Extended Data Fig. 10: Specificity assessment of PAMmla-derived enzymes.

a, Quantification of GUIDE-seq2 double-stranded oligodeoxynucleotide (dsODN) tag integration at on-target sites in nuclease-based experiments with MRRWMR, SpG, and SpRY and sgRNAs targeting two different endogenous sites in HEK 293T cells. SpCas9 variant enzymes are named based on their amino acids at each of the six positions in the SpCas9(6AA) library. dsODN integration efficiency was assessed by targeted amplicon sequencing and modified reads were analyzed using CRISPResso2; mean, standard deviation, and individual datapoints are shown for n = 3 technical replicates. b, Venn diagram representations of the GUIDE-seq-2 detected off-target sites that are shared between or unique to MRRWMR, SpG, and SpRY nucleases using the two sgRNAs targeted to sites with NGTG PAMs (similar to the RHO P23H on-target site). c, Fraction of GUIDE-seq-2 reads attributed to on- and off-target sites for MRRWMR, SpG, and SpRY from experiments using the NGTG-2 or NGTG-3 sgRNAs. d, Quantification of GUIDE-seq-2 dsODN tag integration at the on-target site for experiments in the homozygous RHO P23H cell line, when using the RHO P23H sgRNA and SpCas9-MRRWMR and -KRHWMR, SpG, and SpRY expression plasmids. dsODN integration efficiency was assessed by targeted amplicon sequencing and modified reads were analyzed using CRISPResso2; mean, standard deviation, and individual datapoints are shown for n = 3 technical replicates. e, GUIDE-seq-2 genome-wide specificity outputs for SpCas9-MRRWMR and -KRHWMR, SpG, and SpRY nucleases using the RHO P23H targeted sgRNA in homozygous RHO P23H HEK 293T cells. Mismatched positions in the spacers of the off-target sites are highlighted in color; GUIDE-seq read counts from consolidated unique molecular events for each variant are shown to the right of the sequence plots. f, Venn diagram representation of the GUIDE-seq-2 detected off-target sites that are shared between or unique to SpCas9-MRRWMR and -KRHWMR, SpG, and SpRY nucleases using the RHO P23H sgRNA. g, Unidentifiable sequencing reads unattributable to either WT or P23H alleles due to deletions spanning the base harboring the mutation, for data from heterozygous RHO P23H mice shown in Fig. 5l, h, Ratio of in vivo editing efficiencies observed on mutant (P23H) versus WT RHO alleles, for each SpCas9 nuclease tested in Fig. 5l.