CLC number:
On-line Access: 2022-02-17
Received: 2021-02-27
Revision Accepted: 2021-06-20
Crosschecked: 0000-00-00
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Xinyi LI, Bing SUN, Hongrun QIAN, Jinrong MA, Magdalena PAOLINO, Zhiying ZHANG. A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system[J]. Journal of Zhejiang University Science B,in press.Frontiers of Information Technology & Electronic Engineering,in press.https://doi.org/10.1631/jzus.B2100196 @article{title="A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system", %0 Journal Article TY - JOUR
基于同源介导修复(HDR)的CRISPR/Cas9双等位编辑系统的构建创新点:通过引入双药物和双荧光筛选系统,提高双等位基因编辑效率的基础上,进一步通过剪切肽缩短表达盒长度,提高了CRISPR/Cas9双等位编辑的效率。 方法:将嘌呤霉素抗性基因及博莱霉素抗性基因同绿色荧光蛋白及红色荧光蛋白分别配对组成筛选表达盒,插入带有基因组同源臂区域和突变序列的供体载体中组成双等位编辑供体组合。利用CRISPR/Cas9打靶系统在基因组靶位点处造成双链断裂,经HDR方式将突变序列引入基因组中,并经过荧光显微镜和双药物抗性筛选富集细胞克隆。采用聚合酶链式反应(PCR)、限制性酶切和DNA测序对细胞基因组编辑阳性率进行鉴定和统计。 结论:成功构建了三套高效、普适的基因组双等位敲入系统,为基因组双等位精确编辑提供了新的思路。此方法的建立可为通过CRISPR/Cas9双等位编辑鉴定疾病发生过程中的纯合基因功能奠定基础。 关键词组: Darkslateblue:Affiliate; Royal Blue:Author; Turquoise:Article
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