Increasing genome editing efficiency and specificity

with optimized CRISPR-Cas9 guide RNAs

Ashley Jacobi

Senior Staff Scientist – Molecular Genetics

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Outline

• Background – CRISPR genome editing

• Developments and improvements of guide RNAs (gRNAs)

– In vitro transcribed single guide RNA (sgRNA)

– Chemically synthesized 2-part crRNA + tracrRNA

– Chemically synthesized sgRNA

– Benefits of introducing chemical modifications

– Which gRNA to use and when?

• Optimal delivery with different sources of Cas9

• Off-target analysis of different gRNA and Cas9 formats

• Brief introduction of newest Alt-R CRISPR reagent

• 2

Common CRISPR RNA-guided Restriction Endonucleases

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AsCas12a (Cpf1)SpCas9

“NGG” PAM site

dsDNA cut with blunt ends

Separate crRNA + tracrRNA (annealed)

20 nt protospacer

“TTTV” PAM site

5 base staggered cuts

Single, short crRNA

21-24 nt protospacer

Implementing CRISPR-Cas9 gene editing

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(RNP)

Alt-R CRISPR-Cas9 RNP System—fast, highly efficient deliveryRNP delivery with 2-part guide RNA (crRNA + tracrRNA)

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Alt-R CRISPR-Cas9 RNP System—fast, highly efficient deliveryRNP delivery with sgRNA

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Chemically synthesized guide RNAs

Developments and improvements

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Transfection of IVT sgRNAs can be toxic to cells

8

• Successful gene editing

• Transfection of IVT sgRNAs sometimes result in:

– Large-scale cell death

– Induction of innate immune response

HEK-293 cells only 30 nM IVT sgRNA 30 nM 2-part gRNA

IFNa stimulation in human PBMCs using unmodified gRNAs

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PBMC = peripheral blood mononuclear cell

Schubert et al. (2018) J Cytokine Biol 3:1

No IFN response with modified,

chemically synthesized Alt-R

gRNAs

Benefits of chemically synthesized gRNAs

• Chemical modifications can be added

– Improve nuclease stability

– Increase efficiency

– Reduce risk of triggering innate immune response

• Experiment-ready reagents are easily used

– crRNAs (36 nt) have the target specific sequence, can be synthesized by the

thousands in small scale, can be provided as libraries

– tracrRNAs (67 nt) has the universal sequence, available in larger scales, anneal to

each crRNA

– sgRNAs (full 100 nt) do not require annealing

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Empirically determined tolerance to chemical

modification in crRNA and tracrRNA regions

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5’ C*U*U*AUAUCCAACACuuCGuGGuuuUAGAGCUAU*G*C*U 3’

16-base tracrRNA

binding domain20-base protospacer

guide domain

crRNA

cGGAAuAAaAuuGAACGAUAC*G*A 5’

u| ||

A| ||

gucCguuAUCAACUUG

|||| A

|||| A

AGCCACGGUGAAA

G ||||||

UCGGUGCU*U*U 3’

tracrRNA

AGCU = 2’OMe RNA

agcu = RNA

“*” = PS bond

= Major loss of function with 2’-mod

= Minor loss of function with 2’-mod

= Loss of function varies with sequence

Alt-R gRNA options—crRNA + tracrRNA & sgRNA

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Alt-R

2 part

Alt-R

2 part XT

Alt-R

sgRNAStructure

gRNA format Alt-R CRISPR-Cas9

crRNA & tracrRNA

Alt-R CRISPR-Cas9

crRNA XT & tracrRNA

Alt-R CRISPR-Cas9

sgRNA

ComponentscrRNA

tracrRNA

crRNA XT

tracrRNAsgRNA

Sizes (nt)36

67

36

67 100

Annealing

requiredYes Yes No

Modifications Moderate High Moderate

Applications • Cas9-expressing cells

• RNP in most cell types

• Co-delivery with Cas9 plasmid/Cas9 mRNA

• RNP under difficult experimental conditions

(e.g., high nuclease environments)

