Correcting a PLCζ Mutation in the Human Germ Line to Overcome Hereditary Infertility

Abstract

Human germline gene correction by CRISPR/Cas9 holds great promise to eliminate transmission of genetic mutations. Recent studies have however made troublesome observations of mosaicism (the occurrence of multiple genetic events in one embryo) and loss-of-heterozygosity (LOH, loss of the allele of one of the parents), the latter being either caused by inter-homologue homologous recombination (IH-HR) or chromosome loss events. As a proof-of-concept we targeted a heterozygous base pair substitution mutation in PLCZ1, leading to subfertility. After CRISPR/Cas9 editing, we detected in 26% (7/27) of the targeted embryos that originated from mutant sperm only, wild-type alleles upon analysis, which were not obtained by exogenous repair template use. In these seven embryos, we observed a significant amount of LOH events (14%) that spanned regions beyond the PLCZ1 gene, but did not stretch the complete analysed region, pointing to IH-HR, next to a large proportion of samples (57%) showing potential but non-detectable (i.e. below the resolution of our assay) LOH. In addition, a minority of embryos (29%) showed LOH across the whole analysed region. Remarkably, also 10% of the embryos originating from wild-type sperm showed LOH events, underscoring the subpar specificity of the CRIPSR/Cas9 system. Single-cell analysis demonstrated mosaicism in 80% (4/5) of the targeted embryos, warranting caution for trophectoderm biopsy interpretation in future human embryo editing attempts. Lastly, a correction rate of 75% was observed in our single-cell data which illustrates that the correction rate was possibly underestimated when looking at the whole-embryo level.