Quantifying CRISPR-Cas9 Mutagenesis Frequencies

To quantify mutagenesis frequencies at desired genomic loci, T7E1 assays were performed as previously described^{30 (link)}. Briefly, on- or off-target sites were amplified from ~100 ng of genomic DNA using Phusion Hot-Start Flex DNA Polymerase (New England Biolabs) using the primers listed in Supplementary Table 2. An Agencourt Ampure XP cleanup (Beckman Coulter Genomics) was performed prior to the denaturation and annealing of ~200 ng of the PCR product, followed by digestion with T7E1 (New England Biolabs). Purified digestion products were quantified using a QIAxcel capillary electrophoresis instrument (Qiagen) to approximate the mutagenesis frequencies induced by Cas9-sgRNA complexes. P values for comparisons between SpCas9 variants were calculated using a one-sided t-test with equal variances and adjusted for multiple comparisons using the method of Benjamini and Hochberg (Supplementary Table 3).