Gene silencing by CRISPR interference in mycobacteria

Choudhary, Eira; Thakur, Preeti; Pareek, Madhu; Agarwal, Nisheeth

doi:10.1038/ncomms7267

Cited by 214 publications

(220 citation statements)

References 37 publications

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“…1B). This is consistent with the previous observation that sgRNAs farther from the 5= end of the transcription start site caused weaker repression (42). In the uppP null mutant, depleting bcrC led to xylose sensitivity.…”

Section: Resultssupporting

confidence: 82%

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

et al. 2016

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The integrity of the bacterial cell envelope is essential to sustain life by countering the high turgor pressure of the cell and providing a barrier against chemical insults. In Bacillus subtilis, synthesis of both peptidoglycan and wall teichoic acids requires a common C 55 lipid carrier, undecaprenyl-pyrophosphate (UPP), to ferry precursors across the cytoplasmic membrane. The synthesis and recycling of UPP requires a phosphatase to generate the monophosphate form Und-P, which is the substrate for peptidoglycan and wall teichoic acid synthases. Using an optimized clustered regularly interspaced short palindromic repeat (CRISPR) system with catalytically inactive ("dead") CRISPR-associated protein 9 (dCas9)-based transcriptional repression system (CRISPR interference [CRISPRi]), we demonstrate that B. subtilis requires either of two UPP phosphatases, UppP or BcrC, for viability. We show that a third predicted lipid phosphatase (YodM), with homology to diacylglycerol pyrophosphatases, can also support growth when overexpressed. Depletion of UPP phosphatase activity leads to morphological defects consistent with a failure of cell envelope synthesis and strongly activates the M -dependent cell envelope stress response, including bcrC, which encodes one of the two UPP phosphatases. These results highlight the utility of an optimized CRISPRi system for the investigation of synthetic lethal gene pairs, clarify the nature of the B. subtilis UPP-Pase enzymes, and provide further evidence linking the M regulon to cell envelope homeostasis pathways. IMPORTANCEThe emergence of antibiotic resistance among bacterial pathogens is of critical concern and motivates efforts to develop new therapeutics and increase the utility of those already in use. The lipid II cycle is one of the most frequently targeted processes for antibiotics and has been intensively studied. Despite these efforts, some steps have remained poorly defined, partly due to genetic redundancy. CRISPRi provides a powerful tool to investigate the functions of essential genes and sets of genes. Here, we used an optimized CRISPRi system to demonstrate functional redundancy of two UPP phosphatases that are required for the conversion of the initially synthesized UPP lipid carrier to Und-P, the substrate for the synthesis of the initial lipid-linked precursors in peptidoglycan and wall teichoic acid synthesis.

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Section: Resultssupporting

confidence: 82%

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

et al. 2016

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“…In agreement with the previous study (42), employing an sgRNA targeting the template strand resulted in insignificant repression, as it was reported that the Cas9-sgRNA heteroduplex recognizes the PAM on the nontemplate DNA strand (40,52). These results corroborate well with previously reported CRISPRi-mediated transcriptional repression in E. coli (42) and Mycobacterium tuberculosis (68), where significant levels of transcriptional repression were observed.…”

Section: Discussionsupporting

confidence: 82%

“…4). The difference in the level of CCR relief between the two gene targets may be associated with either the biochemical mechanism of CCR or the targeting efficiency of dCas9, which is knowingly affected by the sgRNA secondary structure and the location of the sgRNA target sequence within the gene (42,68). Our results suggest the potential applicability of CRISPRi in manipulating or even optimizing complex and multigenic phenotypes in C. acetobutylicum for industrial purposes.…”

