Skip to main content

Thank you for visiting You are using a browser version with limited support for CSS. To obtain the best experience, we recommend you use a more up to date browser (or turn off compatibility mode in Internet Explorer). In the meantime, to ensure continued support, we are displaying the site without styles and JavaScript.

Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform


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.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.


All prices are NET prices.

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.


  1. 1

    Ehrt, S. et al. Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor. Nucleic Acids Res. 33, e21 (2005).

    Article  Google Scholar 

  2. 2

    Kim, J. H. et al. Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase. Nucleic Acids Res. 39, 2210–2220 (2011).

    CAS  Article  Google Scholar 

  3. 3

    Wei, J.-R. et al. Depletion of antibiotic targets has widely varying effects on growth. Proc. Natl Acad. Sci. USA 108, 4176–4181 (2011).

    CAS  Article  Google Scholar 

  4. 4

    Zhang, Y. J. et al. Global assessment of genomic regions required for growth in Mycobacterium tuberculosis. PLoS Pathogens 8, e1002946 (2012).

    Article  Google Scholar 

  5. 5

    Qi, L. S. et al. Repurposing CRISPR as an RNA-guided platform for sequence-specific control of gene expression. Cell 152, 1173–1183 (2013).

    CAS  Article  Google Scholar 

  6. 6

    Rath, D., Amlinger, L., Hoekzema, M., Devulapally, P. R. & Lundgren, M. Efficient programmable gene silencing by Cascade. Nucleic Acids Res. 43, 237–246 (2015).

    CAS  Article  Google Scholar 

  7. 7

    Bikard, D. et al. Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system. Nucleic Acids Res. 41, 7429–7437 (2013).

    CAS  Article  Google Scholar 

  8. 8

    Wright, A. V., Nuñez, J. K. & Doudna, J. A. Biology and applications of CRISPR systems: harnessing nature's toolbox for genome engineering. Cell 164, 29–44 (2016).

    CAS  Article  Google Scholar 

  9. 9

    Gasiunas, G., Barrangou, R., Horvath, P. & Siksnys, V. Cas9-crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria. Proc. Natl Acad. Sci. 109, E2579–86 (2012).

    CAS  Article  Google Scholar 

  10. 10

    Garneau, J. E. et al. The CRISPR/Cas bacterial immunesystem cleaves bacteriophage andplasmid DNA. Nature 468, 67–71 (2010).

    CAS  Article  Google Scholar 

  11. 11

    Jinek, M. et al. A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 337, 816–821 (2012).

    CAS  Article  Google Scholar 

  12. 12

    Deltcheva, E. et al. Crispr RNA maturation by trans-encoded small RNA and host factor RNase III. Nature 471, 602–607 (2011).

    CAS  Article  Google Scholar 

  13. 13

    Horvath, P. et al. Diversity, activity, and evolution of CRISPR loci in Streptococcus thermophilus. J. Bacteriol. 190, 1401–1412 (2008).

    CAS  Article  Google Scholar 

  14. 14

    Deveau, H. et al. Phage response to CRISPR-encoded resistance in Streptococcus thermophilus. J. Bacteriol. 190, 1390–1400 (2008).

    CAS  Article  Google Scholar 

  15. 15

    Peters, J. M. et al. A comprehensive, CRISPR-based functional analysis of essential genes in bacteria. Cell 165, 1493–1506 (2016).

    CAS  Article  Google Scholar 

  16. 16

    Choudhary, E., Thakur, P., Pareek, M. & Agarwal, N. Gene silencing by CRISPR interference in mycobacteria. Nat. Commun. 6, 6267 (2015).

    CAS  Article  Google Scholar 

  17. 17

    Singh, A. K. et al. Investigating essential gene function in Mycobacterium tuberculosis using an efficient cRISPR interference system. Nucleic Acids Res. 44, e143 (2016).

    Article  Google Scholar 

  18. 18

    Nielsen, A. A. & Voigt, C. A. Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks. Mol. Syst. Biol. 10, 763–763 (2014).

    Article  Google Scholar 

  19. 19

    Anders, C., Niewoehner, O., Duerst, A. & Jinek, M. Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease. Nature 513, 569–573 (2014).

