bacterial genome editing with crispr-cas9: deletion ... · pdf file1 bacterial genome editing...

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Bacterial genome editing with CRISPR-Cas9: deletion, integration, single nucleotide 1

modification, and desirable ‘clean’ mutant selection in Clostridium beijerinckii as an 2

example 3

4

Yi Wanga,b,ǂ

, Zhong-Tian Zhangc, Seung-Oh Seo

a,b, Patrick Lynn

d, Ting Lu

b,e, Yong-Su Jin

a,b 5

& Hans P. Blascheka,b,f,*

6

aDepartment of Food Science and Human Nutrition, University of Illinois at Urbana-7

Champaign, Urbana, IL 61801, USA 8

bCarl R. Woese Institute for Genomic Biology, University of Illinois at Urbana-Champaign, 9

Urbana, IL 61801, USA 10

cDepartment of Chemistry, University of Illinois at Urbana-Champaign, Urbana, IL 61801, 11

USA 12

dDepartment of Chemical and Biomolecular Engineering, University of Illinois at Urbana-13


eDepartment of Bioengineering, University of Illinois at Urbana-Champaign, Urbana, IL 15

61801, USA 16

fThe Integrated Bioprocessing Research Laboratory (IBRL), University of Illinois at Urbana-17


ǂPresent address: 215 Tom E. Corley Building, Biosystems Engineering Department, Auburn 19

University, Auburn, AL 36849, USA 20

*To whom correspondence should be addressed: 21

Hans P. Blaschek, The Integrated Bioprocessing Research Laboratory (IBRL) 22

University of Illinois at Urbana-Champaign, 23

1207 W Gregory Drive, Urbana, IL 61801, USA 24

Tel: 1-217-333-8224; Fax: 1-217-244-2517; 25

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E-mail: [email protected] 26

Supporting Information 27

METHODS 28

Plasmid construction (Additional detailed construction procedures for other vectors). 29

A general vector pYW19-BseRI was constructed in the same manner as for constructing 30

pYW19-pta, except that gBLK05 (including thlT, a 45bp random sequence containing two 31

BseRI sites fused with the gRNA sequence (same as that in gBLK01), and a transcription 32

terminator derived from S. pyogenes) was used in place of gBLK04 as for pYW19-pta. 33

Vector pYW19-BseRI on one hand was used as a control of pYW19-pta for the 34

transformation, on the other served as the mother vector for constructing vectors targeting on 35

other specific chromosomal sites. For example, vector pYW19-pta2 was constructed based 36

on pYW19-BseRI as follows. First, the insert PCR fragment was amplified using primers P09 37

and P21 (Table S2) with C. beijerinckii gDNA as template; then pYW19-BseRI was digested 38

with BseRI and the obtained PCR fragment was inserted into the vector through Gibson 39

Assembly. 40

pYW27-pta was constructed in the same manner as pYW19-pta, except that the spoIIE 41

(Cbei_0097) promoter (amplified using primers P36 and P37 from C. beijerinckii gDNA) 42

was used to replace the thlP used in pYW19-pta, and Cas9 ORF was amplified from the 43

plasmid pMJ806 1 using primers P38 (instead of P19 for pYW19-pta) and P20 for Gibson 44

Assembly purpose. For constructing pYW27-ptaE3, pYW27-pta was digested with NotI, and 45

the 2kb DNA editing template (amplified with primers P39 and P40 using ptaE1 as template, 46

Table S2) was inserted through Gibson Assembly. A general vector pYW27-BseRI was 47

constructed in the same manner as pYW19-BseRI except that the spoIIE promoter was 48

employed to in place of thlP that was used in pYW19-BseRI. Then the control vector 49

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pYW27-BseRI-ptaE3 was constructed by inserting ptaE3 editing template into the NotI site 50

of pYW27-BseRI. 51

pYW34-pta was constructed in the same manner as pYW27-pta (and pYW19-pta), 52

except that the lactose induce promoter was amplified using primers P43 and P44 from 53

plasmid pKO_mazF 2 and used to replace the spoIIE promoter used in pYW27-pta, and Cas9 54

ORF was amplified from the plasmid pMJ806 1 using primers P45 (instead of P19 for 55

pYW19-pta) and P20 for Gibson Assembly purpose. Then pYW34-ptaE3 was constructed by 56

inserting the DNA editing template ptaE3 into the NotI site of pYW34-pta through Gibson 57

Assembly. The vector pYW34-ptaE7 was constructed in the same manner except that the 2kb 58

DNA editing template ptaE7 was amplified through SOEing PCR using two pairs of primers 59

