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RNA processing enables predictable programming of gene expression

Nature Biotechnology volume 30, pages 1002–1006 (2012)Cite this article

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Abstract

Complex interactions among genetic components often result in variable systemic performance in designed multigene systems1,2. Using the bacterial clustered regularly interspaced short palindromic repeat (CRISPR) pathway3,4 we develop a synthetic RNA-processing platform, and show that efficient and specific cleavage of precursor mRNA enables reliable and predictable regulation of multigene operons. Physical separation of linked genetic elements by CRISPR-mediated cleavage is an effective strategy to achieve assembly of promoters, ribosome binding sites, cis-regulatory elements, and riboregulators into single- and multigene operons with predictable functions in bacteria. We also demonstrate that CRISPR-based RNA cleavage is effective for regulation in bacteria, archaea and eukaryotes. Programmable RNA processing using CRISPR offers a general approach for creating context-free genetic elements and can be readily used in the bottom-up construction of increasingly complex biological systems in a plug-and-play manner.

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Figure 1: The CRISPR RNA-processing system allows engineering of standard genetic elements in various contexts.
Figure 2: RNA processing enables design of operonic systems.
Figure 3: Applications of CRISPR RNA processing to the predictable engineering of complex regulatory systems.

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Acknowledgements

We thank members of BIOFAB, International Open Facility Advancing Biotechnology, for distributing genetic parts, and J. Dueber, M. Lee and H. Lee (University of California, Berkeley) for providing the yeast vector. This work was supported by the US National Science Foundation (SynBERC, NSFEEC-0540879, L.Q. and A.P.A.), Department of Energy through Laboratory Directed Research and Development (DE-AC02-05CH11231d, L.Q., W.S. and A.P.A.) and Howard Hughes Medical Institute (R.E.H. and J.A.D.).

Author information

Author notes

  1. Lei Qi & Rachel E Haurwitz

    Present address: Present addresses: Center for Systems and Synthetic Biology, University of California San Francisco, San Francisco, California, USA (L.Q.) and Caribou Biosciences, Inc., Berkeley, California, USA. (R.E.H.).,

Authors and Affiliations

  1. Department of Bioengineering, University of California Berkeley, Berkeley, California, USA

    Lei Qi & Adam P Arkin

  2. Department of Molecular and Cell Biology, University of California Berkeley, Berkeley, California, USA

    Rachel E Haurwitz, Wenjun Shao & Jennifer A Doudna

  3. Howard Hughes Medical Institute, University of California Berkeley, Berkeley, California, USA

    Jennifer A Doudna

  4. Department of Chemistry, University of California, Berkeley, Berkeley, California, USA

    Jennifer A Doudna

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

    Jennifer A Doudna & Adam P Arkin

  6. California Institute for Quantitative Biosciences (QB3), Berkeley, California, USA

    Adam P Arkin

Authors
  1. Lei Qi
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  2. Rachel E Haurwitz
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  3. Wenjun Shao
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  4. Jennifer A Doudna
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  5. Adam P Arkin
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Contributions

L.Q., A.P.A. and J.A.D. conceived of the research; L.Q., R.E.H., W.S., J.A.D. and A.P.A. designed the study; L.Q., R.E.H. and W.S. performed the experiments; L.Q., R.E.H., J.A.D. and A.P.A. analyzed the data and wrote the manuscript.

Corresponding author

Correspondence to Adam P Arkin.

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Competing interests

L.Q., R.E.H., J.A.D. and A.P.A. have filed a related patent application (US application 61/679,397, filed on 3 August 2012).

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Supplementary Methods and Supplementary Figures 1–8 (PDF 7970 kb)

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Qi, L., Haurwitz, R., Shao, W. et al. RNA processing enables predictable programming of gene expression. Nat Biotechnol 30, 1002–1006 (2012). https://doi.org/10.1038/nbt.2355

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