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 dCas9Sth1CRISPRi system will have broad utility for functional genomics, genetic interaction mapping and drug-target profiling in M. tuberculosis.
Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.
Jeremy M
JM
Rock
Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.
Forrest F
FF
Hopkins
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts 02138, USA.|Department of Pathology, Massachusetts General Hospital, Boston, Massachusetts 02114, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
Alejandro
A
Chavez
Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.
Marieme
M
Diallo
Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.
Michael R
MR
Chase
Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.
Elias R
ER
Gerrick
Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.
Justin R
JR
Pritchard
Wyss Institute for Biologically Inspired Engineering, Harvard University, Cambridge, Massachusetts 02138, USA.|Department of Genetics, Harvard Medical School, Boston, Massachusetts 02115, USA.
George M
GM
Church
Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.
Eric J
EJ
Rubin
Department of Microbiology and Physiological Systems, University of Massachusetts Medical School, Worcester, Massachusetts 01655, USA.
Christopher M
CM
Sassetti
Department of Microbiology and Immunology, Weill Cornell Medical College, New York, New York 10065, USA.
Dirk
D
Schnappinger
Department of Immunology and Infectious Diseases, Harvard T.H. Chan School of Public Health, Boston, Massachusetts 02115, USA.|The Ragon Institute of MGH, Harvard and MIT, Cambridge, Massachusetts 02139, USA.
Sarah M
SM
Fortune
D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism*; D000076987:CRISPR-Associated Protein 9; D064113:CRISPR-Cas Systems; D064112:Clustered Regularly Interspaced Short Palindromic Repeats; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D015870:Gene Expression; D055785:Gene Knockdown Techniques; D020868:Gene Silencing; D005821:Genetic Techniques; D009161:Mycobacterium / Q000235:genetics*; D009169:Mycobacterium tuberculosis / Q000235:genetics; D009876:Operon; D013297:Streptococcus pyogenes / Q000235:genetics; D048249:Streptococcus thermophilus / Q000235:genetics; D024363:Transcription Initiation Site; D014158:Transcription, Genetic
D001426:Bacterial Proteins; D000076987:CRISPR-Associated Protein 9; C000606107:Cas9 endonuclease Streptococcus pyogenes; D004720:Endonucleases