Genome engineering of Clostridium difficile using the CRISPR-Cas9 system

Wang, S.; Hong, Wei; Dong, Sheng; Zhang, Z.-T.; Zhang, J.; Wang, L.; Wang, Y.

doi:10.1016/j.cmi.2018.03.026

Cited by 42 publications

(38 citation statements)

References 26 publications

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“…Similarly, the location of targeting has an effect on the efficiency of repression. Target sites that are farther away CRISPR-Cas9 system are higher than that of Cpf1, few genes were targeted and the same genes were not targeted between the studies (Table 1) (55,62). As for C. beijerinckii, the same mutation efficiencies were reported for two genes, spo0A and pta, using either Cas9 or Cpf1 as the endonuclease (61).…”

Section: Dcas9/crisprimentioning

confidence: 73%

“…In some organisms, including clostridia, the expression of the Cas9 endonuclease has a high cost on the bacterial cell in terms of toxicity (12,13,(47)(48)(49)(50). Despite this cost, CRISPR-Cas9 systems have been successfully applied in C. acetobutylicum (13,14,51), Clostridium autoethanogenum (52), Clostridium beijerinckii (12,13,50,53), C. cellulolyticum (48,54), C. difficile (15,55), Clostridium ljungdahlii (56), Clostridium pasteurianum (49,51), and Clostridium saccharoperbutylacetonicum (57) ( Table 1) by tightly regulating the expression of the cas9 gene. Specifically, the implementation of inducible promoters (e.g., tetracycline-, xylose-, or lactose-inducible promoters) has helped to circumvent the issue of Cas9 toxicity (14,15,50,55,57).…”

Section: Wild-type Cas9mentioning

confidence: 99%

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CRISPR Genome Editing Systems in the Genus Clostridium : a Timely Advancement

McAllister

Sorg

2019

J Bacteriol

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The genus Clostridium is composed of bioproducers, which are important for the industrial production of chemicals, as well as pathogens, which are a significant burden to the patients and on the health care industry. Historically, even though these bacteria are well known and are commonly studied, the genetic technologies to advance our understanding of these microbes have lagged behind other systems. New tools would continue the advancement of our understanding of clostridial physiology. The genetic modification systems available in several clostridia are not as refined as in other organisms and each exhibit their own drawbacks. With the advent of the repurposing of the CRISPR-Cas systems for genetic modification, the tools available for clostridia have improved significantly over the past four years. Several CRISPR-Cas systems such as using wild-type Cas9, Cas9n, dCas9/CRISPR interference (CRISPRi) and a newly studied Cpf1/Cas12a, are reported. These have the potential to greatly advance the study of clostridial species leading to future therapies or the enhanced production of industrially relevant compounds. Here we discuss the details of the CRISPR-Cas systems as well as the advances and current issues in the developed clostridial systems.

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Section: Dcas9/crisprimentioning

confidence: 73%

Section: Wild-type Cas9mentioning

confidence: 99%

CRISPR Genome Editing Systems in the Genus Clostridium : a Timely Advancement

McAllister

Sorg

2019

J Bacteriol

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“…Generally, the endogenous HDR mechanism in most bacterial species follows a similar manner as that in E. coli . Thus, taking advantage of the intrinsic HDR machinery, a relatively simple design, carrying a CRISPR machinery (SpCas9 + sgRNA or FnCas12a + crRNA) and a homologous repair template (1–2 kB) either on the same plasmid or on separate plasmids, is able to manage the genome editing in many genera, such as Bacillus , Clostridium , Corynebacterium , Lactobacillus , Lactococcus , Methylococcus , Pseudomonas , Staphylococcus , Streptococcus , and Streptomyces (Table S1, Supporting Information). However, minor variance in the molecular detail of HDR can cause a large difference in recombination efficiency, since HDR is a sophisticated procedure, requiring the collaboration of multiple proteins .…”

Section: Repair Of Dna Double‐strand Breakmentioning

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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“…Most surviving cells will have undergone a HR event thereby escaping CRISPR-Cas mediated killing. (Jiang et al 2013; Wang et al 2015, 2018). Genome editing approaches using HR and CRISPR-Cas9 have been used for numerous bacterial species, including Gram-positive lactic acid bacteria (Mougiakos et al 2016; Leenay et al 2019).…”

Section: Introductionmentioning

confidence: 99%

CRISPR-Cas9 mediated genome editing in vancomycin resistantEnterococcus faecium

Maat

Stege

Dedden

et al. 2019

Preprint

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The Gram-positive bacterium Enterococcus faecium is becoming increasingly prevalent as a cause of hospital-acquired, antibiotic-resistant infections. There is thus an urgent need to mechanistically characterize the traits that contribute to the emergence of E. faecium as a multidrug resistant opportunistic pathogen. A fundamental part of research into E. faecium biology relies on the ability to generate targeted mutants, but this process is currently labour-intensive and time-consuming, taking 4 to 5 weeks per mutant. In this report we describe a method relying on the high recombination rates of E. faecium and the application of the Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR)-Cas9 genome editing tool to more efficiently generate targeted mutants in the E. faecium chromosome. Using this tool and the multi-drug resistant clinical E. faecium strain E745, we generated a deletion mutant in the lacL gene, which encodes the large subunit of the E. faecium β-galactosidase. Blue/white screening using 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside (X-gal) could be used to distinguish between the wild-type and lacL deletion mutant. We also inserted two copies of gfp into the intrinsic E. faecium macrolide resistance gene msrC to generate stable green fluorescent cells. We conclude that CRISPR-Cas9 can be used to generate targeted genome modifications in E. faecium in 3 weeks, with limited hands-on time. This method can potentially be implemented in other Gram-positive bacteria with high intrinsic recombination rates.

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Genome engineering of Clostridium difficile using the CRISPR-Cas9 system

Cited by 42 publications

References 26 publications

CRISPR Genome Editing Systems in the Genus Clostridium : a Timely Advancement

CRISPR Genome Editing Systems in the Genus Clostridium : a Timely Advancement

Strategies for Developing CRISPR‐Based Gene Editing Methods in Bacteria

CRISPR-Cas9 mediated genome editing in vancomycin resistantEnterococcus faecium

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