Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells View Full Text


Ontology type: schema:ScholarlyArticle     


Article Info

DATE

2016-12

AUTHORS

Yunqing Ma, Jiayuan Zhang, Weijie Yin, Zhenchao Zhang, Yan Song, Xing Chang

ABSTRACT

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. More... »

PAGES

1029-1035

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  • Identifiers

    URI

    http://scigraph.springernature.com/pub.10.1038/nmeth.4027

    DOI

    http://dx.doi.org/10.1038/nmeth.4027

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    PUBMED

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