Improved and Flexible HDR Editing by Targeting Introns in iPSCs.
Improved and Flexible HDR Editing by Targeting Introns in iPSCs.
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通过靶向 iPSC 中的内含子改进和灵活的 HDR 编辑
DOI：
10.1007/s12015-022-10331-1
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发表时间：
2022-06
影响因子：
4.8
通讯作者：
Sun, Chang-Kai
中科院分区：
文献类型：
--
作者：
Fu, Juan;Fu, Ya-Wen;Zhao, Juan-Juan;Yang, Zhi-Xue;Li, Si-Ang;Li, Guo-Hua;Quan, Zi-Jun;Zhang, Feng;Zhang, Jian-Ping;Zhang, Xiao-Bing;Sun, Chang-Kai
目前CRISPR-Cas9基因敲除(KO)编辑已在IPSCs中实现，但同源定向修复(HDR)介导的精确基因敲入(KI)和高水平表达仍是IPSCs临床应用的瓶颈。在这里，我们开发了一种针对内含子的新编辑策略。通过靶向终止密码子之前的内含子，这种方法可以容忍由非同源末端连接(NHEJ)介导的INDELs引起的读码移码突变，从而在不损害非HDR编辑的等位基因的情况下保持基因的完整性。此外，为了增加灵活性和筛选最好的内含子靶向sgRNA，我们设计了一个HDR供体，用人工内含子代替内源内含子。人工内含子的存在，特别是携带增强子元件的内含子的存在，与内含子缺失的对照相比，显著增加了IPSCs中报告基因的表达水平。此外，小分子M3814和曲古抑素A(TSA)的组合通过抑制NHEJ显著提高HDR效率。这些结果应该会在基因治疗和基础研究中得到应用，比如创建报告细胞系。网上版载有补充材料，可在10.1007/s12015-022-10331-1查阅。
Highly efficient gene knockout (KO) editing of CRISPR–Cas9 has been achieved in iPSCs, whereas homology-directed repair (HDR)-mediated precise gene knock-in (KI) and high-level expression are still bottlenecks for the clinical applications of iPSCs. Here, we developed a novel editing strategy that targets introns. By targeting the intron before the stop codon, this approach tolerates reading frameshift mutations caused by nonhomologous end-joining (NHEJ)-mediated indels, thereby maintaining gene integrity without damaging the non-HDR-edited allele. Furthermore, to increase the flexibility and screen for the best intron-targeting sgRNA, we designed an HDR donor with an artificial intron in place of the endogenous intron. The presence of artificial introns, particularly an intron that carries an enhancer element, significantly increased the reporter expression levels in iPSCs compared to the intron-deleted control. In addition, a combination of the small molecules M3814 and trichostatin A (TSA) significantly improves HDR efficiency by inhibiting NHEJ. These results should find applications in gene therapy and basic research, such as creating reporter cell lines. The online version contains supplementary material available at 10.1007/s12015-022-10331-1.
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影响因子：
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作者：
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影响因子：
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作者：
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DOI：
10.1126/science.1232033
发表时间：
2013-02-15
期刊：
Science (New York, N.Y.)
影响因子：
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作者：
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通讯作者：
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