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- Title
Whole genome analysis for 163 gRNAs in Cas9-edited mice reveals minimal off-target activity.
- Authors
Peterson, Kevin A.; Khalouei, Sam; Hanafi, Nour; Wood, Joshua A.; Lanza, Denise G.; Lintott, Lauri G.; Willis, Brandon J.; Seavitt, John R.; Braun, Robert E.; Dickinson, Mary E.; White, Jacqueline K.; Lloyd, K. C. Kent; Heaney, Jason D.; Murray, Stephen A.; Ramani, Arun; Nutter, Lauryl M. J.
- Abstract
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. An analysis of off-target activities for 163 gRNAs in Cas9-edited mice shows that Cas9-induced mutagenesis is rare compared to natural genetic variation.
- Subjects
WHOLE genome sequencing; GENOMES; GENOME editing; GENETIC variation; NUCLEOTIDE sequencing; MICE
- Publication
Communications Biology, 2023, Vol 6, Issue 1, p1
- ISSN
2399-3642
- Publication type
Article
- DOI
10.1038/s42003-023-04974-0