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.
Department of Cellular and Molecular Medicine, University of California at San Diego, La Jolla, La Jolla, California, USA.
Nat Methods. 2016 Dec;13(12):1029-1035. doi: 10.1038/nmeth.4027. Epub 2016 Oct 10.
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.
大量的遗传变异与人类疾病有关。然而，缺乏遗传多样化的方法阻碍了我们在哺乳动物细胞中研究遗传变异功能的能力。目前的筛选方法只能用于破坏一个基因或改变其表达。在这里，我们报告了激活诱导胞嘧啶脱氨酶（AID）与无核酸酶活性的簇状规则间隔短回文重复序列（CRISPR）相关蛋白 9（dCas9）的融合，用于高效的遗传多样化，从而实现了功能变体的高通量筛选。在单指导 RNA（sgRNA）的指导下，dCas9-AID-P182X（AIDx）可独立于 AID 热点基序直接将胞嘧啶或鸟嘌呤转换为其他三个碱基，在所需基因座产生大量变体库。与尿嘧啶-DNA 糖基化酶抑制剂结合使用时，dCas9-AIDx 可特异性地将靶向的胞嘧啶转换为胸腺嘧啶，从而产生特定的点突变。通过靶向 BCR-ABL 与 dCas9-AIDx，我们有效地鉴定了慢性髓性白血病细胞中赋予伊马替尼耐药性的已知和新突变。因此，靶向 AID 介导的诱变（TAM）提供了一种正向遗传工具，可在碱基分辨率下筛选获得功能的变体。
