Fig. 4

Evaluation of various CRISPR-Cas systems in HDR-mediated genome editing using symmetric ssODN donor templates and perfectly matched spacers. a Intended DNA changes at the A3 (in ALK), A11 (in EGFR), B8 (in EGFR), and B18 (in STAG2) target sites. Each red vertical line indicates the cleavage site of Cas9 nucleases, which occurs 3 bp upstream of their PAM. Each blue vertical line indicates the cleavage site of Cpf1 nucleases on one DNA strand, which occurs 18 nt downstream of their PAM. The HindIII restriction site is indicated in green. b Extent of correctly incorporating the HindIII recognition sequence into the A3, A11, or B18 target locus. Donor ssODNs with 27-nt homology arm lengths were used. The donor templates were complementary to the target DNA strand. Cells were harvested for deep sequencing analysis 72 h post-transfection. Both the Cpf1 endonucleases consistently exhibited higher levels of precise gene targeting than SpCas9. Data represent mean ± standard error of the mean (s.e.m.; n ≥ 5). ***P < 0.001; Student’s t-test. c Extent of precise gene editing by SpCas9, AsCpf1, and LbCpf1 at the B8 locus when ssODNs of different homology arm lengths (17–27 nt inclusive) were used. The donor templates were complementary to the target DNA strand. 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 ≥ 3). *P < 0.05, **P < 0.01, ***P < 0.001; Student’s t-test. d Concurrent T7E1 assays and RFLP analysis of cells co-transfected with the indicated CRISPR plasmids and donor ssODNs containing 27 nt long homology arms for the A3, A11, B8, and B18 target sites. Overall, the cleavage efficiencies of SpCas9 were comparable to those of AsCpf1 and LbCpf1, as determined by the T7E1 assays. However, the extent of HindIII integration into the target sites was lower for SpCas9 compared to AsCpf1 and LbCpf1, as determined by RFLP analysis