Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes

Copeland, Matthew F.; Politz, Mark C.; Pfleger, Brian F.

doi:10.1016/j.copbio.2014.02.010

Cited by 34 publications

(22 citation statements)

References 71 publications

(108 reference statements)

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“…Hence, implementing genome engineering with more capacities, such as genome editing and transcriptional regulation, can be useful in developing complex phenotypes (22)(23)(24). Similar to the state of genome editing, transcriptional modulation techniques in Clostridium are generally lacking despite significant advancement of sophisticated trans-acting elements, such as transcription activator-like effectors (TALEs) and zinc finger (ZF) DNA-binding domains fused to various activator and repressor domains in eukaryotes and, more recently, in prokaryotes (3,22,(25)(26)(27). Antisense RNA (asRNA) technology (3,23), in which an RNA molecule complementary to a target mRNA is transcribed for in vivo hybridization with the target mRNA (28), remains the most prevalent transcriptional silencing mechanism employed in Clostridium, particularly for metabolic engineering applications (3).…”

Section: Importancementioning

confidence: 99%

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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Section: Importancementioning

confidence: 99%

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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“…chemical production instead of growth). Trans acting tools that supersede native regulation in specific environments are therefore desirable additions to the synthetic biology toolbox (Copeland et al, 2014). One such tool, termed CRISPR interference (CRISPRi), takes advantage of the adaptive RNA-based defense system that in many bacteria targets and cleaves foreign nucleic acids such as viruses and plasmids (Qi et al, 2013).…”

Section: 1 Introductionmentioning

confidence: 99%

CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002

Gordon¹,

Korosh²,

Cameron

et al. 2016

Metabolic Engineering

164

158

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Trans-acting regulators provide novel opportunities to study essential genes and regulate metabolic pathways. We have adapted the clustered regularly interspersed palindromic repeats (CRISPR) system from Streptococcus pyogenes to repress genes in trans in the cyanobacterium Synechococcus sp. strain PCC 7002 (hereafter PCC 7002). With this approach, termed CRISPR interference (CRISPRi), transcription of a specific target sequence is repressed by a catalytically inactive Cas9 protein recruited to the target DNA by base-pair interactions with a single guide RNA that is complementary to the target sequence. We adapted this system for PCC 7002 and achieved conditional and titratable repression of a heterologous reporter gene, yellow fluorescent protein. Next, we demonstrated the utility of finely tuning native gene expression by downregulating the abundance of phycobillisomes. In addition, we created a conditional auxotroph by repressing synthesis of the carboxysome, an essential component of the carbon concentrating mechanism cyanobacteria use to fix atmospheric CO2. Lastly, we demonstrated a novel strategy for increasing central carbon flux by conditionally downregulating a key node in nitrogen assimilation. The resulting cells produced 2-fold more lactate than a baseline engineered cell line, representing the highest photosynthetically generated productivity to date. This work is the first example of titratable repression in cyanobacteria using CRISPRi, enabling dynamic regulation of essential processes and manipulation of flux through central carbon metabolism. This tool facilitates the study of essential genes of unknown function and enables groundbreaking metabolic engineering capability, by providing a straightforward approach to redirect metabolism and carbon flux in the production of high-value chemicals.

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“…By targeting the sgRNA to other regions of the DNA, systems biologists have coopted this system for genome editing. 160,161 More recently, a fluorescently labeled endonuclease-deficient Cas9 (dCas9) has been used for single-molecule imaging in live mouse and human cells. 158,162 While this technique has yet to be applied to imaging in bacteria, success with bacterial genome editing suggests that it should be possible to fluorescently label specific genomic loci in the bacterial chromosome with dCas9.…”

Section: ■ Biomolecule Labeling Methodsmentioning

confidence: 99%

Unveiling the Inner Workings of Live Bacteria Using Super-Resolution Microscopy

Tuson

Biteen

2014

Anal. Chem.

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Application of TALEs, CRISPR/Cas and sRNAs as trans-acting regulators in prokaryotes

Cited by 34 publications

References 71 publications

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

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

CRISPR interference as a titratable, trans-acting regulatory tool for metabolic engineering in the cyanobacterium Synechococcus sp. strain PCC 7002

Unveiling the Inner Workings of Live Bacteria Using Super-Resolution Microscopy

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