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

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.

Fig 1. Analysis of CRISPR off-targets by whole genome sequencing.

(a) Experimental design: Four sets of C57BL/6N parents gave rise to 9 control embryos (3 “no injection”, 3 “sham injection” with water only and 3 “Cas9 only”), and 10 treated embryos (5 were injected with Cas9 and Tyr2F gRNA and 5 were injected with Cas9 and Tyr2R gRNA). (b) Whole genome sequencing: All 27 mice / embryos were subjected to whole genome sequencing with median depth 39.5x and an average of 3.4% of bases with read depth less than 11x. (c) Variant calling and filtering: starting from the joint variant call (bcftools mpileup + bcftools call), a sequence of filter steps were performed to detect de novo mutations and remove likely false positives arising from low-level parental mosaicism and alignment errors at repeat regions. Parental-noise: alternate-allele reads present in either parent. Cross-noise: alternate reads from all other (non-parental) samples. (d) Filtered SNV and Indel counts are not significantly different within control groups, within treatment groups, or between control and treatment groups.