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Mol Syst Biol. 2014 Nov 24;10:763. doi: 10.15252/msb.20145735.

Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks.

Author information

1
Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA.
2
Synthetic Biology Center, Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA cavoigt@gmail.com.

Abstract

Genetic circuits require many regulatory parts in order to implement signal processing or execute algorithms in cells. A potentially scalable approach is to use dCas9, which employs small guide RNAs (sgRNAs) to repress genetic loci via the programmability of RNA:DNA base pairing. To this end, we use dCas9 and designed sgRNAs to build transcriptional logic gates and connect them to perform computation in living cells. We constructed a set of NOT gates by designing five synthetic Escherichia coli σ70 promoters that are repressed by corresponding sgRNAs, and these interactions do not exhibit crosstalk between each other. These sgRNAs exhibit high on-target repression (56- to 440-fold) and negligible off-target interactions (< 1.3-fold). These gates were connected to build larger circuits, including the Boolean-complete NOR gate and a 3-gate circuit consisting of four layered sgRNAs. The synthetic circuits were connected to the native E. coli regulatory network by designing output sgRNAs to target an E. coli transcription factor (malT). This converts the output of a synthetic circuit to a switch in cellular phenotype (sugar utilization, chemotaxis, phage resistance).

KEYWORDS:

CRISPR; TALE; TetR homologue; genetic compiler; synthetic biology

PMID:
25422271
PMCID:
PMC4299604
[Indexed for MEDLINE]
Free PMC Article

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