1. Modified Cas9 with higher specificity
Sniper Cas9 :
Modified Cas9 with higher specificity
2018

2. Increasing specificity of CRISPR-Cas9: Towards SNP differentiation
Genome Technology
Increasing specificity of CRISPR-Cas9: Towards SNP differentiation
Cell
Strategies to improve specificity of CRISPR-Cas9
sg-RNA
- Addition of G at 5’ end of sgRNA
- Truncation of spacer sequence from 20 to 17-mer
Cas9 protein
- Site-directed mutation of Cas9 protein
Off-target: SNP
Off-target effect more important
Improved Cas9 protein need to lower off-targeting notably without sacrificing on-targeting.
It can work well with improved gRNA as well.
On-target

3. Rational design vs Directed evolution
Library construction
Crystal structure
Site directed mutation
Activity test
Shuffling
Screening system

4. Selection Scheme: Sniper-Screen can select Cas9 variant with high specificity
(induces ccdB)
(induces sgRNA and Cas9)

5. No position overlaps with eSpCas9, HF-Cas9 or Hypa-Cas9, evo Cas9 or
Mutant location of Sniper Cas9s
No position overlaps with eSpCas9, HF-Cas9 or Hypa-Cas9, evo Cas9 or
xCas9 3.7
Sniper 1 showed highest activity
Extensive site directed mutation study  original Sniper 1 is the best
Comparison of Sniper 1 with other engineered Cas9s

6. Compare data : Sniper Cas9 with other Cas9 variants in FANCF01
Specificity ratio = on-target activity / off-target activity (normalized to WT Cas9 activity)

7. Compare data : Sniper Cas9 with other Cas9 variants in AAVS

8. Grey box: range above 70% of WT Cas9 activity
Sniper Cas9 do not sacrifice on-target activity
Grey box: range above 70% of WT Cas9 activity

9. Six targets under specificity ratio 10 Ribonucleoprotein (RNP) form
Sniper Cas9 works well both plasmis form and RNP form
Six targets under specificity ratio 10
Ribonucleoprotein (RNP) form

10. hEMX1 base editing via BE3: C to T
Sniper Cas9 works well with Base Editors
hEMX1 base editing via BE3: C to T
Sniper-BE3 with truncated sgRNA: near background level without loss of on-target activity
aAGTCCGAGgAGAgGAAGAAAGG
GAGTCCaAGCAGtAGAgGAAGGG
GAGTCCtAGCAGgAGAAGAAGAG
GAaTCCaAGCAGAAGAAGAgAAG
GAGTCtaAGCAGAAGAAGAAGAG
GAGTCCGAGCAGAAGAAGAAGGG

11. Sniper Cas9 shows same specificity in iPS and primary cells
On-target specificity of sniper Cas9 paired with gX19 targeting AAVS in iPS and T cells

12. - is compatible with truncated or extended sgRNAs. Sniper Cas9
Summary
SniperCas9
- is compatible with both matching (G) and mismatching (g) 5’ G of sgRNAs.
- is compatible with truncated or extended sgRNAs.
Sniper Cas9
- shows the highest specificity among various SpCas9 variants without sacrificing on-target
activity.
Sniper Cas9 works well in both plasmid form and RNP form.
Sniper Cas9 works well with BE3
- reduces off-targeting near background level with truncated sgRNA.
Sniper Cas9 shows the same specificity in iPS and primary cells as cell lines.