P46 & P47, and P48 & P49. 60

For constructing pYW34-ptaE8, the insert fragment ptaE8 was amplified as follows. 61

Using C. beijerinckii gDNA as template, the first fragment (F1) was amplified with primers 62

P52 & P53, the second fragment (F2, promoter-ORF-terminator of Cbei_2181) with primers 63

P54 & P55, and the third fragment (F3) with P56 & P49. Then the three fragments were 64

assembled with Gibson Assembly and the product was used as the template for the second 65

round of PCR with primer P52 & P49 to obtain ptaE8 for insertion. Finally, ptaE8 was 66

inserted into the NotI site of pYW34-pta to generate the vector pYW34-ptaE8. 67

For constructing pYW34-ptaE9, the insert fragment ptaE9 was amplified (using primers 68

P39, P58-P61 & P40) in a similar manner as that for synthesizing ptaE8. Finally, ptaE9 was 69

inserted into the NotI site of pYW34-pta to generate pYW34-ptaE9. For obtaining pYW34-70

ptaE11 from pYW34-pta, the insert fragment ptaE11 was amplified through SOEing PCR 71

using two pairs of primers P79 & P58 and P59 & P80 with C. beijerinckii gDNA as template. 72

Then, ptaE11 was inserted into the NotI site of pYW34-pta to generate pYW34-ptaE11. 73

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For easily changing the 20-nt protospacer to target on alternative locus of the 74

chromosome, two general mother vectors pYW34-BtgZI (using Erm maker) and pYW35-75

BtgZI (using Sp maker) were constructed (Figure S3). To construct pYW34-BtgZI from 76

pYW34-pta by merely changing the guiding sequence region, in vitro Cas9 digestion was 77

employed for digesting on two sites. First, two single stranded oligonucleotides P63 and P64 78

were annealed together and a PCR reaction followed to amplify the DNA fragment serving as 79

the template for in vitro gRNA synthesis containing 20-nt guiding sequence (5’-80

CCATTCCATCTGCATCATGG-3’). Then, gRNA was synthesized with HiScribe™ T7 Quick 81

High Yield RNA Synthesis Kit (NEB) following the manufacturer’s protocol. Similarly, 82

another template DNA fragment was amplified based on oligonucleotides P65 and P64, and 83

gRNA was synthesized containing guiding sequence (5’-AGAAATAGCAAGTTAAAATA-84

3’). Afterwards, using these two synthesized gRNAs along with Cas9 nuclease (NEB), in 85

vitro digestion was carried out on pYW34-pta. The insert fragment was amplified through 86

two rounds of PCR. First round was conducted with primers P65 and P66 using C. 87

beijerinckii gDNA as template; then the PCR product was used as the template for the second 88

round of PCR with primers P65 and P67 to obtain the final insert fragment. The insert 89

fragment was assembled with the Cas9-digested product from pYW34-pta, and the obtained 90

construct was named as pYW34-BtgZI. 91

The vector pYW35-BtgZI was constructed from pYW34-BtgZI by replacing the Erm 92

maker with Sp marker following an in vitro double digestion with Cas9 nuclease. 93

Oligonucleotides P69 and P70 were paired with P64 respectively for synthesizing the two 94

gRNAs for Cas9 digestion purpose as described above. The Sp marker gene was synthesized 95

by Invitrogen (now part of Thermo Fisher Scientific, Grand Island, NY) 3. Pairs of primers 96

P70 & P71 (C. beijerinckii gDNA as template to synthesize pta promoter), P72 & P73 97

(synthesized Sp marker gene as template), and P74 & 75 (C. beijerinckii gDNA as template 98

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to amplify ack terminator) were used to amplify three fragments. Then the three fragments 99

were assembled through Gibson Assembly and the product was used as the template for the 100

second round of PCR with primer P70 & P75 to obtain the final insert fragment to be 101

assembled with Cas9-digested pYW34-BtgZI to get pYW35-BtgZI. 102

For constructing pYW35-pta3 (with 20-nt protospacer sequence (5’-103

CAATTGCAATAGCTACTGCA-3’) ), the insert fragment was amplified with primers P09 104

and P76 (Table S2) using C. beijerinckii gDNA as template; then pYW35-BtgZI was digested 105

with BtgZI and the PCR fragment obtained above was inserted into the vector through Gibson 106

Assembly. For obtaining pYW35-ptaE10 based on pYW35-pta3, the insert fragment ptaE10 107

was amplified through SOEing PCR using two pairs of primers P39 & P77, and P78 & P40 108

with C. beijerinckii gDNA as template. Finally, ptaE10 was inserted into the NotI site of 109

pYW35-pta3 to generate pYW35-ptaE10. 110

For constructing pYW34-Sp, the insert fragment was amplified with primers P09 and 111

