Whole Genome Analysis for 163 gRNAs in Cas9-Edited Mice Reveals Minimal Off-Target Activity

Authors

Kevin A Peterson
Sam Khalouei
Nour Hanafi
Joshua A Wood
Denise G Lanza
Lauri G Lintott
Brandon J Willis
John R Seavitt
Robert E Braun
Mary E Dickinson
Jacqueline K White
K C Kent Lloyd
Jason D Heaney
Stephen A Murray
Arun Ramani
Lauryl M J Nutter

Language

English

Publication Date

6-10-2023

Journal

Communications Biology

DOI

10.1038/s42003-023-04974-0

PMID

37301944

PMCID

PMC10257658

PubMedCentral® Posted Date

6-10-2023

PubMedCentral® Full Text Version

Post-print

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.

Keywords

Mice, Animals, CRISPR-Cas Systems, Gene Editing, Genome, Mutation, Mutagenesis, Genetics, Biological techniques

Published Open-Access

yes

Recommended Citation

Peterson, Kevin A; Khalouei, Sam; Hanafi, Nour; et al., "Whole Genome Analysis for 163 gRNAs in Cas9-Edited Mice Reveals Minimal Off-Target Activity" (2023). Faculty and Staff Publications. 1147.
https://digitalcommons.library.tmc.edu/baylor_docs/1147