Figure 2
Transient transfection experiments for validating AdV shuttle plasmids pAdSh.PGK.Cas9 and pAdSh.U6.gRNAS1.
AAVS1-specific PCR products amplified from E1-transformed PER.C6 cells co-transfected with hCas9 (9.6 kb) and gRNA_AAVS1-T2 (4 kb), pAdSh.PGK.Cas9 (11.6 kb) and pAdSh.U6.gRNAS1 (7.3 kb), hCas9 and “empty gRNA” construct gRNA_Cloning Vector (3.9 kb), hCas9_D10A (9.6 kb) and gRNA_AAVS1-T2 or mock-transfected. Marker, Gene Ruler DNA Ladder (Fermentas). Plasmids hCas9 and hCas9_D10A express Cas9 nucleases which induce a DSB and a nick at AAVS1, respectively. After amplicon denaturation and reannealing, the presence of mismatches derived from NHEJ-mediated repair of site-specific DSBs in cellula was probed by T7 endonuclease I (T7EI) digestions (upper panel). Negative controls were provided by amplicons not exposed to T7EI (-T7EI) as well as T7EI-treated amplicons (+T7EI) corresponding to mock-transfected cells, to cells co-transfected with hCas9 and gRNA_Cloning Vector and to cells co-transfected with hCas9_D10A and gRNA_AAVS1-T2 encoding the “nickase” mutant version of Cas9 (i.e. Cas9D10A) and gRNAS1, respectively. Solid and open arrowheads indicate the positions of, respectively, undigested and T7EI-digested DNA fragments whose sizes are consistent with DSB formation at the AAVS1 target site. % KO and UN, knockout frequency and undetected, respectively.
