Suppression of Antimicrobial Resistance in MRSA Using CRISPR-dCas9

Wang, Karissa; Nicholaou, Matthew

doi:10.29074/ascls.30.4.207

Cited by 21 publications

(15 citation statements)

References 11 publications

(10 reference statements)

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“…1A ). CRISPRi has been successfully established in bacterial species such as Escherichia coli ( 20 ), Bacillus subtilis ( 21 ), Streptococcus pneumoniae ( 22 ), Staphylococcus aureus ( 23 – 26 ), and Lactococcus lactis ( 27 ). Note that CRISPR-based tools have been used in lactobacilli previously.…”

Section: Introductionmentioning

confidence: 99%

CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes inLactobacillus plantarum

Myrbråten

Wiull

Salehian

et al. 2019

mSphere

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Studies of essential genes in bacteria are often hampered by the lack of accessible genetic tools. This is also the case for Lactobacillus plantarum, a key species in food and health applications. Here, we develop a clustered regularly interspaced short palindromic repeat interference (CRISPRi) system for knockdown of gene expression in L. plantarum. The two-plasmid CRISPRi system, in which a nuclease-inactivated Cas9 (dCas9) and a gene-specific single guide RNA (sgRNA) are expressed on separate plasmids, allows efficient knockdown of expression of any gene of interest. We utilized the CRISPRi system to gain initial insights into the functions of key cell cycle genes in L. plantarum. As a proof of concept, we investigated the phenotypes resulting from knockdowns of the cell wall hydrolase-encoding acm2 gene and of the DNA replication initiator gene dnaA and of ezrA, which encodes an early cell division protein. Furthermore, we studied the phenotypes of three cell division genes which have recently been functionally characterized in ovococcal bacteria but whose functions have not yet been investigated in rod-shaped bacteria. We show that the transmembrane CozE proteins do not seem to play any major role in cell division in L. plantarum. On the other hand, RNA-binding proteins KhpA and EloR are critical for proper cell elongation in this bacterium. IMPORTANCE L. plantarum is an important bacterium for applications in food and health. Deep insights into the biology and physiology of this species are therefore necessary for further strain optimization and exploitation; however, the functions of essential genes in the bacterium are mainly unknown due to the lack of accessible genetic tools. The CRISPRi system developed here is ideal to quickly screen for phenotypes of both essential and nonessential genes. Our initial insights into the function of some key cell cycle genes represent the first step toward understanding the cell cycle in this bacterium.

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Section: Introductionmentioning

confidence: 99%

CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes inLactobacillus plantarum

Myrbråten

Wiull

Salehian

et al. 2019

mSphere

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“…This strategy leads to plasmid curing, thereby reducing the prevalence of antibiotic-resistant bacteria [11,[21][22][23][24][25][26][27]. Researchers have also utilized a catalytically "dead" Cas9 (dCas9) to suppress transcription of the methicillin resistance gene mecA in Staphylococcus aureus [28] and the class 1 integron in Escherichia coli, which is associated with a variety of resistance genes [29]. The diversity of these applications demonstrates how valuable CRISPR/Cas systems could become in the fight against rapidly emerging MDR bacteria.…”

Section: Crispr Antimicrobials: An Overviewmentioning

confidence: 99%

CRISPR-based antimicrobials to obstruct antibiotic-resistant and pathogenic bacteria

2021

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“…Results of the experiments have shown a 77% decrease in gene expression in CRISPR-treated samples; however, 77% was not sufficient to make MRSA clinically susceptible to β-lactam antibiotics. The CRISPR–dCas9 system applied in these experiments did not kill the bacteria, which makes it a viable option for treatment against antibiotic-resistant bacteria [ 91 ]. In a different study, Kang et al introduced a nonviral delivery method for CRISPR (Cr-Nanocomplex), based on a polymer-derivatized Cas9 protein and sgRNA nanocomplex targeting mecA —a major antibiotic resistance gene involved in MRSA.…”

Section: Crispr Applications—antibiotic Resistant Bacteriamentioning

confidence: 99%

Various Aspects of a Gene Editing System—CRISPR–Cas9

Janik

Niemcewicz

Ceremuga

et al. 2020

IJMS

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The discovery of clustered, regularly interspaced short palindromic repeats (CRISPR) and their cooperation with CRISPR-associated (Cas) genes is one of the greatest advances of the century and has marked their application as a powerful genome engineering tool. The CRISPR–Cas system was discovered as a part of the adaptive immune system in bacteria and archaea to defend from plasmids and phages. CRISPR has been found to be an advanced alternative to zinc-finger nucleases (ZFN) and transcription activator-like effector nucleases (TALEN) for gene editing and regulation, as the CRISPR–Cas9 protein remains the same for various gene targets and just a short guide RNA sequence needs to be altered to redirect the site-specific cleavage. Due to its high efficiency and precision, the Cas9 protein derived from the type II CRISPR system has been found to have applications in many fields of science. Although CRISPR–Cas9 allows easy genome editing and has a number of benefits, we should not ignore the important ethical and biosafety issues. Moreover, any tool that has great potential and offers significant capabilities carries a level of risk of being used for non-legal purposes. In this review, we present a brief history and mechanism of the CRISPR–Cas9 system. We also describe on the applications of this technology in gene regulation and genome editing; the treatment of cancer and other diseases; and limitations and concerns of the use of CRISPR–Cas9.

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Suppression of Antimicrobial Resistance in MRSA Using CRISPR-dCas9

Cited by 21 publications

References 11 publications

CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes inLactobacillus plantarum

CRISPR Interference for Rapid Knockdown of Essential Cell Cycle Genes inLactobacillus plantarum

CRISPR-based antimicrobials to obstruct antibiotic-resistant and pathogenic bacteria

Various Aspects of a Gene Editing System—CRISPR–Cas9

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