Whole genome sequencing of CCR5 CRISPR-Cas9-edited Mauritian cynomolgus macaque blastomeres reveals large-scale deletions and off-target edits

Abstract

Introduction: Genome editing by CRISPR-Cas9 approaches offers promise for introducing or correcting disease-associated mutations for research and clinical applications. Nonhuman primates are physiologically closer to humans than other laboratory animal models, providing ideal candidates for introducing human disease-associated mutations to develop models of human disease. The incidence of large chromosomal anomalies in CRISPR-Cas9-edited human embryos and cells warrants comprehensive genotypic investigation of editing outcomes in primate embryos. Our objective was to evaluate on- and off-target editing outcomes in CCR5 CRISPR-Cas9-targeted Mauritian cynomolgus macaque embryos.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.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.

Unintended CRISPR-Cas9 editing outcomes: a review of the detection and prevalence of structural variants generated by gene-editing in human cells

“…An alternative solution is to analyze many individual clones isolated from the original mixed pool of edited cells. Although this would enable the detection of SVs (Alanis-Lobato et al 2021 ; Schmidt et al 2023 ; Simkin et al 2022 ), this method can be expensive, labor-intensive, and would be difficult to achieve sufficient depth to identify rare variants, requiring the analysis of hundreds of cell clones, which makes it unsuitable for use in many settings.…”

“…Quantification requires ddPCR calibration. Does not quantify DNA without SVs (Turchiano et al 2021 ) Whole-genome sequencing Unbiased next-generation sequencing of whole genomic DNA Can detect all types of variants including SNPs, INDELs and SVs at all sites Detection threshold not suitable for pooled DNA (Alanis-Lobato et al 2021 ; Schmidt et al 2023 ; Simkin et al 2022 ) Xdrop Encapsulated PCR of fragmented HMW DNA with primers for a 100–200 bp target, distal (< 5 kb) to the break point. Droplets containing the fragments of interest can be identified with an intercalating fluorescent dye and sorted by flow cytometer droplet sorting.…”

“…Similarly, Alanis-Lobato et al ( 2021 ) demonstrated that segmental deletions of 4 kb to at least 20 kb occurred in 16% of cells from embryos that were edited with a single DSB (Alanis-Lobato et al 2021 ). A recent study in CRISPR-edited macaque embryos also identified large on-target deletions ranging from ~ 0.2 kb to ~ 5 kb, inversions, duplication, and de novo mutations at off-target sites (Schmidt et al 2023 ).…”