Combining Off-flow, a Nextflow-coded program, and whole genome sequencing reveals unintended genetic variation in CRISPR/ Cas-edited iPSCs

Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-Cas nucleases and human induced pluripotent stem cell (iPSC) technology can reveal deep insight into the genetic and molecular bases of human biology and disease. Undesired editing outcomes, both on -target (at the edited locus) and off -target (at other genomic loci) hinder the application of CRISPR-Cas nucleases. We developed Off -flow, a Nextflow-coded bioinformatic workflow that takes a specific guide sequence and Cas protein input to call four separate off -target prediction programs (CHOPCHOP, Cas-Offinder, CRISPRitz, CRISPR-Offinder) to output a comprehensive list of predicted off -target sites. We applied it to whole genome sequencing (WGS) data to investigate the occurrence of unintended effects in human iPSCs that underwent repair or insertion of disease -related variants by homologydirected repair. Off -flow identified a 3 -base -pair -substitution and a mono -allelic genomic deletion at the target loci, KCNQ2, in 2 clones. Unbiased WGS analysis further identified off -target missense variants and a monoallelic genomic deletion at the targeted locus, GNAQ, in 10 clones. On -target substitution and deletions had escaped standard PCR and Sanger sequencing analysis, while missense variants at other genomic loci were not detected by Off -flow. We used these results to filter out iPSC clones for subsequent functional experiments. Offflow, which we make publicly available, works for human and mouse genomes currently and can be adapted for other genomes. Off -flow and WGS analysis can improve the integrity of studies using CRISPR/Cas-edited cells and animal models.