Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK.
Efficient biallelic knock-in in mouse embryonic stem cells by in vivo-linearization of donor and transient inhibition of DNA polymerase θ/DNA-PK.
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DOI：
10.1038/s41598-021-97579-8
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发表时间：
2021-09-13
影响因子：
4.6
通讯作者：
Nakao Y
中科院分区：
文献类型：
--
作者：
Arai D;Nakao Y
CRISPR/Cas9介导的同源定向修复(HDR)用于无差错靶向敲入外源供体DNA。然而，HDR介导的敲入效率较低，阻碍了敲入克隆的建立。在HDR发生之前，CRISPR/Cas9诱导的双链断裂(DSB)优先通过非同源末端连接(NHEJ)或微同源介导的末端连接(MMEJ)修复，从而防止HDR介导的敲入。NHEJ/MMEJ还会导致随机整合，这会引起假阳性敲入事件，或者静默地扰乱基因组。在这项研究中，我们优化了一种HDR介导的小鼠胚胎干细胞(MESCs)的敲入方法。我们成功地提高了hdr介导的基因敲入的效率，同时几乎完全抑制了基于NHEJ/MMEJ的整合，方法是将供体质粒的体内线性化、DNA聚合酶θ的瞬时敲除和M3814对DNA依赖的蛋白激酶(DNA-PK)的化学抑制相结合。这种方法还显著提高了双等位基因敲入的效率；在Rosa26a基因座，95%的HDR介导的敲入克隆是双等位基因。我们将这种方法命名为Bipod(Polθ和DNA-PK抑制辅助的双等位基因敲入)。双足类动物实现了同时高效的双等位基因敲入两个基因座。因此，两足动物能够快速而容易地建立双等位基因敲入mESC品系。
CRISPR/Cas9-mediated homology-directed repair (HDR) is used for error-free targeted knock-in of foreign donor DNA. However, the low efficiency of HDR-mediated knock-in hinders establishment of knock-in clones. Double-strand breaks (DSBs) induced by CRISPR/Cas9 are preferentially repaired by non-homologous end joining (NHEJ) or microhomology-mediated end joining (MMEJ) before HDR can occur, thereby preventing HDR-mediated knock-in. NHEJ/MMEJ also cause random integrations, which give rise to false-positive knock-in events, or silently disrupt the genome. In this study, we optimized an HDR-mediated knock-in method for mouse embryonic stem cells (mESCs). We succeeded in improving efficiency of HDR-mediated knock-in of a plasmid donor while almost completely suppressing NHEJ/MMEJ-based integration by combining in vivo-linearization of the donor plasmid, transient knockdown of DNA polymerase θ, and chemical inhibition of DNA-dependent protein kinase (DNA-PK) by M3814. This method also dramatically improved the efficiency of biallelic knock-in; at the Rosa26a locus, 95% of HDR-mediated knock-in clones were biallelic. We designate this method BiPoD (Biallelic knock-in assisted by Pol θ and DNA-PK inhibition). BiPoD achieved simultaneous efficient biallelic knock-in into two loci. BiPoD, therefore, enables rapid and easy establishment of biallelic knock-in mESC lines.
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