P81 (Table S2) using C. beijerinckii gDNA as template; then pYW34-BtgZI was digested 112


Assembly. For constructing pYW35-Erm, the insert fragment was amplified with primers P09 114

and P82 (Table S2) using C. beijerinckii gDNA as template; then pYW35-BtgZI was digested 115


Assembly. 117

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Table S1 Strains and plasmids used in this study

Strains Description Sources

C. beijerinckii

NCIMB 8052 Wild type Lab stock

E. coli

DH5α Electrocompetent cell NEBa

Turbo Electrocompetent cell NEBa

10-beta Electrocompetent cell NEBa

ER2925 Electrocompetent cell NEBa

Plasmids Description & Relevant characteristics References

pTJ1 Apr, Ermr, E. coli-C. beijerinckii shuttle vectorb 4

pMJ806 pET-derived His6-MBP expression vector with S. pyogenes Cas9 1

pMJ841 pET-derived His6-MBP expression vector with S. pyogenes Cas9 (D10A/H840A double mutant) 1

pYW19gRNA-BseRI pTJ1-derived general vector for screening promoters for gRNA expression This study

pYW19gRNA-Cbe0075p Test Cbe_0075 promoter for gRNA expression This study

pYW19gRNA-Cbe0144p1 Test 284 bp Cbe_0144 (RNA polymerase gene) promoter for gRNA expression This study

pYW19gRNA-Cbe0144p2 Test 330 bp Cbe_0144 (RNA polymerase gene) promoter for gRNA expression This study



pYW19gRNA-sCbe0761p Test sCbei_0761 (small RNA) promoter for gRNA expression This study



pYW19-pta thlP for Cas9 expression; sCbei_5830 promoter for gRNA expression; 20-nt protospacer

sequence: 5’-GATGCAGATGGAATGGTATC-3’. This study

pYW19-pta2 Same as pYW19-pta, but targeting on an alternate site of pta (5’-

GCAGAAAAAATACAAAAATT-3’) This study

pYW19-BseRI General Cas9 vector, same as pYW19-pta, but without the 20-nt protospacer sequence; with two This study

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BseRI sites for easy re-targeting purposes.

pYW27-pta Same as pYW19-pta, except that spoIIE promoter (instead of thlP) was used for Cas9 expression This study

pYW27-BseRI General Cas9 vector, same as pYW27-pta, but without the 20-nt protospacer sequence; with two

BseRI sites for easy re-targeting purposes. This study

pYW27-ptaE3 Based on pYW27-pta, 2kb DNA editing template (for deleting 50bp of pta ORF) was integrated

into the NotI site of the vector. This study

pYW27-BseRI-ptaE3 Same as pYW27-ptaE3, except that sCbei_5830 promoter and 20-nt protospacer sequence are

not included; serving as a control vector for pYW27-ptaE3. This study

pYW34-pta Same as pYW19-pta and pYW27-pta, except that a lactose inducible promoter was used for

Cas9 expression This study

pYW34-ptaE3 Based on pYW34-pta, 2kb DNA editing template (for deleting 50bp of pta ORF) was integrated

into the NotI site of the vector This study

pYW34-ptaE7 Based on pYW34-pta, 2kb DNA editing template (for deleting 1.5kb around pta ORF) was

integrated into the NotI site of the vector This study

pYW34-ptaE8 Based on pYW34-pta, 2kb DNA editing template (for integrating Cbei_2181 (adhE) into the pta

ORF to replace 849bp at the locus) was integrated into the NotI site of the vector This study

pYW34-ptaE9

Based on pYW34-pta, 2kb DNA editing template (with the first two-step approach for single

nucleotide modification, to generate the intermediate mutant with ‘GAA’ changed to ‘GGG’ as an

artificial PAM, and meanwhile changed the used PAM ‘AGG’ to ‘ATT’) was integrated into the

NotI site of the vector.

This study

pYW34-BtgZI General Cas9 vector, same as pYW34-pta, but without the 20-nt protospacer sequence; with two

BtgZI sites for easy re-targeting purposes. This study

pYW35-BtgZI General Cas9 vector, same as pYW35-pta, but without the 20-nt protospacer sequence; with two

BtgZI sites for easy re-targeting purposes. This study

pYW35-pta3

Same as pYW34-pta, except that the Erm maker in pYW34-pta has been replaced with Sp

marker; meanwhile, used an alternative 20-nt protospacer sequence (5’-

CAATTGCAATAGCTACTGCA-3’) for Cas9 targeting purpose.

