Wang Bei, Wang Zishi, Wang Daqi, Zhang Baolong, Ong Sang-Ging, Li Mingqing, Yu Wenqiang, Wang Yongming
1MOE Key Laboratory of Contemporary Anthropology at School of Life Sciences and Zhongshan Hospital, Fudan University, Shanghai, 200438 China.
2Shanghai Public Health Clinical Center & Laboratory of RNA Epigenetics, Institute of Biomedical Sciences, Shanghai Medical College, Fudan University, Shanghai, 201508 China.
J Biol Eng. 2019 Apr 24;13:35. doi: 10.1186/s13036-019-0150-y. eCollection 2019.
CRISPR/Cas9 system is a powerful tool for knocking out genes in cells. However, genes essential for cell survival cannot be directly knocked out. Traditionally, generation of conditional knockout cells requires multiple steps.
In this study, we developed an easy and efficient strategy to generate conditional knockout cells by using double episomal vectors - one which expresses gRNA and Cas9 nuclease, and the other expresses an inducible rescue gene. Using this system which we named "krCRISPR" (knockout-rescue CRISPR), we showed that essential genes, and , can be efficiently knocked out. When cells reach a desired confluency, the exogenous rescue genes can be silenced by the addition of doxycycline. Furthermore, the krCRISPR system enabled us to study the effects of the essential gene mutations on cells. We showed that the P507L mutation in led to downregulation of global DNA methylation in cells, indicating that it is a disease-causing mutation.
The krCRISPR system offers an easy and efficient platform that facilitates the study of essential genes' function.
CRISPR/Cas9系统是一种在细胞中敲除基因的强大工具。然而，细胞存活所必需的基因不能被直接敲除。传统上，生成条件性敲除细胞需要多个步骤。
在本研究中，我们开发了一种简单高效的策略，通过使用双附加体质粒载体来生成条件性敲除细胞——一个载体表达gRNA和Cas9核酸酶，另一个载体表达可诱导的拯救基因。使用我们命名为“krCRISPR”（敲除-拯救CRISPR）的这个系统，我们证明了必需基因 和 可以被有效敲除。当细胞达到所需的汇合度时，通过添加强力霉素可以使外源拯救基因沉默。此外，krCRISPR系统使我们能够研究必需基因突变对细胞的影响。我们证明 中的P507L突变导致细胞中整体DNA甲基化下调，表明它是一个致病突变。
krkrRkrCRISPR系统提供了一个简单高效的平台，便于研究必需基因的功能。
