No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice

PLoS Genet. 2018 Jul 9;14(7):e1007503. doi: 10.1371/journal.pgen.1007503. eCollection 2018 Jul.

Abstract

CRISPR-Cas9 technologies have transformed genome-editing of experimental organisms and have immense therapeutic potential. Despite significant advances in our understanding of the CRISPR-Cas9 system, concerns remain over the potential for off-target effects. Recent studies have addressed these concerns using whole-genome sequencing (WGS) of gene-edited embryos or animals to search for de novo mutations (DNMs), which may represent candidate changes introduced by poor editing fidelity. Critically, these studies used strain-matched, but not pedigree-matched controls and thus were unable to reliably distinguish generational or colony-related differences from true DNMs. Here we used a trio design and whole genome sequenced 8 parents and 19 embryos, where 10 of the embryos were mutagenised with well-characterised gRNAs targeting the coat colour Tyrosinase (Tyr) locus. Detailed analyses of these whole genome data allowed us to conclude that if CRISPR mutagenesis were causing SNV or indel off-target mutations in treated embryos, then the number of these mutations is not statistically distinguishable from the background rate of DNMs occurring due to other processes.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biological Variation, Population / genetics
  • CRISPR-Cas Systems / genetics*
  • DNA Mutational Analysis / methods
  • Female
  • Gene Editing / methods*
  • Genome / genetics
  • Hair Color / genetics
  • Male
  • Mice
  • Mice, Inbred C57BL
  • Mice, Transgenic
  • Monophenol Monooxygenase / genetics*
  • Mutagenesis / genetics*
  • Pedigree
  • RNA, Guide, CRISPR-Cas Systems / genetics
  • Research Design
  • Whole Genome Sequencing / methods*

Substances

  • RNA, Guide, CRISPR-Cas Systems
  • Monophenol Monooxygenase