Fig. 7

Evaluation of various CRISPR-Cas systems in HDR-mediated genome editing using linearized plasmid donor templates. a, b Fusing eGFP to the C-terminus of a CLTA and b GLUL via a P2A self-cleaving peptide. In the schematics of the targeted loci, the blue boxes depict the exons (E), the brown horizontal bars indicate the homology arms (1000–1500 nt each), and the small triangle in the left homology arm of the GLUL donor indicates that there are several third base pair (bp) mutations towards the end of the coding region to prevent re-targeting by the Cas nucleases. Within the nucleotide sequences, the red vertical lines indicate the Cas9 cleavage sites, while the blue vertical lines indicate the Cpf1 cleavage sites. Expectedly, the percentages of GFP-positive cells were very low when no sgRNA was transfected (red bars), but showed a clear increase in the presence of the appropriate sgRNA for all three Cas nucleases (green bars). Data represent mean ± standard error of the mean (s.e.m.; n ≥ 5). *P < 0.05, **P < 0.01, N.S. not significant; Student’s t-test. c Rate of indel formation as determined by T7E1 assays. The cleavage efficiencies observed could largely explain the rates of eGFP knockin shown in a, b. Data represent mean ± s.e.m. (n ≥ 8). *P < 0.05, **P < 0.01, N.S. not significant; Student’s t-test