利用CRISPR/Cas9技术构建基因编辑大鼠模型
收稿日期: 2022-11-10
修回日期: 2022-12-23
网络出版日期: 2023-01-01
基金资助
国家自然科学基金杰出青年基金项目编号(32025023);国家自然科学基金面上项目编号(81873685);国家自然科学基金面上项目编号(31971366);国家重点研发计划项目编号(2019YFA0110802);国家重点研发计划项目(2019YFA0802802);国家重点研发计划项目(2019YFA0802802);上海市科技创新行动计划实验项目(20140900200);华东师范大学闵行实验动物中心平台(011)
Generation of genetically modified rat models via the CRISPR/Cas9 technology
Received date: 2022-11-10
Revised date: 2022-12-23
Online published: 2023-01-01
Supported by
Supported by the National Natural Science Foundation of China(32025023);Supported by the National Natural Science Foundation of China(81873685);Supported by the National Natural Science Foundation of China(31971366);the National Key Research and Development Program of China(2019YFA0110802);the National Key Research and Development Program of China(2019YFA0802802);the National Key Research and Development Program of China(2019YFA0802802);the Shanghai Municipal Commission for Science and Technology(20140900200);the Animal Center of East China Normal University(011)
RNA介导的CRISPR/Cas9基因编辑系统由单链引导RNA(sgRNA)与核酸酶Cas9构成。在细胞内，sgRNA能够按照碱基互补配对的原则引导Cas9与靶点结合，由Cas9切割目标DNA，造成双链DNA断裂(double stranded break, DSB)。在随后的DNA修复过程中，细胞主要进行非同源末端连接(non-homologous end joining, NHEJ)或在有修复模板存在的情况下进行重组修复(homology directed repair, HDR)。如果将CRISPR/Cas9系统以及修复模板通过显微注射的方式导入大鼠的胚胎内，就能借助细胞的修复机制实现大鼠胚胎的基因编辑，由此构建各种基因修饰大鼠模型。本文详细介绍了利用CRISPR/Cas9基因编辑技术构建大鼠模型的具体操作步骤，以期为相关领域的科研人员提供一种大鼠基因修饰模型的构建方法。
关键词： 大鼠; CRISPR/Cas9; 基因编辑; 基因敲除
刘梅珍, 王立人, 李咏梅, 马雪云, 韩红辉, 李大力 . 利用CRISPR/Cas9技术构建基因编辑大鼠模型[J]. 遗传, 2023 , 45(1) : 78 -87 . DOI: 10.16288/j.yczz.22-354
The RNA-guided CRISPR/Cas9 genomic editing system consists of a single guide RNA (sgRNA) and a Cas9 nuclease. The two components form a complex in cells and target the genomic loci complementary to the sgRNA. The Cas9 nuclease cleaves the target site creating a double stranded DNA break (DSB). In mammalian cells, DSBs are often repaired via error prone non-homologous end joining (NHEJ) or via homology directed repair (HDR) with the presence of donor DNA templates. Micro-injection of the CRISPR/Cas9 system into the rat embryos enables generation of genetically modified rat models. Here, we describe a detailed protocol for creating gene knockout or knockin rat models via the CRISPR/Cas9 technology.
Key words： rat; CRISPR/Cas9; genome editing; gene knockout
| [1] | Geurts AM, Moreno C. Zinc-finger nucleases: new strategies to target the rat genome. Clin Sci Lond Engl, 2010, 119(8): 303-311. |
| [2] | Kottaisamy CPD, Raj DS, Prasanth Kumar V, Sankaran U. Experimental animal models for diabetes and its related complications-a review. Lab Anim Res, 2021, 37(1): 23. |
| [3] | Qiu Z, Liu M, Chen Z, Shao Y, Pan H, Wei G, Yu C, Zhang L, Li X, Wang P, Fan HY, Du B, Liu B, Liu M, Li D. High-efficiency and heritable gene targeting in mouse by transcription activator-like effector nucleases. Nucleic Acids Res, 2013, 41(11): e120-e120. |
| [4] | Li D, Qiu Z, Shao Y, Chen Y, Guan Y, Liu M, Li Y, Gao N, Wang L, Lu X, Zhao Y, Liu M. Heritable gene targeting in the mouse and rat using a CRISPR-Cas system. Nat Biotechnol, 2013, 31(8): 681-683. |
| [5] | Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science, 2012, 337(6096): 816-821. |
| [6] | Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F. Multiplex genome engineering using CRISPR/Cas systems. Science, 2013, 339(6121): 819-823. |
| [7] | Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-mediated genome engineering. Cell, 2013, 153(4): 910-918. |
| [8] | Zheng R, Li Y, Wang L, Fang X, Zhang J, He L, Yang L, Li D, Geng H.CRISPR/Cas9-mediated metabolic pathway reprogramming in a novel humanized rat model ameliorates primary hyperoxaluria type 1. Kidney Int, 2020, 98(4): 947-957. |
| [9] | Shao Y, Guan Y, Wang L, Qiu Z, Liu M, Chen Y, Wu L, Li Y, Ma X, Liu M, Li D. CRISPR/Cas-mediated genome editing in the rat via direct injection of one-cell embryos. Nat Protoc, 2014, 9(10): 2493-2512. |
| [10] | Guan Y, Ma Y, Li Q, Sun Z, Ma L, Wu L, Wang L, Zeng L, Shao Y, Chen Y, Ma N, Lu W, Hu K, Han H, Yu Y, Huang Y, Liu M, Li D. CRISPR/Cas9-mediated somatic correction of a novel coagulator factor IX gene mutation ameliorates hemophilia in mouse. EMBO Mol Med, 2016, 8(5): 477-488. |
/
| 〈 |
|
〉 |
www.chinagene.cn
备案号：京ICP备09063187号-4
总访问:,今日访问:,当前在线:
