene sequence Y, wetested candidate cgRNAs with designed single-stranded sequence inserts of each of three lengths (15, 25, and 35 nt) at each offour insert sites (50-extension, Cas9 handle loop, terminator loop 1, terminator loop 2; Figure S39b) or at pairwise combinationsof insert sites. In order to develop highly functional cgRNA mechanisms, the goal is to identify insert locations that:

• are well-tolerated by dCas9 so that the ON state is similar to the standard gRNA, and• upon hybridization to a cognate trigger inactivate the cgRNA to permit a clean OFF state.

Insert sequences were designed for each of the candidate cgRNAs and their corresponding triggers using the reaction pathwayengineering tools within NUPACK employing a design formulation analogous to that of the splinted switch mechanism (Sec-tion S1.1.2). Methods for plasmid construction and molecular cloning, bacterial culture, and flow cytometry were identical tothose for the splinted switch mechanism (Sections S1.2 and S3.2). Using silencing dCas9, the ideal ON state is measured usingthe standard gRNA. To check whether the inserts in a given candidate cgRNA are tolerated by dCas9, we measure fluorescencefor a cgRNA-only strain (ON state). To check for conditional inactivation of the cgRNA by the cognate trigger (OFF state), wemeasure fluorescence in a cgRNA + trigger strain. The ideal OFF state is measured in a strain expressing a no-target gRNAlacking the target-binding region. Autofluorescence (AF) is measured in a strain expressing no fluorescent protein reporters.A total of 71 strains were used to quantify performance of single and double inserts: AF, no-target gRNA, standard gRNA, 16single-insert cgRNA-only (no trigger), 16 single-insert cgRNA + cognate trigger, 18 double-insert cgRNA-only (no trigger), 18double-insert cgRNA + cognate trigger.

The raw data are displayed for all insert combinations in Figure S39c-e. Table S16 quantifies ON, OFF, fold change(OFF/ON), dynamic range (OFF − ON), and fractional dynamic range ([OFF − ON]/[ideal OFF − ideal ON]). For the cgRNAwithout trigger, all modifications appear well-tolerated by dCas9, with strong ON state silencing activity comparable to thestandard gRNA. For each candidate cgRNA, the cognate trigger corresponds to the expressed reverse complement of the inserteddesigned sequence plus a synthetic terminator (see splinted switch plasmid design, Section S3.2). Expression of the cognateRNA trigger is intended to toggle the cgRNA from ON!OFF leading to an increase in fluorescence. The candidate cgRNA withthe highest fractional dynamic range was a double modification with 35 nt inserts in the dCas9 handle loop and the terminatorloop 1. This corresponds to the splinted switch mechanism (Figure 3). An alternative splinted switch cgRNA concept with35 nt (or 25 nt) inserts in the 50 extension and dCas9 handle also exhibits a large dynamic range and merits further investigation.

S58

d

a

i) 5’

ext

ensi

on

ii) H

andl

e lo

op

iii) T

erm

inat

or lo

op 1

iv) T

erm

inat

or lo

op 2

Mean fluorescence (au)

Trig

ger

5’ E

xten

sion

Han

dle

Loop

Term

inat

or L

oop

1Te

rmin

ator

Loo

p 2

+-

+-

+-

+-

+-

+-

+-

+-

+-

+-

+-

+-

1525

35-

--

--

--

--

--

--

--

--

--

--

--

--

--

--

--

--

--

-15

2535

15b

25b

3515

a25

a35

Auto

Standard gRNANo-target gRNA

--

-0

1000

0

2000

0

c

Can

dida

te c

gRN

A +

cogn

ate

trigg

er (O

FF s

tate

) N

o-ta

rget

gR

NA

(idea

l OFF

)

Can

dida

te c

gRN

A (O

N s

tate

) St

anda

rd g

RN

A (id

eal O

N)

