Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach

Mückl, Andrea; Schwarz-Schilling, Matthaeus; Fischer, Katrin; Simmel, Friedrich C.

doi:10.1371/journal.pone.0198058

Cited by 30 publications

(24 citation statements)

References 47 publications

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“…Since sgRNAs act as a NOT gate and two identical sgRNAs act as a NOR gates, all other basic logic gates like AND and OR can be constructed. As outputs, metabolic production is discussed in (131), replication in (49), filamentation in (132), biofilm formation in (112) and cell shape in (136). Table 1.…”

Section: Resultsmentioning

confidence: 99%

CRISPR Tools To Control Gene Expression in Bacteria

Vigouroux

Bikard

2020

Microbiol Mol Biol Rev

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SUMMARY CRISPR-Cas systems have been engineered as powerful tools to control gene expression in bacteria. The most common strategy relies on the use of Cas effectors modified to bind target DNA without introducing DNA breaks. These effectors can either block the RNA polymerase or recruit it through activation domains. Here, we discuss the mechanistic details of how Cas effectors can modulate gene expression by blocking transcription initiation or acting as transcription roadblocks. CRISPR-Cas tools can be further engineered to obtain fine-tuned control of gene expression or target multiple genes simultaneously. Several caveats in using these tools have also been revealed, including off-target effects and toxicity, making it important to understand the design rules of engineered CRISPR-Cas effectors in bacteria. Alternatively, some types of CRISPR-Cas systems target RNA and could be used to block gene expression at the posttranscriptional level. Finally, we review applications of these tools in high-throughput screens and the progress and challenges in introducing CRISPR knockdown to other species, including nonmodel bacteria with industrial or clinical relevance. A deep understanding of how CRISPR-Cas systems can be harnessed to control gene expression in bacteria and build powerful tools will certainly open novel research directions.

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Section: Resultsmentioning

confidence: 99%

CRISPR Tools To Control Gene Expression in Bacteria

Vigouroux

Bikard

2020

Microbiol Mol Biol Rev

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“…Depletion of FtsZ in rod-shaped bacteria prevents division, leading to the formation of long filaments that eventually lyse. The appearance of highly filamentous cells provides a simple visual read-out that has been used to demonstrate successful inhibition of ftsZ expression by CRISPRi in E. coli (35, 36), B. subtilis (23), and Pseudomonas aeruginosa (22). In the coccus Streptococcus pneumoniae , CRISPRi knockdown of ftsZ expression causes swelling rather than filamentation (19).…”

Section: Resultsmentioning

confidence: 99%

A xylose-inducible expression system and a CRISPRi-plasmid for targeted knock-down of gene expression inClostridioides difficile

Müh¹,

Pannullo²,

Weiss³

et al. 2018

Preprint

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17Here we introduce plasmids for xylose-regulated expression and repression of genes in 18 Clostridioides difficile. The xylose-inducible expression vector allows for ~100-fold 19 induction of an mCherryOpt reporter gene. Induction is titratable and uniform from cell-20 to-cell. The gene repression plasmid is a CRISPR-interference (CRISPRi) system 21 based on a nuclease-defective, codon-optimized allele of the Streptococcus pyogenes 22 Cas9 protein (dCas9) that is targeted to a gene of interest by a constitutively-expressed 23 single guide RNA (sgRNA). Expression of dCas9 is induced by xylose, allowing 24 investigators to control the timing and extent of gene-silencing, as demonstrated here 25 by dose-dependent repression of a chromosomal gene for a red fluorescent protein 26 (maximum repression ~100-fold). To validate the utility of CRISPRi for deciphering gene 27 function in C. difficile, we knocked-down expression of three genes involved in 28 biogenesis of the cell envelope: the cell division gene ftsZ, the S-layer protein gene slpA 29 and the peptidoglycan synthase gene pbp-0712. CRISPRi confirmed known or expected 30 phenotypes associated with loss of FtsZ and SlpA, and revealed that the previously 31 uncharacterized peptidoglycan synthase PBP-0712 is needed for proper elongation, cell 32 division and protection against lysis. 33 34 Importance 35 Clostridioides difficile has become the leading cause of hospital-acquired diarrhea in 36 developed countries. A better understanding of the basic biology of this devastating 37 pathogen might lead to novel approaches for preventing or treating C. difficile infections.38 3 Here we introduce new plasmid vectors that allow for titratable induction (Pxyl) or 39 knockdown (CRISPRi) of gene expression. The CRISPRi plasmid allows for easy 40 depletion of target proteins in C. difficile. Besides bypassing the lengthy process of 41 mutant construction, CRISPRi can be used to study the function of essential genes, 42 which are particularly important targets for antibiotic development.43 44 48 diarrhea (1). The symptoms are caused by toxins that damage the intestinal epithelium 49 and can range in severity from mild diarrhea to life threatening conditions such as 50 pseudomembranous colitis and toxic megacolon (2-4). A recent study estimated that C.

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“…The author has used CRISPRi to inhibit the expression of a transcription factor MetJ which globally repressed the SAM synthesis pathway, leading to a significantly improved yield of the final product, peonidin 3-O-glucoside. Similar strategy has been verified in many genera for manufacturing many biochemicals and pharmaceutic precursors, such as poly-3-hydroxbutyrate (PHB), [166][167][168][169] hyaluronic acid, [170] N-acetylglucosamine, [171] l-lysine, [172] l-glutamate, [172] citrulline, [173] flavonoid, [174] malate, [175] butanol, [176] 1,4-butanediol, [177] 3-hydroxypropionic acid, [178] mevalonate, [179] and epothilones. [180]…”

Section: Metabolic Engineering With Crispr-dcas Toolsmentioning

confidence: 99%

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

Wang

Zhang

et al. 2019

Small Methods

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Gene editing is a fundamental technique for the identification of the linkages from genetic determinants to significant biological phenotypes, and the engineering of industrial strains to produce fine chemicals. Recently, the primitive bacterial immunity systems, clustered regularly interspaced short palindromic repeat (CRISPR)‐Cas systems, have been engineered as programmable nucleases to deliver double‐strand breaks (DSBs) at user‐defined loci. The DSB is repaired via either homology‐direct repair or the non‐homologous end joining pathway, generating a mutant in various organisms, and leading a revolution in the field of gene editing. This paper reviews the structural and molecular details of CRISPR‐Cas systems, describing their applications and challenges of genome targeting in bacterial cells. Moreover, the DSB repair pathways in bacteria are summarized and a guideline for selecting repair machines and maximizing the recombination efficiency is presented. In addition, the plasmid curing approaches in bacteria are outlined for iterative gene editing or downstream biological applications. Furthermore, CRISPR‐based transcriptional regulation, base editing, and transposition are also introduced. Finally, this paper prospects the future directions for improving CRISPR‐based gene editing methods in bacteria.

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Filamentation and restoration of normal growth in Escherichia coli using a combined CRISPRi sgRNA/antisense RNA approach

Cited by 30 publications

References 47 publications

CRISPR Tools To Control Gene Expression in Bacteria

CRISPR Tools To Control Gene Expression in Bacteria

A xylose-inducible expression system and a CRISPRi-plasmid for targeted knock-down of gene expression inClostridioides difficile

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

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