Correction of disease-causing mutations in human embryos holds the potential to reduce the burden of inherited genetic disorders and improve fertility treatments for couples with disease-causing mutations in lieu of embryo selection. Here, we evaluate repair outcomes of a Cas9-induced double-strand break (DSB) introduced on the paternal chromosome at the EYS locus, which carries a frameshift mutation causing blindness. We show that the most common repair outcome is microhomology-mediated end joining, which occurs during the first cell cycle in the zygote, leading to embryos with non-mosaic restoration of the reading frame. Notably, about half of the breaks remain unrepaired, resulting in an undetectable paternal allele and, after mitosis, loss of one or both chromosomal arms. Correspondingly, Cas9 off-target cleavage results in chromosomal losses and hemizygous indels because of cleavage of both alleles. These results demonstrate the ability to manipulate chromosome content and reveal significant challenges for mutation correction in human embryos.

纠正人类胚胎中的致病突变具有减轻遗传遗传疾病负担并改善对具有致病突变的夫妇进行育种替代胚胎选择的潜力。在这里，我们评估了在EYS父系染色体上引入Cas9诱导的双链断裂（DSB）的修复结果位点，其携带移码突变导致失明。我们显示最常见的修复结果是微同源介导的末端连接，这发生在合子的第一个细胞周期中，导致胚胎的阅读框非镶嵌式恢复。值得注意的是，大约有一半的断裂未修复，导致无法检测的父本等位基因，并且在有丝分裂后，失去了一个或两个染色体臂。相应地，由于两个等位基因均被切割，Cas9脱靶切割导致染色体丢失和半合插入缺失。这些结果证明了操纵染色体含量的能力，并揭示了人类胚胎突变校正的重大挑战。