This study

pYW35-ptaE10 Based on pYW35-pta, 2kb DNA editing template (to generate the final desirable mutant with

single nucleotide modification) was integrated into the NotI site of the vector. This study

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pYW34-ptaE11

Based on pYW34-pta, 2kb DNA editing template (with the second approach for single

nucleotide modification, to generate the intermediate mutant with 200bp deleted and an artificial

PAM ‘GGG’ created) was integrated into the NotI site of the vector.

This study

pYW34-Sp Generated from pYW34-BtgZI (containing Erm marker) targeting on the Sp marker region to

eliminate the mutants containing integrated vector. This study

pYW35-Erm Generated from pYW35-BtgZI (containing Sp marker) targeting on the Erm marker region to

eliminate the mutants containing integrated vector. This study

a New England Biolabs Inc., Ipswich, MA

b Ap, ampicillin; Erm, erythromycin

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Table S2 DNA oligonucleotide sequences used in this study

IDT gBlocks Sequence (5’-3’)

gBLK01

AAGGTTACTATGATAATTCTCATGGTAACCTTTTTTTATTAAATAAGAGTATAAAATAAAGTTAAAAGAAGAAAATAGAAAT

CAAGCACACTCCTCAGTCTCGGAAGCTCAAAGAGGAGGAACCTGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

CTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTATGGAGAAATCTAGATCAGCATGATGTCTG

ACTAGTACAGATACATTATTATGTATCAAAATATAAAATACTTGCTCAAAGGTTACTTAAGTATTTGTTCTGTTCAATTTTGAA

AGACTAAGTCTTTCAATGTTCTTTGAAAATTGCACATAGATTTAATGTATATAAAATACAACAAAGCCAAGAATAAATATTCT

TTGTGATATGACTAATAATTAGCGGCCGCCAGTGTGATGGTTACTAGT

gBLK02

AAGGTTACTATGATAATTCTCATGGTAACCTTTTTTTATTAAATAAGAGTATAAAATAAAGTTAAAAGAAGAAAATAGAAAT

TGCTTGTTAGGTAAATAGGTATTTTATATAGCTTTATAACTGAAAAGAATTATTCAATAATATTTATTGACAAGAAAAATATCC

TATGATATTATAATATAATGTATTATTCTCTATGGAATACTATATATTTATAGTGGAAATAAGGAGTATAATCTGGTGAATAATGG

TTATACTATAGAAAGACGCTGTCCGAAAGCAAAGTCCTTAGGGAAAGTATGCTTTTGGCTATTTTAGTTTATTTTATACAAG

GGGTGAAAATTCGATGCAGATGGAATGGTATCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAA

CTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTATGGAGAAATCTAGATCAGCATGATGTCTGACTAGTACAGATACA

TTATTATGTATCAAAATATAAAATACTTGCTCAAAGGTTACTTAAGTATTTGTTCTGTTCAATTTTGAAAGACTAAGTCTTTC

AATGTTCTTTGAAAATTGCACATAGATTTAATGTATATAAAATACAACAAAGCCAAGAATAAATATTCTTTGTGATATGACTA

ATAATTAGCGGCCGCCAGTGTGATGGTTACTAGT

gBLK03

AAGGTTACTATGATAATTCTCATGGTAACCTTTTTTTATTAAATAAGAGTATAAAATAAAGTTAAAAGAAGAAAATAGAAATT

AAACTCTAAGCACTGGAAAAGCTTAGTTTATTTTAAAATATATTTTAAGAAAATTATTCATAAAAAAGTATAATTTGAAAATT

CATTAACAAATTTTTAAAAGATGCAGATGGAATGGTATCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTATGGAGAAATCTAGATCAGCATGATGTCTGACTAGTAC