+-

+-

+-

+-

+-

+-

+-

+-

+-

1525

3515

2535

1525

35-

--

15a

25a

3515

a25

a35

--

--

--

1525

35-

--

--

--

--

Auto


--

-

Alte

rnat

ive

splin

ted

switc

hco

ncep

t

Con

stitu

tivel

yAc

tive

cgR

NA

XY

sile

ncin

g dC

as9

Inte

nded

(ON

O

FF) l

ogic

for

cand

idat

e cg

RN

A m

echa

nism

s

b

+-

+-

+-

+-

+-

+-

+-

+-

+-

--

--

--

--

--

--

15b

25b

35-

--

15a

25a

3515

2535

1525

35-

--

15b

25b

3515

b25

b35

Auto


--

--

--

-25

a-

-15

a-

15b

25b

--

--

-+-

+-

+-

+-

Splin

ted

switc

hm

echa

nism

of

Figu

re 3

e

Figu

reS3

9:Pe

rfor

man

ceof

sing

lean

ddo

uble

sequ

ence

inse

rts

for

cons

truc

tion

ofal

lost

eric

cgR

NA

sin

E.c

oli.

(a)

Inte

nded

(ON

!O

FF)

cond

ition

allo

gic

for

cand

idat

ecg

RN

Am

echa

nism

s.(b

)Sc

hem

atic

depi

ctin

gse

quen

cein

sert

loca

tions

(blu

e).

Des

igne

dse

quen

ces

of15

nt,2

5nt

,and

35nt

wer

ein

sert

edin

toth

est

anda

rdgR

NA

sequ

ence

atei

ther

one

ortw

oof

the

four

inse

rtsi

tes:

i)50

exte

nsio

n(s

eque

nce

adde

dto

the

50en

dof

the

gRN

A;n

oot

hers

eque

nce

repl

aced

);ii)

Cas

9ha

ndle

loop

(des

igne

dse

quen

cere

plac

ing

the

4nt

stan

dard

gRN

Aha

ndle

loop

sequ

ence

[GAAA

]);i

ii)Te

rmin

ator

loop

1(d

esig

ned

sequ

ence

repl

acin

gth

e4

ntst

anda

rdgR

NA

sequ

ence

ofth

efir

stS

.p

yogen

es

term

inat

orlo

op[GAAA

]);i

v)Te

rmin

ator

loop

2(d

esig

ned

sequ

ence

repl

acin

gth

e3

ntst

anda

rdgR

NA

sequ

ence

ofth

ese

cond

S.p

yogen

es

term

inat

orlo

op[AGT

]).A

llte

sted

sing

lean

ddo

uble

inse

rtco

mbi

natio

nsar

elis

ted

inTa

ble

S17.

(c)S

ingl

ein

sert

cond

ition

alre

spon

se(1

5,25

,and

35nt

atea

chof

four

inse

rtsi

tes)

.(d

)D

oubl

ein

sert

cond

ition

alre

spon

se(1

5,25

,and

35nt

at50

exte

nsio

npl

ussa

me

leng

thof

inse

rtat

each

ofth

ree

othe

rin

sert

site

s).(

e)D

oubl

ein

sert

cond

ition

alre

spon

se(1

5,25

,and

35nt

inse

rts

forr

emai

ning

com

bina

tions

oftw

oin

sert

site

s)pl

usal

tern

ativ

esi

ngle

inse

rtse

quen

ces

for1

5nt

and

25nt

hand

lelo

opan

dte

rmin

ator

loop

1.In

duce

dex

pres

sion

(aT

c)of

sile

ncin

gdC

as9

and

cons

titut

ive

expr

essi

onof

sfG

FPta

rget

gene

Yan

dei

ther

:no

-tar

getg

RN

A,s

tand

ard

gRN

A,c

gRN

Aw

ithou

ttr

igge

r,cg

RN

A+

cogn

ate

trig

ger.

Aut

ofluo

resc

ence

:ce

llsw

ithno

sfG

FP.R

awflu

ores

cenc

evi

aflo

wcy

tom

etry

:m

ean±

estim

ated

stan

dard

erro

rba

sed

onth

em

ean

sing

le-c

ellfl

uore

scen

ceov

er20

,000

cells

fore

ach

ofN

=3

repl

icat

ew

ells

.Dat

afo

reac

hpa

nelc

olle

cted

ondi

ffer

entd

ays.