    CAS  Article  Google Scholar 

  20. 20

    Park, S. W. et al. Evaluating the sensitivity of Mycobacterium tuberculosis to biotin deprivation using regulated gene expression. PLoS Pathogens 7, e1002264 (2011).

    CAS  Article  Google Scholar 

  21. 21

    Leblanc, C. et al. 4'-Phosphopantetheinyl transferase PptT, a new drug target required for mycobacterium tuberculosis growth and persistence in vivo. PLoS Pathogens 8, e1003097 (2012).

    CAS  Article  Google Scholar 

  22. 22

    Lin, K. et al. Mycobacterium tuberculosis thioredoxin reductase is essential for thiol redox homeostasis but plays a minor role in antioxidant defense. PLoS Pathogens 12, e1005675 (2016).

    Article  Google Scholar 

  23. 23

    Reddy, B. K. K. et al. Assessment of Mycobacterium tuberculosis pantothenate kinase vulnerability through target knockdown and mechanistically diverse inhibitors. Antimicrob. Agents Chemother. 58, 3312–3326 (2014).

    Article  Google Scholar 

  24. 24

    Fonfara, I. et al. Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems. Nucleic Acids Res. 42, 2577–2590 (2014).

    CAS  Article  Google Scholar 

  25. 25

    Ran, F. A. et al. In vivo genome editing using Staphylococcus aureus Cas9. Nature 520, 186–191 (2015).

    CAS  Article  Google Scholar 

  26. 26

    Esvelt, K. M. et al. Orthogonal Cas9 proteins for RNA-guided gene regulation and editing. Nat. Methods 10, 1116–1121 (2013).

    CAS  Article  Google Scholar 

  27. 27

    Ma, E., Harrington, L. B., O'Connell, M. R., Zhou, K. & Doudna, J. A. Single-stranded DNA cleavage by divergent CRISPR-Cas9 enzymes. Mol. Cell 60, 398–407 (2015).

    CAS  Article  Google Scholar 

  28. 28

    Barrangou, R. et al. CRISPR provides acquired resistance against viruses in prokaryotes. Science 315, 1709–1712 (2007).

    CAS  Article  Google Scholar 

  29. 29

    Rock, J. M. et al. DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader. Nat. Genet. 47, 677–681 (2015).

    CAS  Article  Google Scholar 

  30. 30

    Chari, R., Mali, P., Moosburner, M. & Church, G. M. Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach. Nat. Methods 12, 823–826 (2015).

    CAS  Article  Google Scholar 

  31. 31

    Kleinstiver, B. P. et al. Engineered CRISPR-Cas9 nucleases with altered PAM specificities. Nature 523, 481–485 (2015).

    Article  Google Scholar 

  32. 32

    Leenay, R. T. et al. Identifying and visualizing functional PAM diversity across CRISPR-Cas systems. Mol. Cell 62, 137–147 (2016).

    CAS  Article  Google Scholar 

  33. 33

    Karvelis, T. et al. Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements. Genome Biol. 16, 253 (2015).

    Article  Google Scholar 

  34. 34

    Caspi, R. et al. The MetaCyc database of metabolic pathways and enzymes and the BioCyc collection of pathway/genome databases. Nucleic Acids Res. 36, D623–D631 (2007).

    Article  Google Scholar 

  35. 35

    Larson, M. H. et al. CRISPR interference (CRISPRi) for sequence-specific control of gene expression. Nat. Protoc. 8, 2180–2196 (2013).

    CAS  Article  Google Scholar 

  36. 36

    Minato, Y. et al. Mycobacterium tuberculosis folate metabolism and the mechanistic basis for para-aminosalicylic acid susceptibility and resistance.  Antimicrob. Agents Chemother. 59, 5097–5106 (2015).

    CAS  Article  Google Scholar 

  37. 37

    Bushby, S. Trimethoprim-sulfamethoxazole: in vitro microbiological aspects. J. Infect. Dis. 128 (Suppl), 442–462 (1973).

    Article  Google Scholar 

  38. 38

    White, E. L., Ross, L. J., Cunningham, A. & Escuyer, V. Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase. FEMS Microbiol. Lett. 232, 101–105 (2004).

    CAS  Article  Google Scholar 

  39. 39

    Forgacs, P. et al. Tuberculosis and trimethoprim-sulfamethoxazole. Antimicrob. Agents Chemother. 53, 4798–4793 (2009).