AGATACATTATTATGTATCAAAATATAAAATACTTGCTCAAAGGTTACTTAAGTATTTGTTCTGTTCAATTTTGAAAGACTAA

GTCTTTCAATGTTCTTTGAAAATTGCACATAGATTTAATGTATATAAAATACAACAAAGCCAAGAATAAATATTCTTTGTGAT

ATGACTAATAATTAGCGGCCGCCAGTGTGATGGTTACTAGT

gBLK04

GTCAGCTAGGAGGTGACTGATATAAATTAAGATTTAAAAAGGTTACTATGATAATTCTCATGGTAACCTTTTTTTATTAAATA

AGAGTATAAAATAAAGTTAAAAGAAGAAAATAGAAATATAATCTTTAATTTGAAAAGATTTAAGGCTTATTTAAATAAAAAA

TATGAGGGAAGAATTGATATAAATTTAATTTTGTTATTGTATTATGGTATGTATGGAATAAATTTAACATAAAGACAGTAATAA

TGTTCTTGAATTTAGACTTTTTATGTGTTATCATTAACAAGTATCAAAAATGACATTTAATAAATTAATAATAATTTTAAAAAT

ATATTTTTGATAAAAGCAATGATTAACATGGTTTGACGTCTGAGAAGAGACGATTTTCTCAATAGGAGAAATTAAGGTGCA

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AACCCTTATCATTCCACCATGATGCAGATGGAATGGTATCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCG

TTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCTTTTTTTCTATGGAGAAATCTAGATCAGCATGATGTCTGACTAGTAC

AGATACATTATTATGTATCAAAATATAAAATACTTGCTCAAAGGTTACTTAAGTATTTGTTCTGTTCAATTTTGAAAGACTAA

GTCTTTCAATGTTCTTTGAAAATTGCACATAGATTTAATGTATATAAAATACAACAAAGCCAAGAATAAATATTCTTTGTGAT

ATGACTAATAATTAGCGGCCGCCAGTGTGATGGTTACTAGT

gBLK05

GTCAGCTAGGAGGTGACTGATATAAATTAAGATTTAAAAAGGTTACTATGATAATTCTCATGGTAACCTTTTTTTATTAAATA

AGAGTATAAAATAAAGTTAAAAGAAGAAAATAGAAATCAAGCACACTCCTCAGTCTCGGAAGCTCAAAGAGGAGGAACC

TGCGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGGCTAGTCCGTTATCAACTTGAAAAAGTGGCACCGAGTCGGTGCT

TTTTTTCTATGGAGAAATCTAGATCAGCATGATGTCTGACTAGTACAGATACATTATTATGTATCAAAATATAAAATACTTGCT

CAAAGGTTACTTAAGTATTTGTTCTGTTCAATTTTGAAAGACTAAGTCTTTCAATGTTCTTTGAAAATTGCACATAGATTTAA

TGTATATAAAATACAACAAAGCCAAGAATAAATATTCTTTGTGATATGACTAATAATTAGCGGCCGCCAGTGTGATGGTTACT

AGT

Primers Sequence (5’-3’)

