A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.
A new age in functional genomics using CRISPR/Cas9 in arrayed library screening.
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DOI：
10.3389/fgene.2015.00300
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发表时间：
2015
影响因子：
3.7
通讯作者：
Ketteler R
中科院分区：
文献类型：
--
作者：
Agrotis A;Ketteler R
CRISPR技术迅速改变了生物研究的面貌，在其最初开发的几年内，精确的基因组编辑现在已成为许多实验室的常规。CRISPR/Cas9之所以具有革命性意义，是因为它能够通过设计一个特定的短互补核苷酸序列，与一个共同的支架序列一起，构成连接蛋白质和DNA的引导RNA，从而将蛋白质（Cas9）定位到精确的基因组位点。野生型Cas9在其目标序列上切割两条DNA链，但这种蛋白质也可以被修饰以发挥许多其他功能。例如，通过将活化结构域连接到催化失活的Cas9上并靶向启动子区域，可以刺激特定内源基因的表达。原则上，任何基因组区域都可以被靶向，最近的努力已经成功地生成了高覆盖率的人类、小鼠和果蝇基因组编码和调控区域的汇集向导RNA文库，从而促进了功能表型筛选。在这篇综述中，我们将重点介绍基于crispr的功能基因组学领域的最新进展，并讨论潜在的未来方向，特别关注哺乳动物细胞系统和阵列文库筛选。
CRISPR technology has rapidly changed the face of biological research, such that precise genome editing has now become routine for many labs within several years of its initial development. What makes CRISPR/Cas9 so revolutionary is the ability to target a protein (Cas9) to an exact genomic locus, through designing a specific short complementary nucleotide sequence, that together with a common scaffold sequence, constitute the guide RNA bridging the protein and the DNA. Wild-type Cas9 cleaves both DNA strands at its target sequence, but this protein can also be modified to exert many other functions. For instance, by attaching an activation domain to catalytically inactive Cas9 and targeting a promoter region, it is possible to stimulate the expression of a specific endogenous gene. In principle, any genomic region can be targeted, and recent efforts have successfully generated pooled guide RNA libraries for coding and regulatory regions of human, mouse and Drosophila genomes with high coverage, thus facilitating functional phenotypic screening. In this review, we will highlight recent developments in the area of CRISPR-based functional genomics and discuss potential future directions, with a special focus on mammalian cell systems and arrayed library screening.