Evaluating CRISPR/Cas9 and TALENs Using the Surveyor Nuclease Assay.

The pgRNA_DsRed and pCas9_GFP (Addgene plasmid 44719) and the left and right TALEN pairs were cotransfected into HEK293 cells transfected with a partial COL7A1 gene, including the mutation site, using Fugene HD transfection reagent. After transfection, cells were cultured for 2 days at 32 °C and 37 °C, and whole gDNA was extracted using the Qiagen DNeasy Blood and Tissue Kit. The targeted mutant sequence was amplified using intron 108-F and GFP-R primers, whereas the normal sequence was amplified using intron 108-F and intron 110-2-R primers. The amplified PCR products were subjected to Surveyor nuclease treatment as described previously (15). The digestion products were loaded onto a 1.5% agarose gel stained with ethidium bromide, and images were captured with a ChemiDoc MP Imaging System (Bio-Rad). Densitometry was performed using ImageJ as described previously (15).