S59

cgR

NA

ON

OFF

Fold

Cha

nge

Dyn

amic

Ran

geFr

actio

nalD

ynam

icR

ange

aId

eal

59

±12

19970±

140

340

±70

19910

±140

150

exte

nsio

n:15

28

±11

200±

12

7±

3172

±6

0.0086

±0.0003

50ex

tens

ion:

2520

±11

246±

11

13

±7

226

±4

0.0114

±0.0002

50ex

tens

ion:

3524

±10

149±

11

6±

3125

±2

0.00629±

0.00013

Han

dle

loop

:15b

66

±10

81±

13

1.2

±0.3

15

±9

0.0008

±0.0004

Han

dle

loop

:25b

48

±11

223±

11

4.6

±1.0

175

±5

0.0088

±0.0003

Han

dle

loop

:35

47

±10

350±

20

8±

2310

±12

0.0154

±0.0006

Term

inat

orlo

op1:

15a

30

±11

53±

11

1.8

±0.7

23

±3

0.0012

±0.0002

Term

inat

orlo

op1:

25a

40

±12

71±

13

1.8

±0.6

30.5

±9.9

0.0015

±0.0005

Term

inat

orlo

op1:

3521

±11

233±

11

11

±6

213

±5

0.0107

±0.0002

Term

inat

orlo

op2:

1541

±11

24±

11

0.6

±0.3

−18

±5

−0.0009

±0.0003

Term

inat

orlo

op2:

2538

±10

146±

11

3.9

±1.1

108

±5

0.0054

±0.0002

Term

inat

orlo

op2:

3551

±11

73±

11

1.4

±0.4

22

±4

0.0011

±0.0002

bId

eal

65

±10

18200±

700

280

±50

18000

±700

150

exte

nsio

n:15

/Han

dle

loop

:15a

72

±9

276±

13

3.9

±0.5

200

±12

0.0113

±0.0008

50ex

tens

ion:

25/H

andl

elo

op:2

5a59

±8

1380±

20

23

±3

1320

±15

0.073

±0.003

50ex

tens

ion:

35/H

andl

elo

op:3

577

±8

1770±

30

23

±3

1690

±28

0.093

±0.004

50ex

tens

ion:

15/T

erm

loop

1:15

a95

±8

402±

13

4.2

±0.4

310

±11

0.0170

±0.0009

50ex

tens

ion:

25/T

erm

loop

1:25

a58.0

±9.8

443±

97.6

±1.3

385

±8

0.0213

±0.0010

50ex

tens

ion:

35/T

erm

loop

1:35

144

±8

545±

93.8

±0.2

401

±6

0.0222

±0.0010

50ex

tens

ion:

15/T

erm

loop

2:15

43

±8

186±

84.3

±0.8

143

±3

0.0079

±0.0003

50ex

tens

ion:

25/T

erm

loop

2:25

81

±8

422±

95.2

±0.6

341

±7

0.0188

±0.0008

50ex

tens

ion:

35/T

erm

loop

2:35

178

±9

178±

81.00±

0.07

0±

50.0000

±0.0003

cId

eal

90

±5

21000±

300

232

±13

20900

±270

1H

andl

elo

op:1

5b/T

erm

loop

1:15

a100

±7

178±

61.77±

0.13

78

±5

0.0037

±0.0003

Han

dle

loop

:25b

/Ter

mlo

op1:

25a

92

±6

599±

12

6.5

±0.4

510

±11

0.0243

±0.0006

Han

dle

loop

:35

/Ter

mlo

op1:

35123

±5

5100±

50

41

±2

4980

±52

0.238

±0.004

Han

dle

loop

:15b

/Ter

mlo

op2:

1567

±7

107±

81.6

±0.2

40

±7

0.0019

±0.0003

Han

dle

loop

:25b

/Ter

mlo

op2:

25166

±8

359±

82.16±

0.11

193

±8

0.0092

±0.0004

Han

dle

loop

:35

/Ter

mlo

op2:

35307

±9

1015±

13

3.31±

0.11

710

±14

0.0339

±0.0008

Term

loop

1:15

b/T

erm

loop

2:15

43

±6

114±

62.7

±0.4

71

±5

0.0034

±0.0002

Term

loop

1:25

b/T

erm

loop

2:25

243

±6

42±

50.17±

0.02

−201

±3

−0.0096

±0.0002

Term

loop

1:35

/Ter

mlo

op2:

