清脆的
基因
生物
重组酶
Cas9
基因敲除
基因敲除
Cre重组酶
基因靶向
基因组编辑
基因驱动
CRISPR干扰
Cre-Lox重组
计算生物学
突变体
条件基因敲除
遗传学
基因组工程
引导RNA
基因组
同源重组
转基因
表型
重组
转基因小鼠
作者
Tzahi Noiman,Chaim Kahana
出处
期刊:The CRISPR journal
[Mary Ann Liebert]
日期:2018-08-01
卷期号:1 (4): 278-285
被引量:8
标识
DOI:10.1089/crispr.2018.0010
摘要
Gene knockout technologies have contributed fundamentally to our understanding of the cellular functions of various genes. Two prevalent systems used for the efficient elimination of the expression of specific genes are the Cre-LoxP system and the CRISPR-Cas9 system. Here, we present a simple method that combines the use of CRISPR-Cas9 and Cre-LoxP for the conditional deletion of essential genes in mammalian cells. First, an inducible Cre recombinase is stably expressed in the cells. Next, CRISPR-Cas9 is used to knock out an essential gene, whose function is complemented by stable expression of a FLAG-tagged version of the same protein encoded from a floxed transcription unit containing silent mutations, making it refractory to the CRISPR-Cas9 guide. This FLAG-tagged protein can be deleted by activating the expressed Cre protein, enabling evaluation of the cellular consequences of its deletion. We have further used this system to evaluate the ability of phylogenic homologues and of potential mutants to cover functionally for the deleted gene.
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