Fig. 4

Bioinformatic and empirical comparison of NmeCas9 and SpyCas9 off-target sites within the human genome. a Genome-wide computational (CRISPRseek) predictions of off-target sites for NmeCas9 (with N₄GN₃ PAMs) and SpyCas9 (with NGG, NGA, and NAG PAMs) with DTS3, DTS7, and DTS8 sgRNAs. Predicted off-target sites were binned based on the number of mismatches (up to six) with the guide sequences. b GUIDE-Seq analysis of off-target sites in HEK293T cells with sgRNAs targeting DTS3, DTS7, and DTS8, using either SpyCas9 or NmeCas9, and with up to 6 mismatches to the sgRNAs. The numbers of detected off-target sites are indicated at the top of each bar. c Numbers of independent GUIDE-Seq reads for the on- and off-target sites for all six Cas9/sgRNA combinations from (b) (SpyCas9, orange; NmeCas9, blue), binned by the number of mismatches with the corresponding guide. d Targeted deep sequencing analysis of editing efficiencies at on- and off-target sites from (a) or (b) with SpyCas9 (left, orange) or NmeCas9 (right, blue). Data for off-target sites are in grey. For SpyCas9, all off-target sites were chosen from (b) based on the highest GUIDE-Seq read counts for each guide (Additional file 10: Table S3). For NmeCas9, in addition to those candidate off-target sites obtained from GUIDE-Seq (c), we also assayed one or two potential off-target sites (designated with the “-CS” suffix) predicted by CRISPRseek as the closest near-cognate matches with permissive PAMs. Data are mean values ± s.e.m. from three biological replicates performed on different days