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Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform

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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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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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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Supplementary Figures 1–7, Supplementary Tables 1 and 2. (PDF 6096 kb)

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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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