Wellcome Sanger Institute, Wellcome Genome Campus, Hinxton, Cambridge, United Kingdom.
PLoS Genet. 2018 Jul 9;14(7):e1007503. doi: 10.1371/journal.pgen.1007503. eCollection 2018 Jul.
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.
CRISPR-Cas9 技术已经改变了实验生物的基因组编辑方式，并且具有巨大的治疗潜力。尽管我们对 CRISPR-Cas9 系统的理解有了重大进展，但人们仍然对潜在的脱靶效应感到担忧。最近的研究使用基因编辑胚胎或动物的全基因组测序 (WGS) 来寻找新出现的突变 (DNMs)，这些突变可能代表由较差的编辑保真度引起的候选变化。至关重要的是，这些研究使用了与突变体匹配但与品系不匹配的对照，因此无法可靠地区分真正的 DNMs 与世代或群体相关的差异。在这里，我们使用了三体系列设计，并对 8 个亲本和 19 个胚胎进行了全基因组测序，其中 10 个胚胎使用针对毛色酪氨酸酶 (Tyr) 基因座的经过良好表征的 gRNA 进行了诱变。对这些全基因组数据的详细分析使我们能够得出结论，如果 CRISPR 诱变导致处理胚胎中的 SNV 或插入缺失脱靶突变，那么这些突变的数量与由于其他过程而发生的 DNMs 的背景率在统计学上无法区分。
