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Nat Methods. 2016 Dec;13(12):1043-1049. doi: 10.1038/nmeth.4042. Epub 2016 Oct 24.

Complex transcriptional modulation with orthogonal and inducible dCas9 regulators.

Gao Y1,2, Xiong X3,4, Wong S3, Charles EJ1, Lim WA3,4,5,6, Qi LS1,7,8.

Author information

1
Department of Bioengineering, Stanford University, Stanford, California, USA.
2
Cancer Biology Program, Stanford University, Stanford, California, USA.
3
Department of Cellular and Molecular Pharmacology, University of California, San Francisco, San Francisco, California, USA.
4
Howard Hughes Medical Institute, University of California, San Francisco, San Francisco, California, USA.
5
Center for Systems and Synthetic Biology, University of California, San Francisco, San Francisco, California, USA.
6
California Institute for Quantitative Biomedical Research, University of California, San Francisco, San Francisco, California, USA.
7
Department of Chemical and Systems Biology, Stanford University, Stanford, California, USA.
8
ChEM-H, Stanford University, Stanford, California, USA.

Abstract

The ability to dynamically manipulate the transcriptome is important for studying how gene networks direct cellular functions and how network perturbations cause disease. Nuclease-dead CRISPR-dCas9 transcriptional regulators, while offering an approach for controlling individual gene expression, remain incapable of dynamically coordinating complex transcriptional events. Here, we describe a flexible dCas9-based platform for chemical-inducible complex gene regulation. From a screen of chemical- and light-inducible dimerization systems, we identified two potent chemical inducers that mediate efficient gene activation and repression in mammalian cells. We combined these inducers with orthogonal dCas9 regulators to independently control expression of different genes within the same cell. Using this platform, we further devised AND, OR, NAND, and NOR dCas9 logic operators and a diametric regulator that activates gene expression with one inducer and represses with another. This work provides a robust CRISPR-dCas9-based platform for enacting complex transcription programs that is suitable for large-scale transcriptome engineering.

PMID:
27776111
PMCID:
PMC5436902
DOI:
10.1038/nmeth.4042
[Indexed for MEDLINE]
Free PMC Article

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