Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform

Jeremy M. Rock, Forrest F. Hopkins, Alejandro Chavez, Marieme Diallo, Michael R. Chase, Elias R. Gerrick, Justin Pritchard, George M. Church, Eric J. Rubin, Christopher M. Sassetti, Dirk Schnappinger, Sarah M. Fortune

Research output: Contribution to journalArticle

55 Citations (Scopus)

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 (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.

Original languageEnglish (US)
Article number16274
JournalNature Microbiology
Volume2
DOIs
StatePublished - Feb 6 2017

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Clustered Regularly Interspaced Short Palindromic Repeats
Mycobacterium
Mycobacterium tuberculosis
Gene Knockdown Techniques
Streptococcus thermophilus
Streptococcus pyogenes
Gene Silencing
Operon
Genomics
Drug Interactions
Folic Acid
Pharmaceutical Preparations
Dissection
Tuberculosis
Gene Expression
Genes
Proteins

All Science Journal Classification (ASJC) codes

  • Microbiology
  • Immunology
  • Applied Microbiology and Biotechnology
  • Genetics
  • Microbiology (medical)
  • Cell Biology

Cite this

Rock, J. M., Hopkins, F. F., Chavez, A., Diallo, M., Chase, M. R., Gerrick, E. R., ... Fortune, S. M. (2017). Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. Nature Microbiology, 2, [16274]. https://doi.org/10.1038/nmicrobiol.2016.274
Rock, Jeremy M. ; Hopkins, Forrest F. ; Chavez, Alejandro ; Diallo, Marieme ; Chase, Michael R. ; Gerrick, Elias R. ; Pritchard, Justin ; Church, George M. ; Rubin, Eric J. ; Sassetti, Christopher M. ; Schnappinger, Dirk ; Fortune, Sarah M. / Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. In: Nature Microbiology. 2017 ; Vol. 2.
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Rock, JM, Hopkins, FF, Chavez, A, Diallo, M, Chase, MR, Gerrick, ER, Pritchard, J, Church, GM, Rubin, EJ, Sassetti, CM, Schnappinger, D & Fortune, SM 2017, 'Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform', Nature Microbiology, vol. 2, 16274. https://doi.org/10.1038/nmicrobiol.2016.274

Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform. / Rock, Jeremy M.; Hopkins, Forrest F.; Chavez, Alejandro; Diallo, Marieme; Chase, Michael R.; Gerrick, Elias R.; Pritchard, Justin; Church, George M.; Rubin, Eric J.; Sassetti, Christopher M.; Schnappinger, Dirk; Fortune, Sarah M.

In: Nature Microbiology, Vol. 2, 16274, 06.02.2017.

Research output: Contribution to journalArticle

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