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.

[1]  Luke A. Gilbert,et al.  CRISPR interference (CRISPRi) for sequence-specific control of gene expression , 2013, Nature Protocols.

[2]  Sabine Ehrt,et al.  Controlling gene expression in mycobacteria with anhydrotetracycline and Tet repressor , 2005, Nucleic acids research.

[3]  W. Jacobs,et al.  Site-specific integration of mycobacteriophage L5: integration-proficient vectors for Mycobacterium smegmatis, Mycobacterium tuberculosis, and bacille Calmette-Guérin. , 1991, Proceedings of the National Academy of Sciences of the United States of America.

[4]  M. Chase,et al.  DNA replication fidelity in Mycobacterium tuberculosis is mediated by an ancestral prokaryotic proofreader , 2015, Nature Genetics.

[5]  L. Hall,et al.  Tuberculosis and Trimethoprim-Sulfamethoxazole , 2009, Antimicrobial Agents and Chemotherapy.

[6]  Sita J. Saunders,et al.  An updated evolutionary classification of CRISPR–Cas systems , 2015, Nature Reviews Microbiology.

[7]  Meagan E. Sullender,et al.  Rational design of highly active sgRNAs for CRISPR-Cas9–mediated gene inactivation , 2014, Nature Biotechnology.

[8]  Nisheeth Agarwal,et al.  Gene silencing by CRISPR interference in mycobacteria , 2015, Nature Communications.

[9]  Christian Stolte,et al.  TB database: an integrated platform for tuberculosis research , 2008, Nucleic Acids Res..

[10]  Tautvydas Karvelis,et al.  Rapid characterization of CRISPR-Cas9 protospacer adjacent motif sequence elements , 2015, Genome Biology.

[11]  Prashant Mali,et al.  Orthogonal Cas9 Proteins for RNA-Guided Gene Regulation and Editing , 2013, Nature Methods.

[12]  Anirban Ghosh,et al.  Assessment of Mycobacterium tuberculosis Pantothenate Kinase Vulnerability through Target Knockdown and Mechanistically Diverse Inhibitors , 2014, Antimicrobial Agents and Chemotherapy.

[13]  Luke A. Gilbert,et al.  Repurposing CRISPR as an RNA-Guided Platform for Sequence-Specific Control of Gene Expression , 2013, Cell.

[14]  Yusuke Minato,et al.  Mycobacterium tuberculosis Folate Metabolism and the Mechanistic Basis for para-Aminosalicylic Acid Susceptibility and Resistance , 2015, Antimicrobial Agents and Chemotherapy.

[15]  Paramvir S. Dehal,et al.  FastTree 2 – Approximately Maximum-Likelihood Trees for Large Alignments , 2010, PloS one.

[16]  S. Cabantous,et al.  4′-Phosphopantetheinyl Transferase PptT, a New Drug Target Required for Mycobacterium tuberculosis Growth and Persistence In Vivo , 2012, PLoS pathogens.

[17]  S. Ferrari,et al.  Author contributions , 2021 .

[18]  John S. Hawkins,et al.  A Comprehensive, CRISPR-based Functional Analysis of Essential Genes in Bacteria , 2016, Cell.

[19]  A. Sievers,et al.  Mycobacterium tuberculosis and Sulfamethoxazole Susceptibility , 2010, Antimicrobial Agents and Chemotherapy.

[20]  Feng Zhang,et al.  Programmable repression and activation of bacterial gene expression using an engineered CRISPR-Cas system , 2013, Nucleic acids research.

[21]  T. Alber,et al.  Depletion of antibiotic targets has widely varying effects on growth , 2011, Proceedings of the National Academy of Sciences.

[22]  D. Schnappinger,et al.  Mycobacterium tuberculosis Thioredoxin Reductase Is Essential for Thiol Redox Homeostasis but Plays a Minor Role in Antioxidant Defense , 2016, PLoS pathogens.

[23]  Philippe Horvath,et al.  Diversity, Activity, and Evolution of CRISPR Loci in Streptococcus thermophilus , 2007, Journal of bacteriology.

[24]  Philippe Horvath,et al.  The CRISPR/Cas bacterial immune system cleaves bacteriophage and plasmid DNA , 2010, Nature.

[25]  C. Vilchèze,et al.  The Combination of Sulfamethoxazole, Trimethoprim, and Isoniazid or Rifampin Is Bactericidal and Prevents the Emergence of Drug Resistance in Mycobacterium tuberculosis , 2012, Antimicrobial Agents and Chemotherapy.

