Article

Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform

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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.

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Author information

Affiliations

  1. Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, 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, Massachusetts 02138, USA

    • Alejandro Chavez
    •  & George M. Church
  3. Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA

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

    • Alejandro Chavez
    •  & George M. Church
  5. Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA

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

    • Dirk Schnappinger
  7. The Ragon Institute of MGH, Harvard and MIT, Cambridge, Massachusetts 02139, 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.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to Sarah M. Fortune.

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    Supplementary Information

    Supplementary Figures 1–7, Supplementary Tables 1 and 2.