35300

±8

103±

60.34±

0.02

−197

±7

−0.0094

±0.0003

Han

dle

loop

:15a

318

±6

69±

50.22±

0.02

−249

±3

−0.0119

±0.0002

Han

dle

loop

:25a

91

±5

155±

51.69±

0.11

63

±2

0.00303±

0.00010

Term

inat

orlo

op1:

15b

47

±5

143±

63.1

±0.4

96

±4

0.0046

±0.0002

Term

inat

orlo

op1:

25b

58

±5

107±

51.8

±0.2

49

±2

0.00234±

0.00012

Tabl

eS1

6:Q

uant

ifyin

gO

Nst

ate,

OFF

stat

e,fo

ldch

ange

,and

dyna

mic

rang

efo

rca

ndid

ate

cgR

NA

swith

desi

gned

sing

lean

ddo

uble

inse

rtsi

nto

the

stan

dard

gRN

Ast

ruct

ure.

ON

=(c

gRN

A,n

otr

igge

r)−

AF;

OFF

=(c

gRN

A+

cogn

ate

trig

ger)−

AF;

fold

chan

ge=

OFF

/ON

;dyn

amic

rang

e=

OFF

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S60

S6.2.2 Candidate cgRNA and trigger sequences used for single and double insert studies

Part name Sequence of standard gRNA or candidate cgRNA Legend

sfGFP g 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Standard gRNA

NT g 50-GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

No-target gRNAAutofluorescence

i-15 cg 50-TATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

50 extension: 15

i-25 cg 50-ACTATAGACTTATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

50 extension: 25

i-35 cg 50-GCTACTCATTACTATAGACTTATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

50 extension: 35

ii-15a cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCTATTCGAGAAAGTTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Handle loop: 15a

ii-15b cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Handle loop: 15b

ii-25a cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCTATTCGAGAAAGTTTCAGATCCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Handle loop: 25a

ii-25b cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAAAAGTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Handle loop: 25b

ii-35 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCTATTCGAGAAAGTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Handle loop: 35

iii-15a cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Terminator loop 1: 15a

iii-15b cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTCATTCACATAAGCACAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Terminator loop 1: 15b

iii-25a cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATTTCACCATTCAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Terminator loop 1: 25a

iii-25b cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTACAATTTCACCATTCACATAAGCACAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Terminator loop 1: 25b

iii-35 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATTTCACCATTCACATAAGCACAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Terminator loop 1: 35

iv-15 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAACAATCGGTGCTTTTTTT-30


iv-25 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAACAATGAGAACAGAACGGTGCTTTTTTT-30


iv-35 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAACAATGAGAACAGAAGATAGAAATACGGTGCTTTTTTT-30


i-15 ii-15a cg 50-TATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGCTATTCGAGAAAGTTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

50 extension: 15 / Handle loop: 15a

i-25 ii-25a cg 50-ACTATAGACTTATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGCTATTCGAGAAAGTTTCAGATCCCTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

50 extension: 25 / Handle loop: 25a

S61

Part name Sequence of standard gRNA or candidate cgRNA Legend

i-35 ii-35 cg 50-GCTACTCATTACTATAGACTTATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGCTATTCGAGAAAGTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTT-30

50 extension 35 / Handle loop: 35

i-15 iii-15a cg 50-TATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATAAGTGGCACCGAGTCGGTGCTTTTTTT-30

50 extension: 15 / Term loop 1: 15a

i-25 iii-25a cg 50-ACTATAGACTTATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATTTCACCATTCAAGTGGCACCGAGTCGGTGCTTTTTTT-30

50 extension: 25 / Term loop 1: 25a

i-35 iii-35 cg 50-GCTACTCATTACTATAGACTTATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAA ATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATTTCACCATTCACATAAGCACAAGTGGCACCGAGTCGGTGCTTTTTTT-30

50 extension: 35 / Term loop 1: 35

i-15 iv-15 cg 50-TATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAACAATCGGTGCTTTTTTT-30


i-25 iv-25 cg 50-ACTATAGACTTATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAACAATGAGAACAGAACGGTGCTTTTTTT-30


i-35 iv-35 cg 50-GCTACTCATTACTATAGACTTATCATCCATCAACCCATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAACAATGAGAACAGAAGATAGAAATACGGTGCTTTTTTT-30


ii-15b iii-15a cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Handle loop: 15b / Term loop 1: 15a

ii-25b iii-25a cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAAAAGTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATTTCACCATTCAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Handle loop: 25b / Term loop 1: 25a

ii-35 iii-35 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCTATTCGAGAAAGTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATTTCACCATTCACATAAGCACAAGTGGCACCGAGTCGGTGCTTTTTTT-30