All options have a 3–5 day turnaround time

DNA/mRNA Cas9 expression vectors can be co-delivered successfully with

more highly modified gRNA variants (HEK-293 cells)

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12 sites targeting HPRT

Amaxa® Nucleofection® (Lonza) in HEK-293 cells

RNP delivery

• gRNA:Cas9 protein = 1.2:1 ratio

• 3 µM Alt-R Electroporation Enhancer

Cas9 plasmid & mRNA

• Co-delivery with gRNA

• 1 µg of plasmid or mRNA

Indel (insertions/deletions) by NGS

• All gRNA forms work equally

well with RNP delivery

DNA/mRNA Cas9 expression vectors can be co-delivered successfully

with more highly modified gRNA variants (HEK-293 & K562 cells)

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12 sites targeting HPRT

Amaxa Nucleofection in HEK-293 & K562 cells

RNP delivery

• gRNA:Cas9 protein = 1.2:1 ratio

• 3 µM Alt-R Electroporation enhancer

Cas9 plasmid & mRNA

• Co-delivery with gRNA

• 1 µg of plasmid or mRNA

Indel by NGS

• All gRNA forms work equally

well with RNP delivery

Efficiency comparison of gRNA forms in CD34+ cells

• Collaboration with Ayal Hendel Lab (Bar-Ilan University)

• Goal: to efficiently knock-out genes associated with severe combined immunodeficiencies; identify the best gRNA forms to do so with RNP delivery

• Method: Electroporation into CD34+ HSPCs

• Alt-R Cas9 Nuclease V3

• In vitro transcribed sgRNA, Alt-R crRNA:tracrRNA, Alt-R crRNA XT:tracrRNA, Alt-R sgRNA

• Alt-R electroporation enhancer (with or without)

– 100 nt ssDNA

– Non-homologous to human, mouse and rat genomes

– Increases electroporation efficiency of RNP complexes

Titration of RNP complexes

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16

High editing in CD34+ cells using Alt-R RNP complexesRAG2 gRNA variants have similar activity

Alt-R electroporation enhancer significantly boosts editing

levels for all gRNA forms in CD34+ cells.

High editing in CD34+ cells using Alt-R RNP complexesHighly modified gRNAs are more efficient for RAG1

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RNP concentration (µM)

Alt-R electroporation enhancer significantly boosts editing

levels for all gRNA forms in CD34+ cells.

Off-target analysis of different gRNA and Cas9 formats

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CRISPR-Cas9 off-target effects (OTEs)

• Delivering Cas9 in an expression vector increases non-specific

editing due to continued and long-lasting Cas9 synthesis

• Delivery of Cas9 RNP complex reduces off-target editing, but it is not

a total solution

• Other solutions to reduce OTE have significant drawbacks

– crRNA length reduction (18–19 nt)

– Chemical modification

• What about high-fidelity Cas9 proteins?

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Published, rationally-designed, high-fidelity Cas9 mutants

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Bacterial screen to identify novel high-fidelity Cas9 mutants

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22

Nature Medicine (2018) 24:1216–1224

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New IDT HiFi Cas9 mutant retains on-target activity &

increases specificityAlt-R

HiFiCas9

On- & off-target comparison of different gRNA forms

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Sequence Information:

CRISPR-Cas9 off-target detection

• Methods to predict or validate off-target sites

– In silico predictive tools

• Difficult, often miss important sites, over-prediction

– In vitro assays to define possible OTEs

• SITE-seq, CIRCLE-seq, etc.

