Fig. 4. Unbiased and targeted analysis of Cas12f specificity as assessed by Digenome-seq analysis.
a, Tolerance of Cas12f_4.1 to mismatched gRNA. The engineered gRNAs with a singly mismatched base and pairs of mismatched bases were used for the investigation of indel frequencies in HEK293T cells (n = 3). b, Indel frequencies at off-targets identified by OFFinder for AsCas12a, Cas12_ge4.0 and Cas12_ge4.1. An intergene corresponds to target 3 targeted throughout the main text. c, Indel frequencies at previously validated off-targets for AsCas12a, Cas12_ge4.0 and Cas12_ge4.1. The ratio of indel frequency at off-target to that at on-target was considered as an index for specificity (n = 3). Statistical analysis was performed by a two-sided Student’s t-test. NS, not significant. d, IGV files at on-target and off-target loci after in vitro digestion of genomic DNA. The gap indicates a region where sequences were missing in both forward and reverse reads. e, The number of potential off-target loci identified by Digenome-seq analysis for AsCas12a and Cas12f. f, Validation of off-target sites identified by Digenome-seq analysis using Cas12f and AsCas12a as endonucleases. Indel frequencies were measured at both on-target and potential off-target loci after transfection with either Cas12f- or AsCas12a-encoding vector. Control refers to Cas12f-untreated cells (n = 3). All error bars represent the s.d.