Supplementary Figure S1. Genome editing with ShaCas9 and SlutrCas9 for
endogenous loci. (A) Genome editing with ShaCas9 for 16 endogenous loci in
HEK293T cells. n=3. (B) Genome editing with SlutrCas9 for 18 endogenous loci in
HEK293T cells. n=3. 
 
 

 
Supplementary Figure S2. SlugCas9 enables genome editing in diverse cell types,
including (A) A375, (B) HCT116, (C) HeLa, (D) human foreskin fibroblast (HFF) and
(E) N2a cells. n=3. 
 
   
 

 
Supplementary Figure S3. SlugCas9 can be delivered by AAV for genome editing in
diverse cell types, including (A) A375, (B) HCT116, (C) HeLa, (D) human foreskin
fibroblast (HFF) and HEK293T (E) cells. n=3. 
 
   
 

 
Supplementary Figure S4. Compare the activity of wild-type SlugCas9 sgRNA,
engineered SlugCas9 sgRNA and SaCas9 sgRNA. (A) Compare SaCas9 sgRNA,
SlugCas9 sgRNA and engineered SlugCas9 sgRNA sequences and the secondary
RNA structures. Modified “c” is indicated in red. (B) The editing efficiencies of
SlugCas9 used with SaCas9 sgRNA, SlugCas9 sgRNA or engineered SlugCas9
sgRNA for 9 genomic loci in HEK293T cells. Quantification is shown on the right.
ns: not significant. n=3. 
 
   
  
Supplementary Figure S5. Compare the activity of SlutrCas9 sgRNA, ShaCas9
sgRNA and SaCas9 sgRNA. (A) Compare SaCas9 sgRNA, SlutrCas9 sgRNA and
ShaCas9 sgRNA sequences and the secondary RNA structures. (B) The editing
efficiencies of ShaCas9 used with SaCas9 sgRNA or ShaCas9 sgRNA for 12 genomic
loci. Quantification was shown on the right. ns: not significant. n=3. (C) The editing
efficiencies of SlutrCas9 used with SaCas9 sgRNA or SlutrCas9 sgRNA for 12
genomic loci in HEK293T cells. Quantification is shown on the right. ns: not
significant. n=3. 
 
 
  
Supplementary Figure S6. Base editing with SlugCas9. (A) SlugBE4max induces
C to T conversions for 34 human genomic loci in HEK293T cells. Schematic of the
SlugBE4max is shown above. n=3. (B) SlugABEmax induces A to G conversions for
33 human genomic loci in HEK293T cells. Schematic of the SlugABEmax is shown
above. n=3. 
 
   
  
Supplementary Figure S7. Analysis of SlutrCas9 and ShaCas9 specificity.
Schematic of the GFP-reporter assay for specificity analysis in HEK293T cells is
shown on the top. A panel of gRNAs with dinucleotide mutations is shown below.
Each gRNA activity for SlutrCas9 and ShaCas9 is analyzed based on GFP expression.
n=3.
 
   
 

 
Supplementary Figure S8. PAM analysis of engineered SaCas9 orthologs. (A)
WebLogo and PAM wheel for SlugCas9-KH. (B) WebLogo and PAM wheel for
ShaCas9-KKH. (C) WebLogo and PAM wheel for SlutrCas9-KKH. 
 
   
  
Supplementary Figure S9. Comparison of Sa-SlugCas9, SlugCas9-HF, SlugCas9
and SaCas9 ability for stimulating HDR in HEK293T cells. (A) Schematic of
donor DNA design. Target sequences are underlined; PAMs are indicated by yellow
background; EcoRI restriction sites are shown in red. (B) RFLP analysis of HDR rates
after genome editing. The size of DNA bands after digestion is shown by red lines.
Indel frequencies are shown below.  
 
 
 
  
Supplementary Figure S10. Comparison of editing efficiency at on- and
off-targets among 8 SaCas9 orthologs. (A) Comparison of editing efficiency at
on-targets for endogenous loci among 8 SaCas9 orthologs in HEK293T cells. (B)
Comparison of editing efficiency at off-targets based on GFP-activation assay among
8 SaCas9 orthologs in HEK293T cells. The editing efficiency at off-targets is
normalized by on-target efficiency. Low editing efficiency indicates high specificity.
The SauriCas9 and Sa-SauriCas9 data was generated in a previous study 1. One-way
ANOVA test. *p<0.05, **p<0.01, ***p<0.001. 
 

 
Supplementary Figure S11. SaCas9 variants used in this study. Black arrows
indicate the locations of mutations as compared to wild-type Cas9. BH, bridge helix;
PI, PAM-interacting domain; L1 and L2: linker regions. Amino acid position is shown
on the top.
 
 

 
Supplementary Figure S12. Sa-SlugCas9 enables genome editing in mouse
zygotes. (A) gRNA design for Myh6 gene. Schematic of Myh6 gene is shown above.
(B) Gel picture of T7EI digestion for each gRNA-mediated genome editing in N2a
cells. (C) Procedure of microinjection of mouse zygotes. (D) Embryos at day 0.5 and
day 3. (E) Sanger sequencing reveals indels occur. PAM sequences are underlined.  
 
 
1.  Hu,  Z.  et  al.  A  compact  Cas9  ortholog  from  Staphylococcus  Auricularis  (SauriCas9)  expands 
the DNA targeting scope. PLoS biology 18, e3000686 (2020).