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  • Review Article
  • Published:

A versatile framework for microbial engineering using synthetic non-coding RNAs

Nature Reviews Microbiology volume 12, pages 341–354 (2014)Cite this article

Subjects

Key Points

  • This Review focuses on the design, engineering and application of synthetic non-coding RNA (ncRNA) devices for microbial engineering.

  • Recent progress in the development of ncRNA regulators that control gene regulation at the DNA level — in particular, the CRISPR interference (CRISPRi) system — are discussed.

  • Several types of ncRNA regulators that control gene expression at the levels of transcription, translation and mRNA stability are described.

  • In addition, synthetic ncRNA regulators that function as scaffold molecules for protein-level control are described, as well as ncRNA sensors (such as riboswtiches and aptazymes) that respond to chemical or protein ligands.

  • We highlight ncRNA-mediated genetic circuits and their uses in several microbial engineering applications.

Abstract

Synthetic non-coding RNAs have emerged as a versatile class of molecular devices that have a diverse range of programmable functions, including signal sensing, gene regulation and the modulation of molecular interactions. Owing to their small size and the central role of Watson–Crick base pairing in determining their structure, function and interactions, several distinct types of synthetic non-coding RNA regulators that are functional at the DNA, mRNA and protein levels have been experimentally characterized and computationally modelled. These engineered devices can be incorporated into genetic circuits, enabling the more efficient creation of complex synthetic biological systems. In this Review, we summarize recent progress in engineering synthetic non-coding RNA devices and their application to genetic and cellular engineering in a broad range of microorganisms.

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Figure 1: The CRISPR–Cas system for gene repression and activation in bacteria.
Figure 2: ncRNA regulators of transcription and translation.
Figure 3: Ligand-sensing synthetic riboswitches and trans-acting ncRNAs.
Figure 4: Three basic types of ncRNA regulatory circuits.
Figure 5: A universal ncRNA gene regulatory platform.

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Acknowledgements

L.S.Q. acknowledges support from the University of California San Francisco Center for Systems and Synthetic Biology and the US National Institutes of Health (NIH) Office Of The Director (OD). L.S.Q. and A.P.A. acknowledge support from the California Institute for Quantitative Biomedical Research (QB3). This work was supported by NIH Director's Early Independence Award (grant OD017887 to L.S.Q.), NIH R01 (grant DA036858 to L.S.Q.), NIH P50 (grant GM102706 to A.P.A.), and US National Science Foundation (NSF) SynBERC EEC-0540879 (A.P.A.).

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Authors and Affiliations

  1. University of California San Francisco Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, 94158, California, USA

    Lei S. Qi

  2. Department of Cellular and Molecular Pharmacology, University of California San Francisco, San Francisco, 94158, California, USA

    Lei S. Qi

  3. California Institute for Quantitative Biomedical Research, San Francisco, 94158, California, USA

    Lei S. Qi & Adam P. Arkin

  4. Department of Bioengineering, University of California Berkeley, Berkeley, 94720, California, USA

    Adam P. Arkin

  5. Physical Biosciences Division, Lawrence Berkeley National Laboratory, Berkeley, 94720, California, USA

    Adam P. Arkin

Authors
  1. Lei S. Qi
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  2. Adam P. Arkin
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Corresponding authors

Correspondence to Lei S. Qi or Adam P. Arkin.

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Glossary

Toggle switches

Bistable genetic circuits that contain two mutually inhibitory repressors.

Oscillators

Genetic circuits that produce a repetitive and oscillating signal with fixed time periods.

Molecular counters

Circuits that can compute and store the number of times a particular event has occurred.

Non-coding RNAs

(ncRNAs). Functional RNA molecules that are not translated into proteins.

Small RNAs

(sRNAs). Non-coding RNA molecules that are typically 50–250 nucleotides in length.

Antisense RNAs

Single-stranded RNAs that are complementary to an mRNA.

RNA interference

(RNAi). A process in which RNA molecules inhibit gene expression via the degradation of specific mRNA molecules.

MicroRNAs

Small non-coding RNA molecules (usually 22 nucleotides in length) that are often expressed in plants and animals and that control gene expression by transcriptional or post-transcriptional mechanisms.

Riboswitches

Regulatory RNA elements of mRNAs; they bind to small molecules and regulate expression of the mRNA accordingly.

Ribozymes

RNA molecules that are capable of catalysing specific biochemical reactions.

Orthogonally acting RNA elements

RNA elements that have distinct target-binding specificities and do not exhibit crosstalk.

Protospacer adjacent motif

(PAM). A short, conserved DNA sequence (usually 2–5 base pairs) that is adjacent to the binding site of crRNA.

Native elongating transcript sequencing

(NET-seq). A deep sequencing technology used to identify nascent transcripts that are associated with an elongating RNA polymerase.

Operons

Functional units of genomic DNA that contain a cluster of genes under the control of a single promoter.

Seed region

The minimal region on the small guide RNA that enables target recognition and binding.

Insertion sequence

A short DNA sequence that functions as a simple transposable element.

Riboregulator

An RNA molecule that responds to a signal nucleic acid (DNA or RNA) by Watson–Crick base pairing.

Logic evaluators or logic gates

Devices that carry out logical operations using one or more signal inputs to produce a single logical output. For example, a four-input logic gate carries out logical computation using four inputs.

Aptazymes

Complex RNA devices that are composed of a ligand-sensing domain and a ribozyme domain that controls gene expression.

Aptamers

DNA, RNA or peptide molecules that bind to specific ligands. Aptamers that are composed of RNA are called RNA aptamers.

Metabolic flux

The rate of turnover of metabolite molecules through a metabolic pathway.

NOR logic computation

An inverted OR gate. NOR logic is a universal gate that can be combined to form every other type of logic gate.

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Qi, L., Arkin, A. A versatile framework for microbial engineering using synthetic non-coding RNAs. Nat Rev Microbiol 12, 341–354 (2014). https://doi.org/10.1038/nrmicro3244

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