用于哺乳动物细胞基因组激活和抑制的CRISPR技术
CRISPR Technology for Genome Activation and Repression in Mammalian Cells.
作者信息
Du Dan, Qi Lei S
机构信息
Department of Bioengineering, Stanford University, Stanford, California 94305;
Department of Bioengineering, Stanford University, Stanford, California 94305; Department of Chemical and Systems Biology, Stanford University, Stanford, California 94305; ChEM-H; Stanford University, Stanford, California 94305.
出版信息
Cold Spring Harb Protoc. 2016 Jan 4;2016(1):pdb.prot090175. doi: 10.1101/pdb.prot090175.
Targeted modulation of transcription is necessary for understanding complex gene networks and has great potential for medical and industrial applications. CRISPR is emerging as a powerful system for targeted genome activation and repression, in addition to its use in genome editing. This protocol describes how to design, construct, and experimentally validate the function of sequence-specific single guide RNAs (sgRNAs) for sequence-specific repression (CRISPRi) or activation (CRISPRa) of transcription in mammalian cells. In this technology, the CRISPR-associated protein Cas9 is catalytically deactivated (dCas9) to provide a general platform for RNA-guided DNA targeting of any locus in the genome. Fusion of dCas9 to effector domains with distinct regulatory functions enables stable and efficient transcriptional repression or activation in mammalian cells. Delivery of multiple sgRNAs further enables activation or repression of multiple genes. By using scaffold RNAs (scRNAs), different effectors can be recruited to different genes for simultaneous activation of some and repression of others. The CRISPRi and CRISPRa methods provide powerful tools for sequence-specific control of gene expression on a genome-wide scale to aid understanding gene functions and for engineering genetic regulatory systems.
靶向转录调控对于理解复杂的基因网络至关重要，并且在医学和工业应用中具有巨大潜力。除了用于基因组编辑外，CRISPR正成为一种用于靶向基因组激活和抑制的强大系统。本方案描述了如何设计、构建和实验验证序列特异性单导向RNA（sgRNA）的功能，以用于哺乳动物细胞中序列特异性转录抑制（CRISPRi）或激活（CRISPRa）。在这项技术中，将CRISPR相关蛋白Cas9进行催化失活（dCas9），以提供一个用于RNA引导的基因组中任何位点DNA靶向的通用平台。将dCas9与具有不同调控功能的效应域融合，可在哺乳动物细胞中实现稳定且高效的转录抑制或激活。递送多个sgRNA还能够激活或抑制多个基因。通过使用支架RNA（scRNA），可以将不同的效应器招募到不同的基因上，从而同时激活一些基因并抑制另一些基因。CRISPRi和CRISPRa方法为在全基因组范围内对基因表达进行序列特异性控制提供了强大工具，有助于理解基因功能并构建遗传调控系统。
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