• Often over predictive, waste sequencing read space

– GUIDEseq identification of OTEs

• Cell line of interest; mostly quantitative

– rhAmpSeq system for CRISPR: a multiplexed, amplification-based, target

enrichment next-generation sequencing approach

• Quantitative assessment of on-target site and up to 1000 off-target sites in a single

multiplexed reaction

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Identification of potential off-target sites for AR & EMX1

• GUIDEseq into Cas9 cells,

10 µM Alt-R crRNA XT:tracrRNA

• In silico predictions

• rhAmpSeq assay panel designed

and synthesized

• AR panel = 54 assays

• EMX1 panel = 32 assays

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Strand A A T T A T G G G G A T T A C T A G G A GS Reads

- A A T T A T G G G G A T T A C T A G G A 4248

+ C A T T A T G G G G A T T - C T A G G A 359

- T A T T A A G G G A A C T A C T A G G A 328

- G A T G A T G G G G A T T T C T A G G A 136

- T A T T A A G G G G A T T A C T A G G G 121

+ A T T A G A G G G G A C T A C T A G G G 62

+ T T T T A T G G G G A G T A - T A G G A 49

+ T G T T A T A G G G A C C A C T A G G A 43

+ A A T A A T G G G G A T T A C T A G G A 40

+ G A C A C T G G G G A T T A C T A G G A 33

- T G T T A T A G G G A C C A C T A G G A 20

- A T T T A C A G G G A T T A C T T G G T 15

+ T A G T A T G G G A A T T T C T A G G A 15

+ A A T G A T G G G A A C T A C T A G A A 14

- T A T T A T G G G G A A T A C T A G G A 11

- C T T T A T G G G G A T T A C T A T G A 11

+ A A C T A T G G T G A C T A C T A G G A 9

+ A C T T T G G G G G A T T G C T A G G A 8

+ T T G A A T G G G G A T T A C T T G G A 8

+ C A A G T T A G G G A T T A C T A G G A 6

- T A C T A T G G G G A T T A C T A G G A 4

- T G G T A T G G G G G A T A C T A G C A 4

- G A G G A C T G G G A T T A C T A G G G 4

+ A A G T G T G G G G A T T A C - A G G A 3

+ T G T T A A G G G G A T T A C T A G G A 2

+ T A T T A T G G G T A A T A C A A G G A in silico

- G A T T G G A G G G A T T A C T A G G A in silico

+ G A T T A T G G G G A T T A C A A T T A in silico

- G A T T A T T G G A A T T A C T A G G T in silico

- A A T T A T C T G G A T T A C A A G G G in silico

+ A A T T A T G A A G A A T T C T A G G A in silico

+ G A T T A T G G G G A T T A C C A G T T in silico

+ A G T T C T G G G A A T T A C T A G A A in silico

- A G A T A T G G G G G T T A C T A A G A in silico

- T A G T A T G G G A A T T T C T A G G A in silico

Sequence Alignments

More potential off-target sites identified through GUIDEseq

using highly modified gRNAs

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• gRNAs delivered at final concentration of 10 µM into HEK-293 cells expressing Cas9

Guide #1 Guide #2 Guide #3

% = sum of the total unique CRISPR-Cas9 reads

38% 12% 13%

2% 1% 4%

On- & off-target comparison of the gRNA forms

• gRNA variants targeting AR & EMX1

– Transfected by electroporation into HEK-293 cells stably expressing Cas9

• RNP (gRNA variants complexed to Cas9 nuclease [Alt-R WT Cas9 or

Alt-R HiFi Cas9])

– Transfected by electroporation into HEK-293 cells

– Alt-R electroporation enhancer included in transfection

• gDNA extracted after 48 hrs

• On- and off-target editing detected using rhAmpSeq system

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Experimental conditions:

On- & off-target assessment via rhAmpSeq system (AR target)

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• Similar off-target sites and OTE

levels detected across different

gRNA forms.

• Slightly higher OTE levels with

more modified gRNAs (remain

stable in the cell longer).

• OTE levels greatly reduced

when Cas9 delivered as RNP,

rather than expressed form.

• OTEs completely removed when

using Alt-R HiFi Nuclease, while

maintaining on-target editing.

