Fig. 3

Evaluation of various CRISPR-Cas systems in HDR-mediated genome editing using symmetric ssODN donor templates and spacers with overlapping seeds. a, b Extent of XbaI restriction site (depicted in green) insertion into a coding exon of the a CACNA1D or b PPP1R12C gene. The brown horizontal bars represent the 47-nt homology arms of the donor template and NT indicates that the donor is of the non-target strand sequence. Three different spacer lengths (17, 20, and 23 nt) were tested. The cells were harvested 72 h after transfection and the gene-targeting efficiencies were determined by Illumina deep sequencing. Data represent mean ± standard error of the mean (s.e.m.; n ≥ 4). *P < 0.05, **P < 0.01; Student’s t-test. c, d Extent of precise gene editing by SpCas9, AsCpf1, and LbCpf1 when ssODNs of different homology arm lengths (27–47 nt) were used together with 20-nt spacers targeting c CACNA1D or d PPP1R12C. The cells were harvested 72 h after transfection and the gene targeting efficiencies were determined by Illumina deep sequencing. Data represent mean ± s.e.m. (n ≥ 4). *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test. e, f Concurrent T7E1 assays and RFLP analysis of cells co-transfected with the indicated CRISPR plasmids and donor ssODNs containing 47-nt long homology arms. We used 20 or 23 nt long spacers targeting either e CACNA1D or f PPP1R12C. The cells were harvested 72 h after transfection. Overall, the cleavage efficiencies of SpCas9 were comparable to those of AsCpf1 and LbCpf1, as determined by the T7E1 assays. However, the extent of XbaI integration into the target sites was lower for SpCas9 compared to AsCpf1 and LbCpf1, as determined by RFLP analysis