Multiplexed activation of endogenous genes by CRISPR-on, an RNA-guided transcriptional activator system

Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 （dCas9） protein fused with a transcri...

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Bibliographic Details
Published in:Cell research 2013-10, Vol.23 (10), p.1163-1171
Main Authors: Cheng, Albert W, Wang, Haoyi, Yang, Hui, Shi, Linyu, Katz, Yarden, Theunissen, Thorold W, Rangarajan, Sudharshan, Shivalila, Chikdu S, Dadon, Daniel B, Jaenisch, Rudolf
Format: Article
Language:English
Subjects:
Animals
Cloning, Molecular
Clustered Regularly Interspaced Short Palindromic Repeats
Deoxyribonuclease I - genetics
Female
Gene regulation
HEK293 Cells
HeLa Cells
Humans
Mice
Mice, Inbred C57BL
NIH 3T3 Cells
Original
Promoter Regions, Genetic
Recombinant Fusion Proteins - genetics
RNA
RNA, Guide - genetics
Transcription Factors - genetics
Transcriptional Activation
Transgenes
内源基因
基因功能
引导
报告基因
激活系统
表达谱分析
转录激活
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Summary:Technologies allowing for specific regulation of endogenous genes are valuable for the study of gene functions and have great potential in therapeutics. We created the CRISPR-on system, a two-component transcriptional activator consisting of a nuclease-dead Cas9 （dCas9） protein fused with a transcriptional activation domain and single guide RNAs （sgRNAs） with complementary sequence to gene promoters. We demonstrate that CRISPR-on can efficiently activate exogenous reporter genes in both human and mouse cells in a tunable manner. In addition, we show that robust reporter gene activation in vivo can be achieved by injecting the system components into mouse zygotes. Furthermore, we show that CRISPR-on can activate the endogenous IL1RN, SOX2, and OCT4 genes. The most efficient gene activation was achieved by clusters of 3-4 sgRNAs binding to the proximal promoters, suggesting their synergistic action in gene induction. Significantly, when sgRNAs targeting multiple genes were simultaneously introduced into cells, robust multiplexed endogenous gene activation was achieved. Genome-wide expression profiling demonstrated high specificity of the system.
ISSN:1001-0602
1748-7838
DOI:10.1038/cr.2013.122