Dataset Information

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

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 (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.

SUBMITTER: Rock JM

PROVIDER: S-EPMC5302332 | biostudies-literature | 2017 Feb

REPOSITORIES: biostudies-literature

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

Rock Jeremy M JM Hopkins Forrest F FF Chavez Alejandro A Diallo Marieme M Chase Michael R MR Gerrick Elias R ER Pritchard Justin R JR Church George M GM Rubin Eric J EJ Sassetti Christopher M CM Schnappinger Dirk D Fortune Sarah M SM

Nature microbiology 20170206

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 proteot ...[more]

PMID: 28165460

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Project description:Pseudomonas spp. are widely used model organisms in different areas of research. Despite the relevance of Pseudomonas in many applications, the use of protein depletion tools in this host remains limited. Here, we developed the CRISPR interference system for gene repression in Pseudomonas spp. using a nuclease-null Streptococcus pasteurianus Cas9 variant (dead Cas9, or dCas9). We demonstrate a robust and titratable gene depletion system with up to 100-fold repression in β-galactosidase activity in P. aeruginosa and 300-fold repression in pyoverdine production in Pseudomonas putida This inducible system enables the study of essential genes, as shown by ftsZ depletions in P. aeruginosa, P. putida, and Pseudomonas fluorescens that led to phenotypic changes consistent with depletion of the targeted gene. Additionally, we performed the first in vivo characterization of protospacer adjacent motif (PAM) site preferences of S. pasteurianus dCas9 and identified NNGCGA as a functional PAM site that resulted in repression efficiencies comparable to the consensus NNGTGA sequence. This discovery significantly expands the potential genomic targets of S. pasteurianus dCas9, especially in GC-rich organisms.IMPORTANCEPseudomonas spp. are prevalent in a variety of environments, such as the soil, on the surface of plants, and in the human body. Although Pseudomonas spp. are widely used as model organisms in different areas of research, existing tools to deplete a protein of interest in these organisms remain limited. We have developed a robust and inducible gene repression tool in P. aeruginosa, P. putida, and P. fluorescens using the Streptococcus pasteurianus dCas9. This method of protein depletion is superior to existing methods, such as promoter replacements and addition of degradation tags, because it does not involve genomic modifications of the target protein, is titratable, and is capable of repressing multiple genes simultaneously. This gene repression system now enables easy depletion of specific proteins in Pseudomonas, accelerating the study and engineering of this widely used model organism.

Project description:Tools for tuning endogenous gene expression are key to determining the genetic basis of diverse cellular phenotypes. Although synthetic regulatable promoters are available in Toxoplasma, scalable methods for targeted and combinatorial downregulation of gene expression-like RNA interference-have yet to be developed. To investigate the feasibility of CRISPR-mediated transcriptional regulation, we examined the function of two catalytically inactive Cas9 (dCas9) orthologs, from Streptococcus pyogenes and Streptococcus thermophilus, in Toxoplasma. Following the addition of single-guide RNAs (sgRNAs) targeting the promoter and 5' untranslated region (UTR) of the surface antigen gene SAG1, we profiled changes in protein abundance of targeted genes by flow cytometry for transcriptional reporters and immunoblotting. We found that the dCas9 orthologs generated a range of target gene expression levels, and the degree of repression was durable and stably inherited. Therefore, S. pyogenes and S. thermophilus dCas9 can effectively produce intermediate levels of gene expression in Toxoplasma. The distinct sgRNA scaffold requirements of the two dCas9s permit their orthogonal use for simultaneous examination of two distinct loci through transcriptional modulation, labeling for microscopy-based studies, or other dCas9-based approaches. Taking advantage of newly available genomic transcription start site data, these tools will aid in the development of new loss-of-function screening approaches in Toxoplasma. IMPORTANCE Toxoplasma gondii is a ubiquitous intracellular parasite of humans and animals that causes life-threatening disease in immunocompromised patients, fetal abnormalities when contracted during gestation, and recurrent eye lesions in some patients. Despite its health implications, about half of the Toxoplasma genome still lacks functional annotation. A particularly powerful tool for the investigation of an organism's cell biology is the modulation of gene expression, which can produce the subtle phenotypes often required for informing gene function. In Toxoplasma, such tools have limited throughput and versatility. Here, we detail the adaptation of a new set of tools based on CRISPR-Cas9, which allows the targeted downregulation of gene expression in Toxoplasma. With its scalability and adaptability to diverse genomic loci, this approach has the potential to greatly accelerate the functional characterization of the Toxoplasma genome.

Dataset Information

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

Publications

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

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