Section: Discussionmentioning

confidence: 82%

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

Bruder

Pyne

Moo‐Young

et al. 2016

Appl Environ Microbiol

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The discovery and exploitation of the prokaryotic adaptive immunity system based on clustered regularly interspaced short palindromic repeats (CRISPRs) and CRISPR-associated (Cas) proteins have revolutionized genetic engineering. CRISPR-Cas tools have enabled extensive genome editing as well as efficient modulation of the transcriptional program in a multitude of organisms. Progress in the development of genetic engineering tools for the genus Clostridium has lagged behind that of many other prokaryotes, presenting the CRISPR-Cas technology an opportunity to resolve a long-existing issue. Here, we applied the Streptococcus pyogenes type II CRISPR-Cas9 (SpCRISPR-Cas9) system for genome editing in Clostridium acetobutylicum DSM792. We further explored the utility of the SpCRISPR-Cas9 machinery for gene-specific transcriptional repression. For proof-of-concept demonstration, a plasmid-encoded fluorescent protein gene was used for transcriptional repression in C. acetobutylicum. Subsequently, we targeted the carbon catabolite repression (CCR) system of C. acetobutylicum through transcriptional repression of the hprK gene encoding HPr kinase/phosphorylase, leading to the coutilization of glucose and xylose, which are two abundant carbon sources from lignocellulosic feedstocks. Similar approaches based on SpCRISPR-Cas9 for genome editing and transcriptional repression were also demonstrated in Clostridium pasteurianum ATCC 6013. As such, this work lays a foundation for the derivation of clostridial strains for industrial purposes. IMPORTANCEAfter recognizing the industrial potential of Clostridium for decades, methods for the genetic manipulation of these anaerobic bacteria are still underdeveloped. This study reports the implementation of CRISPR-Cas technology for genome editing and transcriptional regulation in Clostridium acetobutylicum, which is arguably the most common industrial clostridial strain. The developed genetic tools enable simpler, more reliable, and more extensive derivation of C. acetobutylicum mutant strains for industrial purposes. Similar approaches were also demonstrated in Clostridium pasteurianum, another clostridial strain that is capable of utilizing glycerol as the carbon source for butanol fermentation, and therefore can be arguably applied in other clostridial strains.

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“…On the other hand, CRISPRi provides excellent transcriptional control and is simple to implement in many organisms. The CRISPRdCas9 system has been applied to genome-scale transcriptional repression (35) and activation (35,36) for interrogation of gene function in human cell lines as well as to genetic and metabolic engineering of Escherichia coli (37,38), Corynebacterium glutamicum (39), and mycobacteria (40).…”

mentioning

confidence: 99%

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

Westbrook

Moo‐Young

Chou

2016

Appl Environ Microbiol

160

156

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The establishment of a clustered regularly interspaced short palindromic repeat (CRISPR)-Cas9 system for strain construction in Bacillus subtilis is essential for its progression toward industrial utility. Here we outline the development of a CRISPR-Cas9 tool kit for comprehensive genetic engineering in B. subtilis. In addition to site-specific mutation and gene insertion, our approach enables continuous genome editing and multiplexing and is extended to CRISPR interference (CRISPRi) for transcriptional modulation. Our tool kit employs chromosomal expression of Cas9 and chromosomal transcription of guide RNAs (gRNAs) using a gRNA transcription cassette and counterselectable gRNA delivery vectors. Our design obviates the need for multicopy plasmids, which can be unstable and impede cell viability. Efficiencies of up to 100% and 85% were obtained for single and double gene mutations, respectively. Also, a 2.9-kb hyaluronic acid (HA) biosynthetic operon was chromosomally inserted with an efficiency of 69%. Furthermore, repression of a heterologous reporter gene was achieved, demonstrating the versatility of the tool kit. The performance of our tool kit is comparable with those of systems developed for Escherichia coli and Saccharomyces cerevisiae, which rely on replicating vectors to implement CRISPR-Cas9 machinery. IMPORTANCEIn this paper, as the first approach, we report implementation of the CRISPR-Cas9 system in Bacillus subtilis, which is recognized as a valuable host system for biomanufacturing. The study enables comprehensive engineering of B. subtilis strains with virtually any desired genotypes/phenotypes and biochemical properties for extensive industrial application.

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Gene silencing by CRISPR interference in mycobacteria

Cited by 214 publications

References 37 publications

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

Depletion of Undecaprenyl Pyrophosphate Phosphatases Disrupts Cell Envelope Biogenesis in Bacillus subtilis

Extending CRISPR-Cas9 Technology from Genome Editing to Transcriptional Engineering in the Genus Clostridium

Development of a CRISPR-Cas9 Tool Kit for Comprehensive Engineering of Bacillus subtilis

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