Handle loop: 35 / Term loop 1: 35

ii-15b iv-15 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAACAATCGGTGCTTTTTTT-30

Handle loop: 15b / Term loop 2: 15

ii-25b iv-25 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAAAAGTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAACAATGAGAACAGAACGGTGCTTTTTTT-30

Handle loop: 25b / Term loop 2: 25

ii-35 iv-35 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGCTATTCGAGAAAGTTTCAGATCCCGTGTTCCGTGTAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGCAAGAACGTAACAATGAGAACAGAAGATAGAAATACGGTGCTTTTTTT-30

Handle loop: 35 / Term loop 2: 35

iii-15b iv-15 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTCATTCACATAAGCACAAGTGGCACCGCAAGAACGTAACAATCGGTGCTTTTTTT-30

Term loop 1: 15b / Term loop 2: 15

iii-25b iv-25 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTACAATTTCACCATTCACATAAGCACAAGTGGCACCGCAAGAACGTAACAATGAGAACAGAACGGTGCTTTTTTT-30

Term loop 1: 25b / Term loop 2: 25

iii-35 iv-35 cg 50-CATCTAATTCAACAAGAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTAAACCTTACCACAATTTCACCATTCACATAAGCACAAGTGGCACCGCAAGAACGTAACAATGAGAACAGAAGATAGAAATACGGTGCTTTTTTT-30

Term loop 1: 35 / Term loop 2: 35

Table S17: Candidate cgRNA sequences used for single and double insert studies in E. coli. For the standard gRNA sfGFP g, nucleotidesshaded orange are constrained by the target gene (sfGFP), and nucleotides shaded black are the standard gRNA subsequences for the handleloop, terminator loop 1, and terminator loop 2 (to illustrate three locations where inserts will be positioned for candidates cgRNAs [inaddition to extensions at the 50 end]). For all candidate cgRNAs, nucleotides shaded blue are NUPACK-designed sequence inserts (positionedas depicted in Figure S39b). In cgRNAs containing no modified sequence in the handle loop, terminator loop 1, and/or terminator loop 2, thestandard gRNA subsequence is used. In cgRNAs containing no modified sequence in the 50 extension, no extra 50 sequence is included. Asin the other bacterial studies, the no-target gRNA contains no target-binding region, and the autofluorescence control strain was transformedwith the no-target gRNA control plasmid (sequence NT g).

S62

Part name Sequence of candidate trigger Legend

i-15 t 50-GGTTGATGGATGATA-30 50 extension: 15 +Triggeri-25 t 50-GGTTGATGGATGATAAGTCTATAGT-30 50 extension: 25 +Triggeri-35 t 50-GGTTGATGGATGATAAGTCTATAGTAATGAGTAGC-30 50 extension: 35 +Triggerii-15a t 50-ACTTTCTCGAATAGC-30 Handle loop: 15a +Triggerii-15b t 50-CACGGAACACGGGAT-30 Handle loop: 15b +Triggerii-25a t 50-GGGATCTGAAACTTTCTCGAATAGC-30 Handle loop: 25a +Triggerii-25b t 50-CACGGAACACGGGATCTGAAACTTT-30 Handle loop: 25b +Triggerii-35 t 50-CACGGAACACGGGATCTGAAACTTTCTCGAATAGC-30 Handle loop: 35 +Triggeriii-15a t 50-ATTGTGGTAAGGTTT-30 Terminator loop 1: 15a +Triggeriii-15b t 50-GTGCTTATGTGAATG-30 Terminator loop 1: 15b +Triggeriii-25a t 50-GAATGGTGAAATTGTGGTAAGGTTT-30 Terminator loop 1: 25a +Triggeriii-25b t 50-GTGCTTATGTGAATGGTGAAATTGT-30 Terminator loop 1: 25b +Triggeriii-35 t 50-GTGCTTATGTGAATGGTGAAATTGTGGTAAGGTTT-30 Terminator loop 1: 35 +Triggeriv-15 t 50-ATTGTTACGTTCTTG-30 Terminator loop 2: 15 +Triggeriv-25 t 50-TTCTGTTCTCATTGTTACGTTCTTG-30 Terminator loop 2: 25 +Triggeriv-35 t 50-TATTTCTATCTTCTGTTCTCATTGTTACGTTCTTG-30 Terminator loop 2: 35 +Triggeri-15 ii-15a t 50-ACTTTCTCGAATAGCGGTTGATGGATGATA-30 50 extension: 15 / Handle loop: 15a +Triggeri-25 ii-25a t 50-GGGATCTGAAACTTTCTCGAATAGCGGTTGATGGATGATA

