Measuring CRISPR-Cas9 Mutagenesis Frequencies

Roughly 72 hr post-transfection, genomic DNA was extracted from U2OS cells using the Agencourt DNAdvance Genomic DNA Isolation Kit (Beckman Coulter Genomics), and T7 endonuclease I (T7E1) assays were performed as previously described^{26 (link)}. Briefly, 600–800 nt amplicons surrounding on-target sites were amplified from ~100 ng of genomic DNA using Phusion Hot-Start Flex DNA Polymerase (New England Biolabs, NEB) using the primers listed in Supplementary Table 3. PCR products were visualized (using a QIAxcel capillary electrophoresis instrument, Qiagen), and purified (Agencourt Ampure XP cleanup, Beckman Coulter Genomics), Denaturation and annealing of ~200 ng of the PCR product was followed by digestion with T7EI (NEB). Digestion products were purified (Ampure) and quantified (QIAxcel) to approximate the mutagenesis frequencies induced by Cas9-sgRNA complexes.