School of Advanced Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo, 169-8555, Japan.
Research Institute for Science and Engineering, Waseda University, 3-4-1 Okubo, Shinjuku-ku, Tokyo, 169-8555, Japan.
Sci Rep. 2021 Sep 13;11(1):18132. doi: 10.1038/s41598-021-97579-8.
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.
CRISPR/Cas9 介导的同源定向修复 (HDR) 可用于无错误的靶向外源供体 DNA 敲入。然而，HDR 介导的敲入效率低，阻碍了敲入克隆的建立。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，然后才能发生 HDR，从而防止 HDR 介导的敲入。NHEJ/MMEJ 还会导致随机整合，从而产生假阳性的敲入事件，或静默破坏基因组。在这项研究中，我们优化了用于小鼠胚胎干细胞 (mESC) 的 HDR 介导的敲入方法。我们通过结合体内线性化供体质粒、瞬时敲低 DNA 聚合酶θ和化学抑制 DNA 依赖性蛋白激酶 (DNA-PK) 来成功提高质粒供体 HDR 介导的敲入效率，同时几乎完全抑制了基于 NHEJ/MMEJ 的整合。该方法还显著提高了双等位基因敲入的效率；在 Rosa26a 基因座上，95%的 HDR 介导的敲入克隆是双等位基因的。我们将这种方法命名为 BiPoD（由 Polθ和 DNA-PK 抑制辅助的双等位基因敲入）。BiPoD 实现了同时高效地将两个基因座进行双等位基因敲入。因此，BiPoD 能够快速、轻松地建立双等位基因敲入 mESC 系。