    Article  Google Scholar 

  40. 40

    Vilcheze, C. & Jacobs, W. R. The combination of sulfamethoxazole, trimethoprim, and isoniazid or rifampin is bactericidal and prevents the emergence of drug resistance in Mycobacterium tuberculosis. Antimicrob. Agents Chemother. 56, 5142–5148 (2012).

    CAS  Article  Google Scholar 

  41. 41

    Ong, W. et al. Mycobacterium tuberculosis and sulfamethoxazole susceptibility. Antimicrob. Agents Chemother. 54, 2748–2749 (2010).

    CAS  Article  Google Scholar 

  42. 42

    Altschul, S. F., Gish, W., Miller, W., Myers, E. W. & Lipman, D. J. Basic local alignment search tool. J. Mol. Biol. 215, 403–410 (1990).

    CAS  Article  Google Scholar 

  43. 43

    Grote, A. et al. JCat: a novel tool to adapt codon usage of a target gene to its potential expression host. Nucleic Acids Res. 33, W526–W531 (2005).

    CAS  Article  Google Scholar 

  44. 44

    Lee, M. H., Pascopella, L., Jacobs, W. R. & Hatfull, G. F. Site-specific integration of mycobacteriophage L5: integration-proficient vectors for mycobacterium smegmatis, mycobacterium tuberculosis, and Bacille Calmette-Guérin. Proc. Natl Acad. Sci. USA 88, 3111–3115 (1991).

    CAS  Article  Google Scholar 

  45. 45

    Doench, J. G. et al. Rational design of highly active sgRNAs for CRISPR-Cas9-mediated gene inactivation. Nat. Biotechnol. 32, 1262–1267 (2014).

    CAS  Article  Google Scholar 

  46. 46

    Barker, L. P., Porcella, S. F., Wyatt, R. G. & Small, P. L. The Mycobacterium marinum G13 promoter is a strong sigma 70-like promoter that is expressed in Escherichia coli and mycobacteria species. FEMS Microbiol. Lett. 175, 79–85 (1999).

    CAS  Article  Google Scholar 

  47. 47

    Morris, P., Marinelli, L. J., Jacobs-Sera, D., Hendrix, R. W. & Hatfull, G. F. Genomic characterization of mycobacteriophage Giles: evidence for phage acquisition of host DNA by illegitimate recombination. J. Bacteriol. 190, 2172–2182 (2008).

    CAS  Article  Google Scholar 

  48. 48

    Makarova, K. S. et al. An updated evolutionary classification of CRISPR-Cas systems. Nat. Rev. Microbiol. 13, 722–736 (2015).

    CAS  Article  Google Scholar 

  49. 49

    Chylinski, K., Makarova, K. S., Charpentier, E. & Koonin, E. V. Classification and evolution of type II CRISPR-Cas systems. Nucleic Acids Res. 42, 6091–6105 (2014).

    CAS  Article  Google Scholar 

  50. 50

    Price, M. N., Dehal, P. S. & Arkin, A. P. Fasttree 2—approximately maximum-likelihood trees for large alignments . PLoS ONE 5, e9490 (2010).

    Article  Google Scholar 

  51. 51

    Reddy, T. B. K. et al. TB database: an integrated platform for tuberculosis research. Nucleic Acids Res. 37, D499–D508 (2009).

    CAS  Article  Google Scholar 

  52. 52

    Fivian-Hughes, A. S., Houghton, J. & Davis, E. O. Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid. Microbiology 158, 308–318 (2012).

    CAS  Article  Google Scholar 

Download references


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.

Author information




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.

Corresponding author

Correspondence to Sarah M. Fortune.

Ethics declarations

Competing interests

The authors declare no competing financial interests.

Supplementary information

Supplementary Information

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

Rights and permissions

Reprints and Permissions

About this article

Verify currency and authenticity via CrossMark

Cite this article

Rock, J., Hopkins, F., Chavez, A. et al. Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nat Microbiol 2, 16274 (2017).

Download citation

Further reading


Quick links

Nature Briefing

Sign up for the Nature Briefing newsletter — what matters in science, free to your inbox daily.

Get the most important science stories of the day, free in your inbox. Sign up for Nature Briefing