Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform
Open accessAbstract
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 (dCas9 Sth1), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9 Sth1-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 dCas9 Sth1 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.
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CRISPRi technology was developed and optimized in M. smegmatis and its application to fine-tune gene repression was demonstrated in M. tuberculosis.
“Like other method breakthroughs, the CRISPRi technology was developed and optimized in M. smegmatis (35) and its application to fine-tune gene repression elegantly demonstrated in M. tuberculosis (36).”
From Mycobacterium smegmatis: The Vanguard of Mycobacterial Research (2023) · cites this paper as [35]
The Results section, “Gene silencing with CRISPRi in M. tuberculosis” (page unavailable), says the system was developed in M. smegmatis and then shown to produce robust knockdown in M. tuberculosis. The Discussion says knockdown magnitude is tunable, but the Results section, “Identification of synergistic interactions in the folate biosynthesis pathway” (page unavailable), describes its explicit demonstration of partial, tuned knockdown in M. smegmatis, not M. tuberculosis. Thus the claimed fine-tuning demonstration in M. tuberculosis is not established by these passages. (LLM, conf=0.84)
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- Authors
- Rock, J. M., Hopkins, F. F., Chavez, A., Diallo, M., Chase, M. R., Gerrick, E. R., Pritchard, J. R., Church, G. M., Rubin, E. J., Sassetti, C. M., Schnappinger, D., & Fortune, S. M.
- Affiliations
- Harvard University (US)
- University of Massachusetts Chan Medical School (US)
- Weill Cornell Medicine (US)
- Corresponding author
- Fortune, Sarah M.
- ORCID
- Venue
- Nature Microbiology
- Published
- 2017-02-06
- Volume
- 2
- Issue
- 4
- Pages
- 16274-16274
- ISSN
- 2058-5276 (ISSN-L)
- PMC
- PMC5302332
- OpenAlex
- W2586637020
- Cited by
- 538
- References
- 53
- Citations by year
- 2026: 61 · 2025: 78 · 2024: 77 · 2023: 59 · 2022: 63 · 2021: 62 · 2020: 59 · 2019: 43 · 2018: 32 · 2017: 4
- FWCI
- 15.41
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Licence
- Locations
- Nature Microbiology (publishedVersion)
- PubMed (publishedVersion)
- Europe PMC (PubMed Central) (acceptedVersion)
- PubMed Central (acceptedVersion)
- Digital Access to Scholarship at Harvard (DASH) (Harvard University) (submittedVersion, cc-by)
- The Journal of the American Medical Association (JAMA) Network (American Medical Association) (submittedVersion)
- Funders
- National Human Genome Research Institute (P50HG005550)
- National Cancer Institute (T32CA009216)
- National Institute of Allergy and Infectious Diseases (R01AI064282, U19AI107774, U19AI109755, R01AI097191)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › CRISPR and Genetic Engineering
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Bacterial Genetics and Biotechnology
- Health Sciences › Medicine › Infectious Diseases › Tuberculosis Research and Epidemiology
- Keywords
- CRISPR interference, Mycobacterium tuberculosis, transcriptional repression, gene knockdown efficiency, proteotoxicity, drug synergy, bacterial operons, tuberculosis drug development, functional genomics
- MeSH terms
- CRISPR-Associated Protein 9, Bacterial Proteins, Endonucleases, Genetic Techniques, Mycobacterium, Mycobacterium tuberculosis, Operon, Streptococcus pyogenes, Transcription, Genetic, Gene Expression, Gene Silencing, Transcription Initiation Site, Streptococcus thermophilus, Gene Knockdown Techniques, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Cas Systems
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