Fig. 1: Schematic and initial results of the double tap method.

a Schematic overview of the double tap method. Cas9 introduces a DSB at a locus of interest using the primary guide RNA. HDR processes a subset of the DSBs into the desired outcome using a donor template (blue sequence). Concurrently, indels are introduced at the DSB site via end-joining pathways (red sequences). These undesired indel sequences are subsequently targeted with secondary gRNAs to improve overall yields of the desired outcome through a second DSB introduction and sequential HDR repair. b Indel sequences and their corresponding introduction efficiencies at the MMACHC site after transfecting HEK293T cells with Cas9 and a non-targeting gRNA (top), the primary gRNA plus a non-targeting gRNA (middle), or the primary gRNA plus a secondary gRNA targeted to the indel sequence indicated with the black arrow (bottom). c HDR-mediated genome editing efficiencies at the FANCF (in which a low-frequency indel was targeted), APOB1, and MMACHC sites when HEK293T cells are transfected with an ssODN and plasmids encoding Cas9, the primary gRNA, and either a non-targeting gRNA (NT, left) or secondary gRNA(s) (DT for “double tap”, right; two secondary gRNAs were used with the FANCF primary gRNA, and one secondary gRNA was used at the other two sites). Plotted are the percent of total DNA sequencing reads with the desired modification introduced (perfect HDR products without indels). d HDR-mediated genome editing efficiencies at the RNF2 locus when HEK293T cells are transfected with an ssODN and plasmids encoding Cas9, the primary gRNA, and either a non-targeting gRNA (NT, far left) or one (1 x DT), two (2 x DT), or three (3 x DT) secondary gRNAs. Values on 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.