Fig. 6: Cleavage specificity and off-target effects of AaCas12b in mammalian genomes.

a Analysis of cleavage specificity of AaCas12b/sgRNA in human and mouse cells using sgRNAs carrying single base-pair mismatches in the guide sequence. Error bars indicate standard errors of the mean (s.e.m.), n = 3. b (Top) Schematic illustration of AaCas12b target sites in the human CCR5 and RNF2 loci, respectively. (Bottom) Indel frequencies induced by AaCas12b directed by sgRNAs targeting endogenous CCR5 and RNF2 sites and their corresponding off-target sites in human 293FT cells. Mutation frequencies were assessed by T7EI assay. Error bars indicate s.e.m., n = 2. c WGS analysis of genomic DNAs of RNF2-targeted 293FT cells. None of 2598 (RNF2-site 1) and 3394 (RNF2-site 2) sites identified with Cas-OFFinder in the reference genome (hg38) that differed from the on-target site by up to five mismatches harbored indels in the mutated genome. d Whole-genome sequencing (WGS) analysis of genomic DNAs of Nrl-mutated mouse gonads. None of the 2657 sites identified using Cas-OFFinder in the reference genome (mm10) that differed from the on-target site by up to five mismatches harbored indels in the mutated genome