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

By A. Cheng, Haoyi Wang, Hui Yang, Linyu Shi, Y. Katz, T. Theunissen, Sudharshan Rangarajan, C. Shivalila, Daniela Dadon, R. Jaenisch. Cell Research, 2013

769 citations recorded by the source corpus.

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.

Subfields: Molecular Biology

View Paper: https://www.semanticscholar.org/paper/871a3350b11affb9b27ba557a6ba220047d6906e

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In Molecular Biology

Citations in the corpus

Dimeric CRISPR RNA-guided FokI nucleases for highly specific genome editing (2014, methodology): “Our Csy4-based system should also prove generally useful for any application requiring the expression of two or more gRNAs (e.g.—the targeting of multiple dCas9-activator proteins to achieve synergistic upregulation of gene expression 20–22 ).”
A protein tagging system for signal amplification in gene expression and fluorescence imaging (2014, positive): “…copies of dCas9-VP64 to a native promoter using multiple nonoverlap- 642 Cell 159, 635–646, October 23, 2014 ª2014 Elsevier Inc. ping sgRNAs also greatly enhanced transcriptional activation (Cheng et al., 2013; Hu et al., 2014; Maeder et al., 2013; Mali et al., 2013; Perez-Pinera et al., 2013).”
sgRNAcas9: A Software Package for Designing CRISPR sgRNA and Evaluating Potential Off-Target Cleavage Sites (2014, background): “The clustered, regularly interspaced short palindromic repeats (CRISPR) in combination with a CRISPR-associated nuclease 9 (Cas9) were recently demonstrated to be versatile tools for genome engineering [1–6].”
Target specificity of the CRISPR-Cas9 system (2014, positive): “Secondly, transcriptome profiling revealed negligible offtarget gene expression change [25,27,55].”
A CRISPR/Cas9 Toolbox for Multiplexed Plant Genome Editing and Transcriptional Regulation1[OPEN] (2015, background): “…such as VP16 or VP64 (Sadowski et al., 1988; Beerli et al., 1998) to a deactivated Cas9 (dCas9) lacking endonuclease activity can up-regulate endogenous gene expression in human cells (Cheng et al., 2013; Gilbert et al., 2013; Maeder et al., 2013; Mali et al., 2013a; Perez-Pinera et al., 2013).”