Interrogating the noncoding genome in a high-throughput fashion.
Interrogating the noncoding genome in a high-throughput fashion.
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以高通量方式询问非编码基因组
DOI：
10.1093/nsr/nwy138
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发表时间：
2019-05
影响因子：
20.6
通讯作者：
Wei W
中科院分区：
文献类型：
--
作者：
Zhou Z;Wei W
哺乳动物基因组的绝大多数由不编码蛋白质序列的DNA组成。几十年来，这些非编码DNA的功能潜力一直知之甚少。大规模的研究，如DNA元素百科全书项目和全基因组关联研究，已经表明非编码基因组在广泛的生物和生理过程中发挥功能[1]。然而，在任何给定的生物环境中，将功能归因于过多的非编码元件在技术上一直是具有挑战性的，这主要是由于缺乏方便的高通量方法。最近开发的簇状规则间隔短回文重复序列(CRISPR)-CAS系统能够有效和精确地扰乱基因组中的DNA序列，从而提供了前所未有的机会将功能或表型与遗传元素联系起来[2]。在具有与靶DNA互补区域的单引导RNA(SgRNA)的指导下，Cas核酸酶在靶点切割基因组DNA以产生双链DNA断裂(DSB)，随后通过容易出错的非同源末端连接(NHEJ)途径修复该断裂，导致经常扰乱基因功能的插入或缺失(INDel)[3]。CRISPR-Cas系统已被进一步设计为通过将催化失活的Cas9(DCas9)与转录激活因子、抑制因子或其他效应物融合，实现转录激活(CRISPR激活，CRISPRa)、抑制(CRISPR干扰，CRISPRi)或表观遗传修饰，从而随意调节基因表达[3]。由于其可编程性和多重性，CRISPR-CAS系统在高通量功能基因组学中尤其有效。为了实现这一点，sgRNAs被设计在电子计算机上，并在克隆到慢病毒载体之前被合成为一个池，以产生一个用于靶细胞转导的病毒库。经过表型选择，如耐药性/敏感性或荧光激活的细胞分选，通过对浓缩或枯竭细胞群体的sgRNA条形码的下一代测序(NGS)分析，发现了负责目标功能的候选基因[4]。尽管集合CRISPR筛查在解剖各种生物过程中的关键基因方面具有强大的能力，但迄今为止大多数此类筛查主要针对蛋白质编码基因。这是因为NHEJ创造的小Indels(<；10bp)不太可能在非编码元件上产生功能丧失的表型。最近，人们通过利用定制的基于CRISPR的屏幕来探索哺乳动物基因组中的非编码区。
The vast majority of the mammalian genome consists of DNAs that do not encode protein sequences. For decades, the functional potentials of these noncoding DNAs have remained poorly understood. Large-scale studies, such as theEncyclopedia of DNAElements project and genome-wide association studies, have suggested that the noncoding genome functions in a wide variety of biological and physiological process [1]. However, it has been technically challenging to attribute functions to a plethora of noncoding elements in any given biological context, largely due to a lack of convenient high-throughput approaches. The recently developed clustered regularly interspaced short palindromic repeats (CRISPR)-Cas system enables efficient and precise perturbation of DNA sequences in the genome, thus offering an unprecedented opportunity to associate functions or phenotypes with genetic elements [2]. Directed by a single-guide RNA (sgRNA) with a region complementary to the target DNA, Cas nuclease cleaves the genomic DNA at the target locus to generate a double-strand DNA break (DSB), which is subsequently repaired through an internal error-prone nonhomologous end-joining (NHEJ) pathway, resulting in an insertion or deletion (indel) that often disrupts gene function [3]. The CRISPR-Cas system has been further engineered to regulate gene expression at will through the fusion of the catalytically inactive Cas9 (dCas9) with transcriptional activators, repressors or other effectors, enabling transcriptional activation (CRISPR activation, CRISPRa), inhibition (CRISPR interference, CRISPRi) or epigenetic modifications [3]. Owing to its programmability and multiplexability, theCRISPR-Cas system is especially potent in high-throughput functional genomics. To achieve this, sgRNAs are designed in silico and synthesized as a pool before being cloned into lentiviral vectors to generate a library of viruses for target cell transduction. After phenotypic selection, such as drug resistance/sensitivity or fluorescenceactivated cell sorting, candidate genes responsible for the functions of interests are revealed through next-generation sequencing (NGS) analysis of sgRNA barcodes from enriched or depleted cell populations [4]. Despite the power of pooled CRISPR screening in the dissection of key genes in a variety of biological processes, the majority of such screens hitherto have mainly targeted protein-coding genes. This is because the small indels (<10 bp) created by NHEJ are unlikely to produce loss-of-function phenotypes on the noncoding elements. Recently, endeavors have been made to probe the noncoding regions in mammalian genome by exploiting customized CRISPR-based screens.
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DOI：
10.1126/science.aah7111
发表时间：
2017-01-06
期刊：
Science (New York, N.Y.)
影响因子：
--
作者：
Liu SJ;Horlbeck MA;Cho SW;Birk HS;Malatesta M;He D;Attenello FJ;Villalta JE;Cho MY;Chen Y;Mandegar MA;Olvera MP;Gilbert LA;Conklin BR;Chang HY;Weissman JS;Lim DA
通讯作者：
Lim DA
影响因子：
48
作者：
Diao Y;Fang R;Li B;Meng Z;Yu J;Qiu Y;Lin KC;Huang H;Liu T;Marina RJ;Jung I;Shen Y;Guan KL;Ren B
通讯作者：
Ren B
影响因子：
56.9
作者：
Jinek, Martin;Chylinski, Krzysztof;Charpentier, Emmanuelle
通讯作者：
Charpentier, Emmanuelle
人类细胞功能基因组学 CRISPR/Cas9 文库的高通量筛选
影响因子：
64.8
作者：
Zhou, Yuexin;Zhu, Shiyou;Wei, Wensheng
通讯作者：
Wei, Wensheng
通过 Cas9 靶向剪接位点对人类细胞中的功能性长非编码 RNA 进行全基因组筛选。
影响因子：
46.9
作者：
Liu, Ying;Cao, Zhongzheng;Wei, Wensheng
通讯作者：
Wei, Wensheng