Fig. 1

Homology-directed DMD-targeting using standard and in trans paired nicking strategies. a Schematics of standard and in trans paired nicking (Nick²) procedures. The former involve DSB formation only at the target sequence; the latter comprise SSB formation at target plus donor sequences. pDonor^DMD and pDonor^DMD.TS have their transgenes flanked by sequences identical to those framing the gRNA^DMD target site (TS). Open and solid magenta arrowheads, position of the phosphodiester bond cleavage induced by Cas9’s RuvC and HNH nuclease domains, respectively. Solid arrowhead, position of the SSB induced by Cas9^D10A. The modified pDonor^DMD.TS differs from pDonor^DMD in that it has the gRNA^DMD TS next to its targeting module. The transgene is formed by human PGK1 promoter, EGFP ORF, and bovine GH1 polyadenylation signal sequences. Cas9:gRNA^DMD and Cas9^D10A:gRNA^DMD are cleaving and nicking RGN complexes, respectively. PAM protospacer adjacent motif. An integrant generated by HR events at both ends of the targeting module is depicted. The amplicons specific for telomere-sided and centromere-sided transgenic-DMD junctions (jT and jC, respectively), are equally shown. Horizontal arrowheads, primers. b Quantification of stable transfection levels by flow cytometry. Flow cytometry of long-term HeLa cell cultures initially exposed to the indicated plasmids. The bars correspond to mean ± s.d. of three independent biological replicates done on different days. **P = 0.006 (two-tailed t-test). c Cumulative molecular characterization of integrants generated by the conventional vs. the in trans paired nicking strategies. The frequencies of clones with random insertions (jT−/jC−), HR-derived telomeric junctions (jT+/jC−), HR-derived centromeric junctions (jT−/jC+) and HR-derived telomeric and centromeric junctions (jT+/jC+) are plotted. The corresponding PCR screening data are presented in Supplementary Fig. 3