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Mol Cell. 2014 May 22;54(4):698-710. doi: 10.1016/j.molcel.2014.04.022. Epub 2014 May 15.

Multiplexed and programmable regulation of gene networks with an integrated RNA and CRISPR/Cas toolkit in human cells.

Author information

1
Synthetic Biology Group, Research Laboratory of Electronics, Department of Biological Engineering and Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02142, USA.
2
Synthetic Biology Group, Research Laboratory of Electronics, Department of Biological Engineering and Electrical Engineering & Computer Science, Massachusetts Institute of Technology, Cambridge, MA 02142, USA. Electronic address: timlu@mit.edu.

Abstract

RNA-based regulation and CRISPR/Cas transcription factors (CRISPR-TFs) have the potential to be integrated for the tunable modulation of gene networks. A major limitation of this methodology is that guide RNAs (gRNAs) for CRISPR-TFs can only be expressed from RNA polymerase III promoters in human cells, limiting their use for conditional gene regulation. We present new strategies that enable expression of functional gRNAs from RNA polymerase II promoters and multiplexed production of proteins and gRNAs from a single transcript in human cells. We use multiple RNA regulatory strategies, including RNA-triple-helix structures, introns, microRNAs, and ribozymes, with Cas9-based CRISPR-TFs and Cas6/Csy4-based RNA processing. Using these tools, we efficiently modulate endogenous promoters and implement tunable synthetic circuits, including multistage cascades and RNA-dependent networks that can be rewired with Csy4 to achieve complex behaviors. This toolkit can be used for programming scalable gene circuits and perturbing endogenous networks for biology, therapeutic, and synthetic biology applications.

PMID:
24837679
PMCID:
PMC4077618
DOI:
10.1016/j.molcel.2014.04.022
[Indexed for MEDLINE]
Free PMC Article

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