Fig. 1. Genome editing and domain architecture of four Staphylococcus Cas9s.

a Activity of wild-type Cas9s on HEK293T loci assayed by amplicon sequencing. Cas9s were tested as RNPs with their respective native tracr sequences and delivered via nucleofection. Spa, Smi, and Slu were tested on two targets with 5′-NNGG-3′ PAM (guide_87 and guide_102 targeting the HBB_R01_T2 and VEGFA_T22 loci, respectively). Because of the distinct PAM for ShyCas9, the corresponding experiments with ShyCas9 required a different set of targets in these loci containing a 5′-NNARMM-3′ PAM (guide_1-4 targeting the HBB_R01_T1, VEGFA_T1 and FANCF_T1, and FANCF_T2 loci). Data are presented as the mean of n = 2 independent biological replicates. Editing values were background subtracted. b Effect of guide length on the efficiency of SluCas9 editing. To assess the optimal protospacer length for SluCas9, a single HBB site (R01_T2) was targeted by synthetic sgRNAs with protospacer lengths varying from 13 to 25 nt (guide_267, 268, and guide_80-90) using RNP nucleofection. The editing efficiency was measured using ddPCR. For negative controls, the nuclease was nucleofected in the absence of sgRNA. Shown are individual measurements with the median as bars plus interquartile range, n = 3 for length 13–19, 21, 24, and 25, n = 5 for length 20, 22, and 23 independent biological replicates. c Domain architecture schematic for SpyCas9⁶² and SauCas9⁶³, and putative domain architecture for SluCas9 based on an alignment with SauCas9. Source data of 1a and 1b are provided in the source data file.