AGTCTATAGT-3050 extension: 25 / Handle loop: 25a +Trigger

i-35 ii-35 t 50-CACGGAACACGGGATCTGAAACTTTCTCGAATAGCGGTTGATGGATGATAAGTCTATAGTAATGAGTAGC-30

50 extension: 35 / Handle loop: 35 +Trigger

i-15 iii-15a t 50-ATTGTGGTAAGGTTTGGTTGATGGATGATA-30 50 extension: 15 / Term loop 1: 15a +Triggeri-25 iii-25a t 50-GAATGGTGAAATTGTGGTAAGGTTTGGTTGATGGATGATA

AGTCTATAGT-3050 extension: 25 / Term loop 1: 25a +Trigger

i-35 iii-35 t 50-GTGCTTATGTGAATGGTGAAATTGTGGTAAGGTTTGGTTGATGGATGATAAGTCTATAGTAATGAGTAGC-30

50 extension: 35 / Term loop 1: 35 +Trigger

i-15 iv-15 t 50-ATTGTTACGTTCTTGGGTTGATGGATGATA-30 50 extension: 15 / Term loop 2: 15 +Triggeri-25 iv-25 t 50-TTCTGTTCTCATTGTTACGTTCTTGGGTTGATGGATGATA

AGTCTATAGT-3050 extension: 25 / Term loop 2: 25 +Trigger

i-35 iv-35 t 50-TATTTCTATCTTCTGTTCTCATTGTTACGTTCTTGGGTTGATGGATGATAAGTCTATAGTAATGAGTAGC-30

50 extension: 35 / Term loop 2: 35 +Trigger

ii-15b iii-15a t 50-ATTGTGGTAAGGTTTCACGGAACACGGGAT-30 Handle loop: 15b / Term loop 1: 15a +Triggerii-25b iii-25a t 50-GAATGGTGAAATTGTGGTAAGGTTTCACGGAACACGGGAT

CTGAAACTTT-30Handle loop: 25b / Term loop 1: 25a +Trigger

ii-35 iii-35 t 50-GTGCTTATGTGAATGGTGAAATTGTGGTAAGGTTTCACGGAACACGGGATCTGAAACTTTCTCGAATAGC-30

Handle loop: 35 / Term loop 1: 35 +Trigger

ii-15b iv-15 t 50-ATTGTTACGTTCTTGCACGGAACACGGGAT-30 Handle loop: 15b / Term loop 2: 15 +Triggerii-25b iv-25 t 50-TTCTGTTCTCATTGTTACGTTCTTGCACGGAACACGGGAT

CTGAAACTTT-30Handle loop: 25b / Term loop 2: 25 +Trigger

ii-35 iv-35 t 50-TATTTCTATCTTCTGTTCTCATTGTTACGTTCTTGCACGGAACACGGGATCTGAAACTTTCTCGAATAGC-30

Handle loop: 35 / Term loop 2: 35 +Trigger

iii-15b iv-15 t 50-ATTGTTACGTTCTTGGTGCTTATGTGAATG-30 Term loop 1: 15b / Term loop 2: 15 +Triggeriii-25b iv-25 t 50-TTCTGTTCTCATTGTTACGTTCTTGGTGCTTATGTGAATG

GTGAAATTGT-30Term loop 1: 25b / Term loop 2: 25 +Trigger

iii-35 iv-35 t 50-TATTTCTATCTTCTGTTCTCATTGTTACGTTCTTGGTGCTTATGTGAATGGTGAAATTGTGGTAAGGTTT-30

Term loop 1: 35 / Term loop 2: 35 +Trigger

Table S18: Trigger sequences used for single and double insert studies in E. coli. Nucleotides shaded blue are NUPACK-designedsequence complementary to the sequence inserts of the corresponding cgRNA of Table S17.

