Figure 1.

Schema of approach. Cas9 and gRNA expression plasmids are transfected into a cell population with or without a DNA donor sequence. Both single stranded oligonucleotide donors (ssODN) and double stranded DNA donors (dsDonor) with 3 substitutions (in green) are used in this study. NHEJ is classified as short deletions ranging from 1–5 bp and Alt-EJ is categorized by at least 5 bp deletion with at least 2 bp MH (representative 2 bp MH shown in red). Following an incubation period of typically 2–3 days, a range of repair pathway usage are assessed using PCR amplification of a 201 bp segment around the break followed by sequencing of the PCR product with paired end 150 × 2 Illumina MiSeq sequencing (Amplicon Sequencing). Alternatively, genomic DNA is used as an input for droplet digital PCR (ddPCR) to measure specific repair products on a Biorad QX200 platform.