Fig. 7: Assessment of off-target editing due to the double tap method.

a HEK293T cells were transfected with Cas9 and gRNA plasmids (non-targeting, primary, or secondary gRNAs). After 72 h, cells were analyzed by NGS at the primary (on-target) and all predicted off-target loci. Shown are total indel rates of all samples. The primary (on-target) loci are labeled as OG, while predicted off-target sites for primary gRNAs are labeled as OG_OT, and predicted off-target sites for secondary gRNAs are labeled as DT_OT on the y axis. The label on the x-axis indicates which gRNA the cells were transfected with; the secondary (DT), non-targeting (NT) or primary (OG). Only one gRNA was used at a time, b HEK293T cells were transfected with plasmids encoding Cas9-p2A-GFP, primary gRNA, and either non-targeting gRNA or secondary gRNA(s). As a control, HEK293T were transfected with plasmids encoding Cas9-P2A-GFP and non-targeting gRNA only. After 72 h cells were stained with propidium iodide to quantify cell viability FACS. The percentage of transfected cells (as determined by GFP fluorescence) that were viable are plotted with respect to the primary gRNA used (RNF2, HBB5, APOB1, and MMACHC). Samples with primary and non-targeting gRNAs are shown in blue, while those with primary and secondary gRNAs are in pink. Three secondary gRNAs were used with the RNF2 and HBB5 primary gRNA, and one secondary gRNA was used at APOB1 and MMACHC sites. The sample with non-targeting gRNA only is in green. Values and error bars represent the mean and standard deviation of viable cells within the transfected population for n = 3 biological replicates.