Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing
Rapid repair of human disease-specific single-nucleotide variants by One-SHOT genome editing
复制标题
DOI：
10.1038/s41598-020-70401-7
复制
发表时间：
2020-08
影响因子：
4.6
通讯作者：
Y. Yokouchi;Shinichi Suzuki;N. Ohtsuki;Keitaro Yamamoto;Satomi Noguchi;Yumi Soejima;Mizuki Goto;Ken Ishioka;I. Nakamura;Satoru Suzuki;S. Takenoshita;T. Era
中科院分区：
文献类型：
--
作者：
Y. Yokouchi;Shinichi Suzuki;N. Ohtsuki;Keitaro Yamamoto;Satomi Noguchi;Yumi Soejima;Mizuki Goto;Ken Ishioka;I. Nakamura;Satoru Suzuki;S. Takenoshita;T. Era
从癌症到遗传性疾病的许多人类疾病都是由关键基因的单核苷酸突变引起的。修复这些突变将显著提高遗传性疾病患者的生活质量。然而，目前修复有害单核苷酸突变的程序并不简单，需要多个步骤，需要几个月的时间才能完成。在目前的研究中，我们的目标是使用单轮基因组编辑修复致病等位基因特异性单核苷酸突变。使用高保真、位点特异的核酸酶AsCas12a/Cpf1，我们试图修复疾病特异性诱导的多能干细胞中的致病单核苷酸变体(SNV)。结果，我们实现了对人癌基因RET中Met918Thr SNV的修复，包括一个单核苷酸标记，然后是绝对无标记、无疤痕的RETSNV修复，没有检测到脱靶效应。然后，这种无标记方法在人类VII型胶原编码基因COL7A1中得到证实。因此，使用这种一次性方法，我们成功地将基因组修复所需的遗传操作从两个连续事件减少到一个，导致等位基因特异性修复可以在3周内完成，无论有没有单核苷酸标记。我们的发现表明，一次性可以用于修复其他类型的突变，其潜力超出了人类医学的范畴。
Many human diseases ranging from cancer to hereditary disorders are caused by single-nucleotide mutations in critical genes. Repairing these mutations would significantly improve the quality of life for patients with hereditary diseases. However, current procedures for repairing deleterious single-nucleotide mutations are not straightforward, requiring multiple steps and taking several months to complete. In the current study, we aimed to repair pathogenic allele-specific single-nucleotide mutations using a single round of genome editing. Using high-fidelity, site-specific nucleaseAsCas12a/Cpf1, we attempted to repair pathogenic single-nucleotide variants (SNVs) in disease-specific induced pluripotent stem cells. As a result, we achieved repair of the Met918Thr SNV in human oncogeneRETwith the inclusion of a single-nucleotide marker, followed by absolute markerless, scarless repair of theRETSNV with no detected off-target effects. The markerless method was then confirmed in human type VII collagen-encoding geneCOL7A1. Thus, using this One-SHOT method, we successfully reduced the number of genetic manipulations required for genome repair from two consecutive events to one, resulting in allele-specific repair that can be completed within 3 weeks, with or without a single-nucleotide marker. Our findings suggest that One-SHOT can be used to repair other types of mutations, with potential beyond human medicine.