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Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform

Nature Microbiology volume 2, Article number: 16274 (2017) Cite this article

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Abstract

The development of new drug regimens that allow rapid, sterilizing treatment of tuberculosis has been limited by the complexity and time required for genetic manipulations in Mycobacterium tuberculosis. CRISPR interference (CRISPRi) promises to be a robust, easily engineered and scalable platform for regulated gene silencing. However, in M. tuberculosis, the existing Streptococcus pyogenes Cas9-based CRISPRi system is of limited utility because of relatively poor knockdown efficiency and proteotoxicity. To address these limitations, we screened eleven diverse Cas9 orthologues and identified four that are broadly functional for targeted gene knockdown in mycobacteria. The most efficacious of these proteins, the CRISPR1 Cas9 from Streptococcus thermophilus (dCas9Sth1), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9Sth1-mediated gene knockdown is robust when targeted far from the transcriptional start site, thereby allowing high-resolution dissection of gene function in the context of bacterial operons. We demonstrate the utility of this system by addressing persistent controversies regarding drug synergies in the mycobacterial folate biosynthesis pathway. We anticipate that the dCas9Sth1 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.

The treatment of drug-susceptible Mycobacterium tuberculosis requires a regimen of four drugs taken for six months. One of the overarching goals of the M. tuberculosis research community is to develop a regimen of novel agents that can treat both drug-susceptible and drug-resistant M. tuberculosis in less than two weeks. This will necessitate the development of new genetic tools to identify drug target synergies that accelerate pathogen clearance while avoiding drug target antagonisms. Current genetic approaches for drug target characterization include promoter replacement and inducible protein degradation systems that allow the regulation of target protein levels over two orders of magnitude1–3, but can take months to target a single gene. Transposon-sequencing (Tn-seq) allows the simultaneous assessment of hundreds of thousands of loss-of-function mutants, but as currently implemented in M. tuberculosis is not conditional and thus does not allow the manipulation of essential genes4. Furthermore, neither method provides a simple mechanism to modulate multiple genes simultaneously and thereby elucidate genetic interactions.

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Figure 1: In vivo screen for functional Cas9 orthologues in mycobacteria.
Figure 2: dCas9Spy sensitizes M. smegmatis to sublethal drug treatment.
Figure 3: In vivo identification of permissive PAM position variants for dCas9Sth1.
Figure 4: dCas9Sth1 CRISPRi is highly active against endogenous genes in mycobacteria.
Figure 5: Functional profiling of the mycobacterial folate synthesis pathway.

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Acknowledgements

The authors thank L. Gilbert for advice. This work was supported by a Helen Hay Whitney fellowship to J.M.R., an NIH Director's New Innovator Award 1DP20D001378, subcontracts from NIAID U19 AI107774 and a Doris Duke Charitable Foundation Grant 2010054 to S.M.F. A.C. was funded by the National Cancer Institute grant no. 5T32CA009216-34. G.M.C. acknowledges support from the US National Institutes of Health National Human Genome Research Institute grant no. P50 HG005550 and the Wyss Institute for Biologically Inspired Engineering.

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Authors and Affiliations

  1. Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, 02115, Massachusetts, USA

    Jeremy M. Rock, Forrest F. Hopkins, Marieme Diallo, Michael R. Chase, Elias R. Gerrick, Justin R. Pritchard, Eric J. Rubin & Sarah M. Fortune

  2. Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, 02138, Massachusetts, USA

    Alejandro Chavez & George M. Church

  3. Department of Pathology, Massachusetts General Hospital, Boston, 02114, Massachusetts, USA

    Alejandro Chavez

  4. Department of Genetics, Harvard Medical School, Boston, 02115, Massachusetts, USA

    Alejandro Chavez & George M. Church

  5. Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, 01655, Massachusetts, USA

    Christopher M. Sassetti

  6. Department of Microbiology and Immunology, Weill Cornell Medical College, New York, 10065, New York, USA

    Dirk Schnappinger

  7. The Ragon Institute of MGH, Harvard and MIT, Cambridge, 02139, Massachusetts, USA

    Sarah M. Fortune

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Contributions

S.M.F. supervised the project. J.M.R. and J.R.P. conceived the study. J.M.R. designed and executed the study. F.F.H. and M.D. cloned CRISPR constructs and assisted with luciferase assays. A.C., M.R.C., E.R.G., G.M.C., E.J.R., C.M.S. and D.S. contributed reagents and expertise. J.M.R. and S.M.F. wrote the manuscript with input from all co-authors.

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Correspondence to Sarah M. Fortune.

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The authors declare no competing financial interests.

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Rock, J., Hopkins, F., Chavez, A. et al. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat Microbiol 2, 16274 (2017). https://doi.org/10.1038/nmicrobiol.2016.274

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