title: Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells.
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.
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences &Shanghai Jiao Tong University School of Medicine (SJTUSM), Chinese Academy of Sciences, Shanghai, China.
authors.forename: Yunqing
authors.initials: Y
authors.lastname: Ma
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences &Shanghai Jiao Tong University School of Medicine (SJTUSM), Chinese Academy of Sciences, Shanghai, China.
authors.forename: Jiayuan
authors.initials: J
authors.lastname: Zhang
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences &Shanghai Jiao Tong University School of Medicine (SJTUSM), Chinese Academy of Sciences, Shanghai, China.
authors.forename: Weijie
authors.initials: W
authors.lastname: Yin
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences &Shanghai Jiao Tong University School of Medicine (SJTUSM), Chinese Academy of Sciences, Shanghai, China.
authors.forename: Zhenchao
authors.initials: Z
authors.lastname: Zhang
authors.affiliation: Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, La Jolla, California, USA.
authors.forename: Yan
authors.initials: Y
authors.lastname: Song
authors.affiliation: Key Laboratory of Stem Cell Biology, Institute of Health Sciences, Shanghai Institutes for Biological Sciences &Shanghai Jiao Tong University School of Medicine (SJTUSM), Chinese Academy of Sciences, Shanghai, China.|Collaborative Innovation Center of Systems Biomedicine, Shanghai Jiao Tong University School of Medicine, Shanghai, China.
authors.forename: Xing
authors.initials: X
authors.lastname: Chang
mesh_terms: D064130:CRISPR-Associated Proteins / Q000737:chemistry / Q000235:genetics*; D018929:Cell Culture Techniques; D064112:Clustered Regularly Interspaced Short Palindromic Repeats / Q000235:genetics*; D003564:Cytidine Deaminase / Q000737:chemistry / Q000235:genetics*; D019008:Drug Resistance, Neoplasm / Q000235:genetics; D016044:Fusion Proteins, bcr-abl / Q000235:genetics; D018390:Gene Targeting; D049452:Green Fluorescent Proteins / Q000737:chemistry / Q000235:genetics; D057809:HEK293 Cells; D006801:Humans; D000068877:Imatinib Mesylate / Q000494:pharmacology; D020014:K562 Cells; D016297:Mutagenesis, Site-Directed; D017354:Point Mutation; D000094704:RNA, Guide, CRISPR-Cas Systems; D011993:Recombinant Fusion Proteins / Q000737:chemistry / Q000235:genetics*; D000098830:AICDA (Activation-Induced Cytidine Deaminase)
chemical_list: D064130:CRISPR-Associated Proteins; D000094704:RNA, Guide, CRISPR-Cas Systems; D011993:Recombinant Fusion Proteins; C485184:enhanced green fluorescent protein; D049452:Green Fluorescent Proteins; D000068877:Imatinib Mesylate; D016044:Fusion Proteins, bcr-abl; D000098830:AICDA (Activation-Induced Cytidine Deaminase); D003564:Cytidine Deaminase