Abstract
Clustered regulatory interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 nuclease (Cas9), the third-generation genome editing tool, has been favored because of its high efficiency and clear system composition. In this technology, the introduced double-strand breaks (DSBs) are mainly repaired by non-homologous end joining (NHEJ) or homology-directed repair (HDR) pathways. The high-fidelity HDR pathway is used for genome modification, which can introduce artificially controllable insertions, deletions, or substitutions carried by the donor templates. Although high-level knock-out can be easily achieved by NHEJ, accurate HDR-mediated knock-in remains a technical challenge. In most circumstances, although both alleles are broken by endonucleases, only one can be repaired by HDR, and the other one is usually recombined by NHEJ. For gene function studies or disease model establishment, biallelic editing to generate homozygous cell lines and homozygotes is needed to ensure consistent phenotypes. Thus, there is an urgent need for an efficient biallelic editing system. Here, we developed three pairs of integrated selection systems, where each of the two selection cassettes contained one drug-screening gene and one fluorescent marker. Flanked by homologous arms containing the mutated sequences, the selection cassettes were integrated into the target site, mediated by CRISPR/Cas9-induced HDR. Positively targeted cell clones were massively enriched by fluorescent microscopy after screening for drug resistance. We tested this novel method on the amyloid precursor protein (APP) and presenilin 1 (PSEN1) loci and demonstrated up to 82.0% biallelic editing efficiency after optimization. Our results indicate that this strategy can provide a new efficient approach for biallelic editing and lay a foundation for establishment of an easier and more efficient disease model.
概要
目的
本研究拟基于同源介导修复 (HDR) 原理, 创建一种高效的双等位基因编辑系统
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.
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Acknowledgments
This work was supported by the National Science and Technology Major Project of China (No. 2018ZX08010-09B), the Swedish Research Council (No. NE 2016-04458), and the Ragnar Söderberg Foundation (No. M21/17), Sweden. We would like to thank all the members of Professor ZHANG’s lab for their excellent technical assistance and helpful discussions.
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Xinyi LI and Zhiying ZHANG conceived and supervised the project. Xinyi LI, Bing SUN, Hongrun QIAN, and Jinrong MA conducted experiments and data analysis. Xinyi LI wrote the manuscript and edited it in collaboration with Magdalena PAOLINO. All authors have read and approved the final manuscript, and therefore, have full access to all the data in the study and take responsibility for the integrity and security of the data.
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Xinyi LI, Bing SUN, Hongrun QIAN, Jinrong MA, Magdalena PAOLINO, and Zhiying ZHANG declare that they have no conflict of interests.
This article does not contain any studies with human or animal subjects performed by any of the authors.
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Li, X., Sun, B., Qian, H. et al. A high-efficiency and versatile CRISPR/Cas9-mediated HDR-based biallelic editing system. J. Zhejiang Univ. Sci. B 23, 141–152 (2022). https://doi.org/10.1631/jzus.B2100196
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DOI: https://doi.org/10.1631/jzus.B2100196