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Increasing Gene Editing Efficiency for CRISPR-Cas9 by Small RNAs in Pluripotent Stem Cells
The CRISPR Journal ( IF 3.7 ) Pub Date : 2021-08-16 , DOI: 10.1089/crispr.2021.0014
Alireza Shahryari 1, 2, 3, 4 , Noel Moya 1, 2 , Johanna Siehler 1, 2, 3 , Xianming Wang 1, 2 , Ingo Burtscher 1, 2 , Heiko Lickert 1, 2, 3
Affiliation  

Gene manipulations of human induced pluripotent stem cells (iPSCs) by CRISPR-Cas9 genome engineering are widely used for disease modeling and regenerative medicine applications. There are two competing pathways, non-homologous end joining (NHEJ) and homology directed repair (HDR) that correct the double-strand break generated by CRISPR-Cas9. Here, we improved gene editing efficiency of gene knock-in (KI) in iPSCs with minimum components by manipulating the Cas9 expression vector. Either we inserted short hairpin RNA expression cassettes to downregulate DNAPK and XRCC4, two main players of the NHEJ pathway, or we increased cell survival by inserting an anti-apoptotic expression cassette of miRNA-21 into the Cas9 vector. For an easy readout, the pluripotency gene SOX2 was targeted with a T2A-tdTomato reporter construct. In vitro downregulating DNAPK and XRCC4 increased the targeting efficiency of SOX2 KI by around twofold. Furthermore, co-expression of miRNA-21 and Cas9 improved the efficiency of SOX2 KI by around threefold. Altogether, our strategies provide a simple and valuable approach for efficient CRISPR-Cas9 gene editing in iPSCs.

中文翻译:


通过小 RNA 提高多能干细胞中 CRISPR-Cas9 的基因编辑效率



通过 CRISPR-Cas9 基因组工程对人类诱导多能干细胞 (iPSC) 进行基因操作广泛用于疾病建模和再生医学应用。有两种竞争途径,即非同源末端连接 (NHEJ) 和同源定向修复 (HDR),可以纠正 CRISPR-Cas9 产生的双链断裂。在这里,我们通过操纵 Cas9 表达载体,以最少的成分提高了 iPSC 中基因敲入 (KI) 的基因编辑效率。我们要么插入短发夹 RNA 表达盒来下调 NHEJ 途径的两个主要参与者DNAPKXRCC4 ,要么通过将 miRNA-21 的抗凋亡表达盒插入 Cas9 载体来增加细胞存活率。为了方便读出,用 T2A-tdTomato 报告基因构建体靶向多能性基因SOX2体外下调DNAPKXRCC4可使SOX2 KI 的靶向效率提高约两倍。此外,miRNA-21 和 Cas9 的共表达将SOX2 KI 的效率提高了约三倍。总而言之,我们的策略为 iPSC 中有效的 CRISPR-Cas9 基因编辑提供了一种简单而有价值的方法。
更新日期:2021-08-19
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