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S6.3 Characterization of splinted switch conditional response to lacI-regulated trigger expressionat different time points in E. coli

In the present work, we focus on end point assays to characterize the conditional response of cgRNAs to cognate and non-cognate RNA triggers. To gain some insight into the temporal response of cgRNAs to trigger expression, here we constitu-tively express a splinted switch cgRNA (BBa J23114 promoter, Table S8) and induce expression of the cognate RNA trigger(BBa R0011 promoter, Table S8) at different time points after dCas9 induction. The sequences used for cgRNA (ii-35 iii-35 cg,Table S17) and trigger (ii-35 iii-35 t , Table S18) were those designed for the double insert studies above, with methods for plas-mid construction and molecular cloning, and flow cytometry identical to those for the splinted switch mechanism (Sections S1.2and S3.2), and using the dCas9+lacI expression plasmid (Section S3.4) in place of the dCas9-only expression plasmid. ControlgRNA and cgRNA-only were expressed under the same constitutive promoter (BBa J23114). The conditional response of thecgRNA with lacI-regulated trigger expression was measured via flow cytometry (Figure S40a), 12 h post induction of dCas9 (2nM aTc) and trigger (5 mM IPTG, also added to strains not expressing trigger). Time course data were collected with the Neo2microplate reader (Biotek) for the conditional response of the cgRNA with induction of trigger (5 mM IPTG) at 4, 5, 6, or 7 hpost dCas9 induction (Figure S40b, 2 nM aTc at t = 0). Induction with IPTG results in the expected increase in observed nor-malized fluorescence, with normalized fluorescence of the un-induced cgRNA + pLac-trigger remaining comparable to that thecgRNA-only strain for the duration of the experiment. Subtraction of the mean normalized fluorescence of un-induced cgRNA+ pLac-trigger from each induction time course (Figure S40c) reveals dependence of the conditional response on induction ofcognate trigger, with a substantial increase in ∆(Fluorescence/A600) observed 1–2 h after trigger induction.

4 6 8 10 12

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Time post dCas9 induction (h)

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cgRNA + pLac-trigger, +IPTG 6hcgRNA + pLac-trigger, +IPTG 5hcgRNA + pLac-trigger, +IPTG 4h

3000

4000a b

c


Figure S40: Characterization of splinted switch response to lacI-regulated trigger expression at different time points in E. coli. (a)Expression of RNA trigger X (70 nt + synthetic terminator) induced by IPTG toggles the cgRNA from ON!OFF, leading to an increasein fluorescence. Single-cell fluorescence intensities via flow cytometry over 20,000 cells. Induced expression (aTc) of silencing dCas9 andconstitutive expression of sfGFP target gene Y and either: standard gRNA (ideal ON state), cgRNA (ON state), cgRNA + RNA trigger X(OFF state; trigger expression is IPTG-induced), no-target gRNA that lacks target-binding region (ideal OFF state). Autofluorescence (AF):cells with no sfGFP. Trigger expression was induced simultaneously with dCas9 expression with 5 mM IPTG for each strain. (b) Time coursemicroplate fluorescence data normalized by A600. Identical color traces represent N = 3 replicate wells. Induction of trigger (5 mM IPTG,time of induction indicated by arrows) at 4, 5, 6, and 7 h post dCas9 induction (2 nM aTc at t = 0) toggles the cgRNA from ON!OFF,leading to an increase in fluorescence. (c) Difference in normalized fluorescence between: 1) cgRNA + trigger induced by IPTG (5 mM IPTG,time of induction indicated by arrows) at 4, 5, 6, and 7 h post dCas9 induction and 2) un-induced cgRNA + trigger (-IPTG). Induction oftrigger expression toggles the cgRNA from ON!OFF, leading to an increase in fluorescence 1–2 h after addition of IPTG. Mean ± estimatedstandard error (with uncertainty propagation) based on the A600-normalized fluorescence per well for each of N = 3 replicate wells.

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