Discussion
This is the first report of WGS analysis of CRISPR-Cas9-targeted nonhuman primate embryonic cells, in which a high editing efficiency was coupled with the incidence of editing errors in cells from two embryos. These data demonstrate that comprehensive sequencing-based methods are warranted for evaluating editing outcomes in primate embryos, as well as any resultant offspring to ensure that the observed phenotype is due to the targeted edit and not due to unidentified off-target mutations.
Methods
DNA isolated from individual blastomeres of two embryos, along with paternal and maternal DNA, was subjected to whole genome sequencing (WGS) analysis.
Results
Large deletions were identified in macaque blastomeres at the on-target site that were not previously detected using PCR-based methods. De novo mutations were also identified at predicted CRISPR-Cas9 off-target sites.