Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.

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.

Genetic and Molecular Quality Control of Genetically Engineered Mice.

Genetically engineered mice are used as avatars to understand mammalian gene function and develop therapies for human disease. During genetic modification, unintended changes can occur, and these changes may result in misassigned gene-phenotype relationships leading to incorrect or incomplete experimental interpretations. The types of unintended changes that may occur depend on the allele type being made and the genetic engineering approach used. Here we broadly categorize allele types as deletions, insertions, base changes, and transgenes derived from engineered embryonic stem (ES) cells or edited mouse embryos. However, the methods we describe can be adapted to other allele types and engineering strategies. We describe the sources and consequ ences of common unintended changes and best practices for detecting both intended and unintended changes by screening and genetic and molecular quality control (QC) of chimeras, founders, and their progeny. Employing these practices, along with careful allele design and good colony management, will increase the chance that investigations using genetically engineered mice will produce high-quality reproducible results, to enable a robust understanding of gene function, human disease etiology, and therapeutic development.