CRISPR/Cas9-mediated gene editing in human tripronuclear zygotes

TLDR

CRISPR/Cas9 tools are widely used for gene editing, yet little is known about DNA repair mechanisms, editing efficiency, or off‑target effects in human early embryos. The study aimed to evaluate CRISPR/Cas9‑mediated gene editing in human tripronuclear (3PN) zygotes. The authors employed 3PN zygotes to assess editing efficiency, homologous recombination rates, mosaicism, and off‑target activity. CRISPR/Cas9 efficiently cleaved the β‑globin gene, but HDR was low, producing mosaic embryos with off‑target cuts, unintended mutations from endogenous δ‑globin competition, and repair mainly via non‑crossover HDR, highlighting the need to improve fidelity for clinical applications.

Abstract

Genome editing tools such as the clustered regularly interspaced short palindromic repeat (CRISPR)-associated system (Cas) have been widely used to modify genes in model systems including animal zygotes and human cells, and hold tremendous promise for both basic research and clinical applications. To date, a serious knowledge gap remains in our understanding of DNA repair mechanisms in human early embryos, and in the efficiency and potential off-target effects of using technologies such as CRISPR/Cas9 in human pre-implantation embryos. In this report, we used tripronuclear (3PN) zygotes to further investigate CRISPR/Cas9-mediated gene editing in human cells. We found that CRISPR/Cas9 could effectively cleave the endogenous β-globin gene (HBB). However, the efficiency of homologous recombination directed repair (HDR) of HBB was low and the edited embryos were mosaic. Off-target cleavage was also apparent in these 3PN zygotes as revealed by the T7E1 assay and whole-exome sequencing. Furthermore, the endogenous delta-globin gene (HBD), which is homologous to HBB, competed with exogenous donor oligos to act as the repair template, leading to untoward mutations. Our data also indicated that repair of the HBB locus in these embryos occurred preferentially through the non-crossover HDR pathway. Taken together, our work highlights the pressing need to further improve the fidelity and specificity of the CRISPR/Cas9 platform, a prerequisite for any clinical applications of CRSIPR/Cas9-mediated editing.