[26]  Lucas B. Harrington,et al.  Single-Stranded DNA Cleavage by Divergent CRISPR-Cas9 Enzymes. , 2015, Molecular cell.

[27]  J. Vogel,et al.  CRISPR RNA maturation by trans-encoded small RNA and host factor RNase III , 2011, Nature.

[28]  David A. Scott,et al.  In vivo genome editing using Staphylococcus aureus Cas9 , 2015, Nature.

[29]  Chase L. Beisel,et al.  Identifying and Visualizing Functional PAM Diversity across CRISPR-Cas Systems. , 2016, Molecular cell.

[30]  E. Davis,et al.  Mycobacterium tuberculosis thymidylate synthase gene thyX is essential and potentially bifunctional, while thyA deletion confers resistance to p-aminosalicylic acid , 2012, Microbiology.

[31]  Philippe Horvath,et al.  Phage Response to CRISPR-Encoded Resistance in Streptococcus thermophilus , 2007, Journal of bacteriology.

[32]  S. Porcella,et al.  The Mycobacterium marinum G13 promoter is a strong sigma 70-like promoter that is expressed in Escherichia coli and mycobacteria species. , 1999, FEMS Microbiology Letters.

[33]  Kira S. Makarova,et al.  Classification and evolution of type II CRISPR-Cas systems , 2014, Nucleic acids research.

[34]  R. Barrangou,et al.  CRISPR Provides Acquired Resistance Against Viruses in Prokaryotes , 2007, Science.

[35]  G. Church,et al.  Unraveling CRISPR-Cas9 genome engineering parameters via a library-on-library approach , 2015, Nature Methods.

[36]  Martin J. Aryee,et al.  Engineered CRISPR-Cas9 nucleases with altered PAM specificities , 2015, Nature.

[37]  Kira S. Makarova,et al.  Phylogeny of Cas9 determines functional exchangeability of dual-RNA and Cas9 among orthologous type II CRISPR-Cas systems , 2013, Nucleic acids research.

[38]  Thomas R. Ioerger,et al.  Global Assessment of Genomic Regions Required for Growth in Mycobacterium tuberculosis , 2012, PLoS pathogens.

[39]  Dieter Jahn,et al.  JCat: a novel tool to adapt codon usage of a target gene to its potential expression host , 2005, Nucleic Acids Res..

[40]  T. Kunkel.  DNA Replication Fidelity* , 2004, Journal of Biological Chemistry.

[41]  A. Singh,et al.  Investigating essential gene function in Mycobacterium tuberculosis using an efficient CRISPR interference system , 2016, Nucleic acids research.

[42]  D. Schnappinger,et al.  Evaluating the Sensitivity of Mycobacterium tuberculosis to Biotin Deprivation Using Regulated Gene Expression , 2011, PLoS pathogens.

[43]  Christopher A. Voigt,et al.  Multi-input CRISPR/Cas genetic circuits that interface host regulatory networks , 2014, Molecular systems biology.

[44]  E. Myers,et al.  Basic local alignment search tool. , 1990, Journal of molecular biology.

[45]  S. Bushby,et al.  Trimethoprim-sulfamethoxazole: in vitro microbiological aspects. , 1973, The Journal of infectious diseases.

[46]  Jennifer A. Doudna,et al.  Biology and Applications of CRISPR Systems: Harnessing Nature’s Toolbox for Genome Engineering , 2016, Cell.

[47]  J. Doudna,et al.  A Programmable Dual-RNA–Guided DNA Endonuclease in Adaptive Bacterial Immunity , 2012, Science.

[48]  V. Escuyer,et al.  Cloning, expression, and characterization of Mycobacterium tuberculosis dihydrofolate reductase. , 2004, FEMS microbiology letters.

[49]  M. Jinek,et al.  Structural basis of PAM-dependent target DNA recognition by the Cas9 endonuclease , 2014, Nature.

[50]  D. Schnappinger,et al.  Protein inactivation in mycobacteria by controlled proteolysis and its application to deplete the beta subunit of RNA polymerase , 2010, Nucleic acids research.

[51]  R. Barrangou,et al.  Cas9–crRNA ribonucleoprotein complex mediates specific DNA cleavage for adaptive immunity in bacteria , 2012, Proceedings of the National Academy of Sciences.

[52]  Magnus Lundgren,et al.  Efficient programmable gene silencing by Cascade , 2014, Nucleic acids research.

[53]  P. Morris,et al.  Genomic Characterization of Mycobacteriophage Giles: Evidence for Phage Acquisition of Host DNA by Illegitimate Recombination , 2008, Journal of bacteriology.