Fig. 6: Installation of disease relevant mutations in the HBB and HEXA genes using the double tap method.

a 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), or primary gRNA and secondary gRNA(s) (DT; three secondary gRNAs were used at the HBB5 site, and one secondary gRNA was used at the HBB1, HEXA2 and HEXA5 sites). b Shown are total indel rates of all samples from (a), with the specific indels targeted by secondary gRNAs shown in yellow, orange, and red (depending on how many secondary gRNAs were used for a particular site, there may only be yellow bars). Blue represents indels not targeted by secondary gRNAs. c HEK293T cells were transfected with plasmids encoding the prime editor and pegRNA only (PE2 sample), or pegRNA and nicking gRNA (PE3 sample) to introduce the same mutations as in (a). After 72 h, cells were analyzed by NGS to determine the efficiencies of introduction of the intended edit. Shown are the percent of DNA sequencing reads with the desired modification introduced (perfectly edited products without indels) for double tap samples from (a) (labeled as DT), PE2 treated cells (labeled as PE2), or PE3 treated cells (labeled as PE3). Values on the whisker plots in (a) and (c) 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. Values and error bars in (b) 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. Data points are marked as circles when the ssODN encoded an extra blocking mutation, and as triangles when no additional mutation was installed. Data points are marked as squares for prime editing samples.