RT Journal Article
SR Electronic
T1 No unexpected CRISPR-Cas9 off-target activity revealed by trio sequencing of gene-edited mice
JF bioRxiv
FD Cold Spring Harbor Laboratory
SP 263129
DO 10.1101/263129
A1 Iyer, Vivek
A1 Boroviak, Katharina
A1 Thomas, Mark
A1 Doe, Brendan
A1 Ryder, Edward
A1 Adams, David
YR 2018
UL http://biorxiv.org/content/early/2018/02/09/263129.abstract
AB CRISPR-Cas 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 induced 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.