Extended Data Fig. 7: Genome-wide off-target analysis of PAMmla predicted enzymes.

a, Quantification of GUIDE-seq2 double-stranded oligodeoxynucleotide (dsODN) tag integration at the on-target site, in nuclease-based experiments with SpG, SpRY, and PAMmla predicted enzymes targeting endogenous target 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 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 PAMmla generated, SpG, and SpRY nucleases. c, Nucleotide composition of PAMs adjacent to off-target spacers detected in GUIDE-seq-2 experiments, not including the on-target reads. The y-axis represents the fraction of total off-target GUIDE-seq-2 reads containing each nucleotide at each position of the PAM. d, Quantification of GUIDE-seq-2 double-stranded oligodeoxynucleotide (dsODN) tag integration at the on-target site, in nuclease-based experiments with KWRQLC and SpG when using the CYBB T362I sgRNA but targeting the wild-type genome of 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. e, GUIDE-seq-2 genome-wide specificity outputs for KWRQLC and SpG nucleases using the CYBB T362I targeted sgRNA; note that HEK 293T cells harbor the wild-type copy of the CYBB gene and are therefore an imperfect match to the sgRNA. 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.