Generation of NPHP1 knockout human pluripotent stem cells by a practical biallelic gene deletion strategy using CRISPR/Cas9 and ssODN
Generation of NPHP1 knockout human pluripotent stem cells by a practical biallelic gene deletion strategy using CRISPR/Cas9 and ssODN
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DOI：
10.1007/s11626-022-00655-0
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发表时间：
2022-02-14
影响因子：
2.1
通讯作者：
Sohara, Eisei
中科院分区：
文献类型：
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作者：
Nakano, Yuta;Susa, Koichiro;Sohara, Eisei
CRISPR/Cas9基因组编辑取得了显著进展，为生命科学的发展做出了重大贡献。诱导多能干细胞（iPSC）也对再生医学，药理学研究和遗传疾病分析做出了相关贡献。然而，使用CRISPR/Cas9的敲除iPSC生成通常难以使用诸如移码突变的方法来复制具有全长或接近全长基因缺失的遗传疾病。此外，剪接和非法翻译可能会使完全敲除变得困难。在iPSCs中的全长基因缺失方法可能会解决这些问题，尽管还没有这种方法的报道。在这项研究中，我们提出了一种实用的两步基因编辑策略，导致iPSCs中全长NPHP 1基因的精确，双等位基因和完全缺失，这是首次使用CRISPR/Cas9和主要通过单链模板修复（SSTR）的单链寡脱氧核苷酸在iPSCs中进行双等位基因（复合杂合）全基因缺失的报道。我们的策略不需要选择或物质来增强SSTR，并且可以用于分析难以通过常规敲除方法复制的遗传疾病。
CRISPR/Cas9 genome editing underwent remarkable progress and significantly contributed to the development of life sciences. Induced pluripotent stem cells (iPSCs) have also made a relevant contribution to regenerative medicine, pharmacological research, and genetic disease analysis. However, knockout iPSC generation with CRISPR/Cas9 in general has been difficult to achieve using approaches such as frameshift mutations to reproduce genetic diseases with full-length or nearly full-length gene deletions. Moreover, splicing and illegitimate translation could make complete knockouts difficult. Full-length gene deletion methods in iPSCs might solve these problems, although no such approach has been reported yet. In this study, we present a practical two-step gene-editing strategy leading to the precise, biallelic, and complete deletion of the full-length NPHP1 gene in iPSCs, which is the first report of biallelic (compound heterozygous) full-gene deletion in iPSCs using CRISPR/Cas9 and single-stranded oligodeoxynucleotides mainly via single-strand template repair (SSTR). Our strategy requires no selection or substances to enhance SSTR and can be used for the analysis of genetic disorders that are difficult to reproduce by conventional knockout methods.