Programmable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.

TitleProgrammable transcriptional repression in mycobacteria using an orthogonal CRISPR interference platform.
Publication TypeJournal Article
Year of Publication2017
AuthorsRock JM, Hopkins FF, Chavez A, Diallo M, Chase MR, Gerrick ER, Pritchard JR, Church GM, Rubin EJ, Sassetti CM, Schnappinger D, Fortune SM
JournalNat Microbiol
Date Published2017 Feb 06
KeywordsBacterial Proteins, Clustered Regularly Interspaced Short Palindromic Repeats, CRISPR-Associated Protein 9, CRISPR-Cas Systems, Endonucleases, Gene Expression, Gene Knockdown Techniques, Gene Silencing, Genetic Techniques, Mycobacterium, Mycobacterium tuberculosis, Operon, Streptococcus pyogenes, Streptococcus thermophilus, Transcription Initiation Site, Transcription, Genetic

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), typically achieves 20- to 100-fold knockdown of endogenous gene expression with minimal proteotoxicity. In contrast to other CRISPRi systems, dCas9-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 CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.

Alternate JournalNat Microbiol
PubMed ID28165460
PubMed Central IDPMC5302332
Grant ListP50 HG005550 / HG / NHGRI NIH HHS / United States
U19 AI109755 / AI / NIAID NIH HHS / United States
U19 AI107774 / AI / NIAID NIH HHS / United States
T32 CA009216 / CA / NCI NIH HHS / United States
R01 AI097191 / AI / NIAID NIH HHS / United States
R01 AI064282 / AI / NIAID NIH HHS / United States

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