P01* ACTAAAACTGAATTGATTGGGCCCTTTATTAAATAAGAGTATAAAATAAAGTTAAAAG

P02* CACTAGTAACCATCACACTGGCGGCCGCTAATTATTAGTCATATCACAAAGAATATTTAT

P03** AGTTAAAAGAAGAAAATAGAAATATTATCTTCTCCTCAAATGT

P04** TTCTAGCTCTAAAACGATACCATTCCATCTGCATCTATTTGTAATCTATAATATTATAAC

P05** AGTTAAAAGAAGAAAATAGAAATGTATGTTAATATAAAATTTTAAA

P06** TTCTAGCTCTAAAACGATACCATTCCATCTGCATCTTATTATTTATTAATATTTTAGTTT

P07** AGTTAAAAGAAGAAAATAGAAATATCTACTACCGCCTCAC

P08** TTCTAGCTCTAAAACGATACCATTCCATCTGCATCCTATTCTTCTAATAATTATAACATA

P09** AAAGTTAAAAGAAGAAAATAGAAATATAATCTTTAATTTGAAAAGATTTAAG

P10** TTGCTATTTCTAGCTCTAAAACGATACCATTCCATCTGCATCATGGTGGAATGATAAGGG

P11** AAAGTTAAAAGAAGAAAATAGAAATAATATTTATTATTTCTTATACTGATATTTTTATTA

P12** TTGCTATTTCTAGCTCTAAAACGATACCATTCCATCTGCATCATATCTAGGCTAATAAAA

P13** AAAGTTAAAAGAAGAAAATAGAAATTTTGTTTTAAAGAAAAAGGTG

P14** TTGCTATTTCTAGCTCTAAAACGATACCATTCCATCTGCATCGAATTTTCACCCCTTGTA

P15** GTTTTAGAGCTAGAAATAGCAAGTTAAAATAAG

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P16** GCACCGACTCGGTGC

P17 AAACTGAATTGATTGGGCCCGAAGAATAGCAGATGCTATATTAAAATATTTT

P18 GCCTATTGAGTATTTCTTATCCATGTTTGACCTCCTAAAATTTTATAGATTATTT

P19 AAAATTTTAGGAGGTCAAACATGGATAAGAAATACTCAATAGGCTT

P20 GTAACCTTTTTAAATCTTAATTTATATCAGTCACCTCCTAGCTGAC

P21 TTGCTATTTCTAGCTCTAAAACAATTTTTGTATTTTTTCTGCATGGTGGAATGATAAGGG

P22*** CACATTGATTGTTTCTTGG

P23*** TAAATCTCCAGTTCATCGTACCAAAATATAGAGG

P24*** TTTTGGTACGATGAACTGGAGATTTATTAAGACCAG

P25*** ATAAAGCTATTGAAAAAACATGC

P26*** CTTATGTTGACATCGTTATATAATG

P27*** TCTATAGAATATCCTTTTTCTTTTACTAAATAAG

P28*** TATACCATCTGCGTTTGTG

P29*** ACCATGAACAACTCTATGTCC

P30*** AAAATTATACTTTTCCTATATTGTTATTTTAC

P31*** TAGCTTCAGCATTTGCAAATC

P32*** TATAGCAGCTGCAGAAAAAATACAA

P33*** AGAGAATGATAACATTGCCACCTT

P34*** AAAGTATATTGAAGCCTTCTATG

P35*** AGATGATCCTGGAACTTGC

P36 ACTGGTATTAGTAATACTAAAACTGAATTGATTGGGCCCTTTTAATAATTAGCCAGATATCTTGATTTA

P37 ACGCTATTTGTGCCGATATCTAAGCCTATTGAGTATTTCTTATCCATATTAGCTCGCCTCATTTCC

P38 TGGAAATGAGGCGAGCTAATATGGATAAGAAATACTCAATAGGCTT

P39 TTTGTGATATGACTAATAATTAGCGGCCGCACCATGAACAACTCTATGTCC

P40 GCTCGGATCCACTAGTAACCATCACACTGGCGGCCGCTATACCATCTGCGTTTGTGTC

P41 GACAAAAGGCGTTACAATGGA

P42 TGCTGCTAATTGATCCTCGTT

P43 ACTGGTATTAGTAATACTAAAACTGAATTGATTGGGCCCTTATATACTTGGTTTATTTACTTGATTATTTC

P44 ACGCTATTTGTGCCGATATCTAAGCCTATTGAGTATTTCTTATCCATTTCAGCCCTCCTGTGAAATT

12

P45 CACAGGAGGGCTGAAATGGATAAGAAATACTCAATAGGCTT

P46 TTTGTGATATGACTAATAATTAGCGGCCGCTAAAACAAACACAACTAATTATATAAATTG

P47 TCTTCCAGAGGAAAAAGCATTTATGTATGCTC

P48 ATGCTTTTTCCTCTGGAAGAACAATTTTTTG

P49 ACTAGTAACCATCACACTGGCGGCCGCAAAGGCTCGGAAACTAAAAC

P50 TAAGTTGTTTGATTAAAATAAAGATTAAGC

P51 TTATGCTACTTTCGACAATTTCTGTG

P52 TTTGTGATATGACTAATAATTAGCGGCCGCGATTTCTTATTTAAGATATCATTTACTTC

P53 GGCAATAAGGATGATCAATGACTTATCAAGAGGTTG

P54 ATAAGTCATTGATCATCCTTATTGCCATATTAACAAC

P55 TGTTCTTCCAGAGATATATATTTAATAACTTAATTTAAAACCTTTAAATATAATATTTC

P56 ATTAAATATATATCTCTGGAAGAACAATTTTTTG

P57 ATCTGTTCCTATTCCTGATATAC

P58 TAGCTACTGCAGGGACAGCTAAGAATTTATGTAACATG

P59 ATTCTTAGCTGTCCCTGCAGTAGCTATTGCAATTG

P60 GGAATGGTATCATTAGCAATTCACACAACTGGAG

P61 GTGTGAATTGCTAATGATACCATTCCATCTGCATC

P62 AGAAATTAATACGACTCACTATAGGGCCATTCCATCTGCATCATGGGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

P63 AAAAAAAGCACCGACTCGGTGCCACTTTTTCAAGTTGATAACGGACTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC

