Journal: Scientific Reports
Article Title: Adenoviral vector delivery of RNA-guided CRISPR/Cas9 nuclease complexes induces targeted mutagenesis in a diverse array of human cells
Figure Lengend Snippet: Transient transfection experiments for validating AdV shuttle plasmids pAdSh.PGK.Cas9 and pAdSh.U6.gRNA S1 . 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.gRNA S1 (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. Cas9 D10A ) and gRNA S1 , 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.
Article Snippet: One fifth of each PCR mixture was incubated in 15-μl reactions with 1 × NEBuffer 2 and 5 U of T7EI (both from New England Biolabs).
Techniques: Transfection, Polymerase Chain Reaction, Amplification, Transformation Assay, Construct, Clone Assay, Plasmid Preparation, Marker, Derivative Assay, Non-Homologous End Joining, Mutagenesis, Knock-Out