Fig. 3: Further characterization of the double tap method.

a Additive effects of double tap and previously developed HDR-improving methods were investigated at the MMACHC site. Shown are the percent of DNA sequencing reads with the desired modification introduced (perfect HDR products without indels) for cells treated with primary gRNA and a non-targeting gRNA (NT, left), or primary gRNA and secondary gRNA (DT, right; only one secondary gRNA was used at the MMACHC site). NT and DT samples were additionally treated with the small molecule HDR enhancer (Alt-R) or with a Cas9-CtIP fusion construct (Cas9-HE). DMSO-treated and no additive samples served as a base line for comparison (the Alt-R molecule is dissolved in a DMSO solution). b Shown are total indel rates of samples from (a), with the specific indels targeted by the secondary gRNA shown in yellow. Blue represents indels not targeted by a secondary gRNA. Values and error bars represent the mean of the number of sequencing reads with indel sequences divided by the total number of sequencing reads ± SD for n = 3 biological replicates. c Double tap improvements using Cas9:gRNA RNP complex at 3 sites. Shown are the percent of DNA sequencing reads with the desired modification introduced (perfect HDR products without indels) for cells treated with primary gRNA and a non-targeting gRNA (NT, left), or primary gRNA and secondary gRNA(s) (DT, right; three secondary gRNAs were used at the RNF2 site, two secondary gRNAs were used at the HEK3 site, and one secondary gRNA was used at the MMACHC site). d Analysis of zygosity of genome edited isogenic cells (n = 41 for each groups) at the MMACHC locus. Shown are the frequency of the indicated genome editing outcomes from each set of edited cells. Samples in (a–c) were analyzed by NGS after 72 h, and samples in (d) were clonally expanded and genotyped by NGS after 3 weeks. a, c Values on the whisker plots represent the lowest observation, lower quartile, median, upper quartile and the highest observation of three independent replicates. Data were analyzed with univariate statistics (one-way ANOVA [one-sided]) and p values are labeled on the graphs.