生物
胞苷脱氨酶
清脆的
遗传学
胞苷
计算生物学
突变
体细胞突变
阿波贝克
基因组编辑
活化诱导(胞苷)脱氨酶
基因
突变
Cas9
基因组
抗体
酶
B细胞
生物化学
作者
Yunqing Ma,Jiayuan Zhang,Weijie Yin,Zhenchao Zhang,Yan Song,Xing Chang
出处
期刊:Nature Methods
[Springer Nature]
日期:2016-10-10
卷期号:13 (12): 1029-1035
被引量:352
摘要
A large number of genetic variants have been associated with human diseases. However, the lack of a genetic diversification approach has impeded our ability to interrogate functions of genetic variants in mammalian cells. Current screening methods can only be used to disrupt a gene or alter its expression. Here we report the fusion of activation-induced cytidine deaminase (AID) with nuclease-inactive clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (dCas9) for efficient genetic diversification, which enabled high-throughput screening of functional variants. Guided by single guide (sg)RNAs, dCas9-AID-P182X (AIDx) directly changed cytidines or guanines to the other three bases independent of AID hotspot motifs, generating a large repertoire of variants at desired loci. Coupled with a uracil-DNA glycosylase inhibitor, dCas9-AIDx converted targeted cytidines specifically to thymines, creating specific point mutations. By targeting BCR-ABL with dCas9-AIDx, we efficiently identified known and new mutations conferring imatinib resistance in chronic myeloid leukemia cells. Thus, targeted AID-mediated mutagenesis (TAM) provides a forward genetic tool to screen for gain-of-function variants at base resolution.
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