Department of Pediatrics and Naomi Berrie Diabetes Center, Columbia University, New York, NY 10032, USA; Columbia University Stem Cell Initiative, New York, NY 10032, USA.
Genomic Prediction Inc., North Brunswick, NJ 08902, USA.
Cell. 2020 Dec 10;183(6):1650-1664.e15. doi: 10.1016/j.cell.2020.10.025. Epub 2020 Oct 29.
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有可能减轻遗传疾病的负担，并改善携带致病突变的夫妇的生育治疗，而无需进行胚胎选择。在这里，我们评估了 Cas9 诱导的在 EYS 基因座上的父本染色体上的双链断裂（DSB）的修复结果，该基因座携带导致失明的移码突变。我们表明，最常见的修复结果是微同源介导的末端连接，它发生在受精卵的第一个细胞周期中，导致胚胎中阅读框的非嵌合性恢复。值得注意的是，大约一半的断裂仍然未修复，导致无法检测到父本等位基因，并且在有丝分裂后，丢失一个或两个染色体臂。相应地，Cas9 的脱靶切割会导致染色体缺失和杂合缺失，因为两个等位基因都被切割。这些结果表明了操纵染色体含量的能力，并揭示了人类胚胎中突变纠正的重大挑战。
