CRISPR-Cas9 Cutting Efficiency Analysis

Analysis was performed on raw reads from Illumina deep sequencing. Sequencing reads were filtered to remove low-quality (Illumina average quality <28) or unmapped reads. For each sequencing read representing a CRISPR-Cas9 cutting event, the cutting genotype was identified and categorized as an insertion or deletion event; overall fractions of insertions and deletions of all lengths were then computed from the two replicates. This was carried out with a published analysis pipeline described in Shen et al. ^{12 (link)}. For indel and 1-bp insertion detection at native human genomic loci, Crispresso2 was used to carry out the alignment of NGS reads to the wild-type amplicon sequence, using default parameters and a Needleman Wunsch gap extended score of 0 for optimal alignment with visual inspection^{39 (link)}. Reads were determined to be modified or unmodified if a 1-bp insertion was located within 2 bp from each sgRNA cleavage (default quantification window for Cas9 Crispresso2).