Homo sapiens Targeted Locus (Loci)

To evaluate the DNA cleavage specificity of fCas9, we measured the modification of Cas9 off-target sites of CLTA, EMX, and VEGF genomic target sites. The target site and its corresponding known off-target sites were amplified from genomic DNA isolated from HEK293 cells treated with fCas9, Cas9 nickase, or wild-type Cas9 and two sgRNAs spaced 19 bp apart targeting the CLTA site, two sgRNAs spaced 23 bp apart targeting the EMX site, two sgRNAs spaced 14 bp apart targeting the VEGF site, or two sgRNAs targeting an unrelated site (GFP) as a negative control.