Journal
NATURE METHODS
Volume 13, Issue 12, Pages 1029-+Publisher
NATURE PUBLISHING GROUP
DOI: 10.1038/NMETH.4027
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Funding
- National Natural Science Foundation of China (NSFC) [31370858]
- Ministry of Science and Technology (China) (MOST) [2014CB943600]
- Shanghai Municipal Science and Technology Committee (SMSTC) [13PJ1409300]
- National Thousand Talents Program for Distinguished Young Scholars (China)
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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) RNRNAs, 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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