P64 AGAAATTAATACGACTCACTATAGGGAGAAATAGCAAGTTAAAATAGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

P65 AAAAAGGTTACTATGATAATTCTC

P66 GAAGGAACTCCTCATTGCGATGAGCTAGGTCAGTATTTCTATTTTCTTCTTTTAACTTTA

P67 TTAACTTGCTATTTCTAGCTCTAAAACCTTGCAGTCATCGCTGAAGGAACTCCTCATTGC

P68 AGAAATTAATACGACTCACTATAGGGATGAGTCGCTTTTGTAAATTGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

P69 AGAAATTAATACGACTCACTATAGGGTCCAATTTCGTAAACGGTATGTTTTAGAGCTAGAAATAGCAAGTTAAAATAAGG

P70 CAATTGAATTTAAAAGAAACCGATATATAAAATAATTTTAAATAAAATTTAATTGTTAAAAATTATAC

P71 ATATCCTCCTCATAATCTATTTATCTCCTCTCTATATCC

P72 ATAAATAGATTATGAGGAGGATATATTTGAATACATAC

P73 CAATTATGCCTTATTATAATTTTTTTAATCTGTTATTTAAATAGTTTATAG

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P74 AAATTATAATAAGGCATAATTGAAAAAATGGTG

P75 TTTAGTAACGTGTAACTTTCCAAATTCTATACTAACTGCTCCCCTTTAC

P76 TTGCTATTTCTAGCTCTAAAACTGCAGTAGCTATTGCAATTGATGGTGGAATGATAAGGG

P77 TAGCTACTGCAGATACAGCTAAGAATTTATGTAACATG

P78 ATTCTTAGCTGTATCTGCAGTAGCTATTGCAATTG

P79 TTTGTGATATGACTAATAATTAGCGGCCGCTGCTAATTTTGTAAATTCATCAAG

P80 ACTAGTAACCATCACACTGGCGGCCGCCATTTCAGCTCTTTTCCATTTATC

P81 TTGCTATTTCTAGCTCTAAAACTTATGGATTCGTCAGAGGAAATGGTGGAATGATAAGGG

P82 TTGCTATTTCTAGCTCTAAAACTCTTCAACAATCAGATAGATATGGTGGAATGATAAGGG

P83 TTAAGATTTAAAAAGGTTACTATGATAATTCTC

P84 CGGCATCAGAGCAGATTGTA

P85 AGAGCCTTACATGCATTTATTCCAATTATATTT

P86 TAGAACTAGATCAACACCATTTTTAATAGCC

Notes:

*P01 & P02: amplifying the insert fragment to construct pYW19gRNA-BseRI, pYW19gRNA-Cbe0144p1 or pYW19gRNA-sCbe2478p using

gBLK01, gBLK02 or gBLK03 respectively as template. These gBlock fragments were originally designed to construct a ‘general’ vector

containing the Cas9 gene expression upstream of the gRNA, and thus partial sequence of a thiolase gene (Cbei_0411) terminator was included

at the beginning of them.

**For amplifying PCR fragments (containing various promoters) being inserted into pYW19gRNA-BseRI in order to achieve the

corresponding vector for screening promoter for gRNA expression:

P03 & P04: Cbei_0075 promoter; P05 & P06: Cbei_1823 promoter; P07 & P08: Cbei_2561 promoter; P09 & P10: sRNA sCbei_5830

promoter; P11 & P12: sRNA sCbei_0761 promoter; P13 & P14: Cbei_0144 promoter (all the 330bp of the intergenic region between

Cbei_0143 and Cbei_0144).

14

***For amplifying the linear DNA editing templates ptaE1 to ptaE6:

P22-P25: amplifying ptaE1 (4kb, with 2kb arms at both ends). The first fragment (upstream 2kb) was amplified using P22 and 23 with C.

beijerinckii 8052 gDNA as template; the second fragment (downstream 2kb) was amplified using P24 and 25 with C. beijerinckii 8052 gDNA

as template. Then, the final 4kb fragment was amplified using Splicing by Overlap Extension (SOEing) PCR using P22 and P25.

P26 & P27: amplifying ptaE2 (3kb, with 1.5kb arms at both ends) using ptaE1 as template.

P28 & P29: amplifying ptaE3 (2kb, with 1kb arms at both ends) using ptaE1 as template.

P30 & P31: amplifying ptaE4 (1kb, with 0.5kb arms at both ends) using ptaE1 as template.

P32 & P33: amplifying ptaE5 (0.5kb, with 0.25kb arms at both ends) using ptaE1 as template.

P34 & P35: amplifying ptaE6 (0.2kb, with 0.1kb arms at both ends) using ptaE1 as template.

15

Table S3 Summary of the testing of vector integration event (VIE) in various transformants.