On- & off-target assessment via rhAmpSeq system (EMX1 target)

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• Similar off-target sites and OTE

levels detected across different

gRNA forms.

• Slightly higher OTE levels with

more modified gRNAs (remain

stable in the cell longer).

• OTE levels reduced when Cas9

delivered as RNP, but still present.

• OTEs greatly reduced when using

Alt-R HiFi Nuclease, while

maintaining on-target editing.

All gRNA and Cas9 forms tested produce identical Indel profiles

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gRNAs targeting EMX1

Conclusions

• Different gRNA formats give similar on-target editing when delivered as RNP.

(Higher modification needed when co-delivered with Cas9 in an expressed form)

• Subset of sequences respond better to higher modification, even with RNP delivery.

(Sequence and cell type dependent effect)

• Chemically synthesized gRNAs allow addition of chemical modifications, which

increase stability and reduce induction of innate immune system. (IVT sgRNAs induce

high levels of IFNα)

• The different gRNA forms result in the similar off-target editing levels at similar

sites. (Slight increase with gRNAs with increased modification)

• Delivering the gRNAs as RNP complexes shows a huge reduction in non-specific

editing. (This editing is even further reduced when using the Alt-R HiFi Cas9, while

maintaining high on-target activity)

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Alt-R CRISPR System — a complete workflow

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Design Cut Repair Analyze

Alt-R CRISPR-Cas9

guide RNA design tool

• Predesigned guides

• Custom designs

• Design checking

• Human, mouse, rat,

zebrafish, and

C. elegans

Coming soon –

HDR design tool

Alt-R guide RNAs

• Cas9 crRNA:tracrRNA

• Cas9 sgRNA

• Cas12a crRNA

Alt-R CRISPR proteins

• WT Cas9

• HiFi Cas9

• D10A nickase

• H840A nickase

• Cas12a (Cpf1)

Alt-R Electroporation

Enhancers

Ultramer

Oligonucleotides

• Up to 200 bases

Megamer ssDNA

Fragments

• Up to 2000 bases

• Sequence-verified

via NGS

Alt-R HDR Enhancer

Genome Editing

Detection Kit

• Simple, fast, T7EI-

based assay

Coming soon –

rhAmpSeq system for

CRISPR

• Multiplexed,

amplification for

Illumina NGS

• Up to 1000-plex

NEW: Alt-R HDR Enhancer increases HDR rates

with Cas9 and Cas12a nucleases

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0

10

20

30

40

50

60

70

MET TNPO3 site1 TNPO3 site 2 HPRT control HPRT site 1 HPRT site 2 HPRT site 3 HPRT site 4

Alt-R S.p. Cas9 nuclease v3 Alt-R A.s. Cas12a (Cpf1) nuclease v3

Eco

RI c

leav

age

(%)

Alt-R HDR Enhancer Neon electroporation, Jurkat cells

4 µM RNP, 3 µM HDR template

No treatment 30 µM HDR Enhancer

HDR enhancer is a small molecule compound that increases HDR efficiency

Take home messages• Standard Alt-R 2-part crRNA + tracrRNA works well for many

applications and is the most cost-effective synthetic option– Cas9 expressing cells

– gRNA screens (libraries)

– RNP delivery

– Reports of greater efficiency than sgRNA in zebrafish

• Increased chemical modifications in Alt-R crRNA XT + tracrRNA or using Alt-R sgRNA can add additional nuclease stability– Co-delivering with Cas9 plasmid or Cas9 mRNA

– Lipid nanoparticle delivery

– High nuclease environments

– Subset of sequence more susceptible to nuclease degradation

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More information and protocols available at: www.idtdna.com/crispr-cas9

(Click on the “Resources” tab for protocols)

THANK YOU

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reserved. Alt-R, Megamer, Ultramer, and rhAmpSeq are

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see www.idtdna.com/trademarks

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