Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity. Commun Biol. 2023;6(1):626
JMG, Mice, Animals, CRISPR-Cas Systems, Gene Editing, Genome, Mutation, Mutagenesis
Commun Biol. 2023;6(1):626
Research reported in this work was supported by the NIH Common Fund, National Human Genome Research Institute UM1 HG006348 (JDH and MED), and National Institutes of Health Office of the Director UM1 OD023221 (KCKL) and UM1 OD023222 (REB, SAM and JKW) and Genome Canada and Ontario Genomics OGI-137 (LMJN). Additional support was provided by the Canadian Center for Computational Genomics (C3G), part of the Genome Technology Platform (GTP), funded by Genome Canada through Genome Québec and Ontario Genomics (AR).
Genome editing with CRISPR-associated (Cas) proteins holds exceptional promise for "correcting" variants causing genetic disease. To realize this promise, off-target genomic changes cannot occur during the editing process. Here, we use whole genome sequencing to compare the genomes of 50 Cas9-edited founder mice to 28 untreated control mice to assess the occurrence of S. pyogenes Cas9-induced off-target mutagenesis. Computational analysis of whole-genome sequencing data detects 26 unique sequence variants at 23 predicted off-target sites for 18/163 guides used. While computationally detected variants are identified in 30% (15/50) of Cas9 gene-edited founder animals, only 38% (10/26) of the variants in 8/15 founders validate by Sanger sequencing. In vitro assays for Cas9 off-target activity identify only two unpredicted off-target sites present in genome sequencing data. In total, only 4.9% (8/163) of guides tested have detectable off-target activity, a rate of 0.2 Cas9 off-target mutations per founder analyzed. In comparison, we observe ~1,100 unique variants in each mouse regardless of genome exposure to Cas9 indicating off-target variants comprise a small fraction of genetic heterogeneity in Cas9-edited mice. These findings will inform future design and use of Cas9-edited animal models as well as provide context for evaluating off-target potential in genetically diverse patient populations.