2016年10月10日，國(guó)際學(xué)術(shù)權(quán)威刊物自然出版集團(tuán)旗下子刊《Nature Methods》雜志在線發(fā)表了上海交通大學(xué)醫(yī)學(xué)院健康科學(xué)研究所常興研究組題為Targeted AID -mediated mutagenesis (TAM) enablesefficient genomic diversification in mammalian cells 的最新研究成果，研究報(bào)道了一種利用靶向性胞嘧啶脫氨酶在體內(nèi)實(shí)現(xiàn)高效率和高通量的DNA堿基編輯的新方法。
單核苷酸的多樣性是遺傳多樣性的主要來(lái)源，是分子進(jìn)化的動(dòng)力和很多疾病的直接誘因。然而由于哺乳動(dòng)物基因組的高度穩(wěn)定性，在哺乳動(dòng)物細(xì)胞內(nèi)很難高效和高通量地誘導(dǎo)單核苷酸的突變，進(jìn)而研究這些突變的功能。雖然通過(guò)CRISPR等基因編輯技術(shù)，可以實(shí)現(xiàn)較高效的DNA切割和基因敲除，但由于同源重組(HDR)的效率低下，現(xiàn)有的CRISPR技術(shù)對(duì)于體內(nèi)構(gòu)建單核苷酸突變?nèi)蕴幱诘托щA段。
靶向性AID介導(dǎo)的核苷酸突變(TAM)這種新的研究方法，有可能改變這一現(xiàn)狀。有別于絕大多數(shù)體細(xì)胞基因組，適應(yīng)性免疫系統(tǒng)在淋巴細(xì)胞發(fā)育過(guò)程中可以進(jìn)行高效編輯，對(duì)抗原受體進(jìn)行高效突變，產(chǎn)生近乎無(wú)限的抗原受體庫(kù)，用以抵御可能的病原體入侵。受這一“突變自我”機(jī)制的啟發(fā)，博士研究生馬云青和張佳元在研究員常興的指導(dǎo)下發(fā)現(xiàn)，當(dāng)把核酸酶缺陷的Cas9蛋白和誘導(dǎo)抗體高頻突變的胞嘧啶脫氨酶AID融合后，在sgRNA靶向的基因組DNA上，胞嘧啶和鳥嘌呤可以隨機(jī)地向其它三個(gè)堿基轉(zhuǎn)變。這一新方法可以對(duì)細(xì)胞內(nèi)的特定DNA序列進(jìn)行多樣化，完成遺傳篩選，從而分析單核苷酸突變的功能。同時(shí)在一種多肽抑制劑的輔助下，dCas9-AID可以誘導(dǎo)特定的胞嘧啶向胸腺嘧啶轉(zhuǎn)變，實(shí)現(xiàn)單堿基的精確編輯。該研究團(tuán)隊(duì)進(jìn)一步證明，利用這一方法可以快速有效地模擬腫瘤細(xì)胞體內(nèi)耐藥機(jī)制的異質(zhì)性，預(yù)測(cè)可能的腫瘤耐藥性突變，進(jìn)而改良小分子抑制劑和研究小分子與蛋白質(zhì)靶點(diǎn)的相互作用。該研究成果為分子進(jìn)化、基因治療和在單堿基水平上分析基因調(diào)控元件等領(lǐng)域提供新的方法。
DNA堿基編輯的新方法
原文鏈接：
Targeted AID-mediated mutagenesis (TAM) enables efficient genomic diversification in mammalian cells
原文摘要：
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.