Plasmid used for transformation VIE colonies/Total

colonies tested

Primer used to test the left joint

(PCR amplicon size in bp)

Primer used to test the right joint

(PCR amplicon size in bp)

pYW27-ptaE3 10/15 P26 & P83 (3239) P84 & P85 (2443)

pYW34-ptaE3 12/15 P26 & P83 (3239) P84 & P85 (2443)

pYW34-ptaE7 0/15 P26 & P83 (2974) P84 & P51 (2748)

pYW34-ptaE8 13/15 P26 & P83 (4584) P84 & P57 (3966)

pYW34-ptaE9 (>300)/(>300)* P26 & P83 (3289) P84 & P85 (2493)

pYW35-ptaE10** (>300)/(>300)* P26 & P83 (3289) P84 & P85 (2493)

pYW34-ptaE11 8/17 P26 & P83 (3164) P84 & P85 (2368)

pYW35-ptaE10*** 16/23 P26 & P83 (3289) P84 & P85 (2493)

*From the transformation with pYW34-ptaE9 or pYW35-ptaE10, more than 99% of the tested colonies from replating had VIE.

**For the first approach of Single Nucleotide Modification (SNM) (Strategy I, by transformation of pYW34-ptaE9 in the first step, and

then pYW35-ptaE10 in the second step).

***For the second approach of Single Nucleotide Modification (SNM) (Strategy II, by transformation of pYW34-ptaE11 in the first step,

and then pYW35-ptaE10 in the second step).

16

a

b

c

Figure S1. (a) Scheme of pYW19-BseRI, a general vector used to construct other vectors (by

inserting sCbei_5830 promoter along with 20-nt guiding sequence upstream of gRNA) to

target on specific loci using CRISPR-Cas9. Two BseRI sites have been included upstream of

the gRNA sequence for easy re-targeting purposes. (b) Scheme of pYW19-pta, with

CRISPR-Cas9 targeting onto the pta gene with 20-nt guiding sequence (5’-

GATGCAGATGGAATGGTATC-3’). (c) Scheme of pYW19-pta2, with CRISPR-Cas9

targeting onto the pta gene (to another site) with 20-nt guiding sequence (5’-

GCAGAAAAAATACAAAAATT-3’).

7

8

9

17

10 Figure S2. The transcriptional dynamics of Cbei_0097 (spoIIE gene)

5. RPKM: Reads Per 11

Kilobase per Million mapped reads, the method of quantifying gene expression from RNA 12

sequencing data by normalizing for total read length and the number of sequencing reads 6. 13

14

15

A

B

16

Figure S3. (a) Scheme of pYW34-BtgZI (with erythromycin, or Erm marker), and (b) 17

Scheme of pYW35-BtgZI (with spectinomycin, or Sp marker). These are two general vectors 18

used to construct other vectors (by inserting sCbei_5830 promoter along with 20-nt 19

protospacer sequence upstream of gRNA) to target on specific loci using CRISPR-Cas9. Two 20

BtgZI sites have been included upstream of the gRNA sequence for easy re-targeting 21

purposes. 22

23

24

0

500

1000

1500

2000

0 3010Time (h)

Gen

e e

xp

ressio

n (

RP

KM

)20

18

25

REFERENCES 26 (1) Jinek, M.; Chylinski, K.; Fonfara, I.; Hauer, M.; Doudna, J. A.; Charpentier, E., A programmable dual-27 RNA–guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337 (6096), 816-821. 28 (2) Al-Hinai, M. A.; Fast, A. G.; Papoutsakis, E. T., Novel system for efficient isolation of Clostridium double-29 crossover allelic exchange mutants enabling markerless chromosomal gene deletions and DNA integration. 30 Applied and Environmental Microbiology 2012, 78 (22), 8112-8121. 31 (3) Heap, J. T.; Kuehne, S. A.; Ehsaan, M.; Cartman, S. T.; Cooksley, C. M.; Scott, J. C.; Minton, N. P., The 32 ClosTron: Mutagenesis in Clostridium refined and streamlined. Journal of Microbiological Methods 2010, 80 33 (1), 49-55. 34 (4) Jesse, T. W. Genetic characterization and manipulation of solvent-producing clostridia. PhD dissertation, 35 University of Illinois at Urbana-Champaign, Urbana, IL, 2003. 36 (5) Wang, Y.; Li, X.; Mao, Y.; Blaschek, H., Genome-wide dynamic transcriptional profiling in Clostridium 37 beijerinckii NCIMB 8052 using single-nucleotide resolution RNA-Seq. BMC Genomics 2012, 13, 102. 38 (6) Mortazavi, A.; Williams, B. A.; McCue, K.; Schaeffer, L.; Wold, B., Mapping and quantifying mammalian 39 transcriptomes by RNA-Seq. Nature Methods 2008, 5 (7), 621-628. 40

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