Reversal of carbapenem-resistance in Shewanella algae by CRISPR/Cas9 genome editing

Wu, Zong‐Yen; Huang, Yao‐Ting; Chao, Wen‐Cheng; Ho, Shu-Peng; Cheng, Jan‐Fang; Liu, Po‐Yu

doi:10.1016/j.jare.2019.01.011

Cited by 38 publications

(29 citation statements)

References 49 publications

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“…These results indicate that CRISPR/Cas9-mediated DSBs and HDR efficiently introduced a site-directed mutation in the MR-1 genome. The frequency of CRISPR/Cas9-mediated mutations observed here is equal to or greater than those of Shewanella algae and other bacteria (Liu et al, 2017;Oh and van Pijkeren, 2014;Wu et al, 2019). A previous study has reported that DNA damage induces the up-regulation of genes involved in homologous recombination in MR-1 (Qiu et al, 2006), supporting that HDR after CRISPR/Cas9-mediated DSB occurs at a high frequency in this strain.…”

Section: Induction Of Dsbs and Hdr In Mr-1 By Crispr/cas9supporting

confidence: 67%

CRISPR/Cas9-mediated genome editing of Shewanella oneidensis MR-1 using a broad host-range pBBR1-based plasmid

Suzuki

Kouzuma

Watanabe

2020

J. Gen. Appl. Microbiol.

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Commonly used CRISPR/Cas9 systems such as the type-II system from Streptococcus pyogenes are composed of an RNA-guided endonuclease (Cas9) and a single-guide RNA (sgRNA) comprising a CRISPR RNA (crRNA) complementary to a 20-nt target DNA sequence as well as a transactivating crRNA (tracrRNA) that recruits the crRNA to Cas9 (Doudna and Charpentier, 2014). The 20-nt target DNA sequence is followed by a 5¢-NGG-3¢ protospacer adjacent motif (PAM) (Choudhary et al., 2015; Nayak and Metcalf, 2017). The Cas9 complex, guided by the sgRNA, cleaves the target sequence approximately three base pairs upstream of the PAM to generate a double-strand DNA break (DSB), which triggers mutations through nonhomologous end joining (NHEJ) repair or homologydirected repair (HDR) (Cho et al., 2018; Ran et al., 2013). Despite extensive use of genome-editing technologies in eukaryotes, their use in bacteria is limited because of the lack of vectors for CRISPR/Cas9 systems. Several cas9-expressing plasmids applicable to genome editing of bacteria are available (

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Section: Induction Of Dsbs and Hdr In Mr-1 By Crispr/cas9supporting

confidence: 67%

CRISPR/Cas9-mediated genome editing of Shewanella oneidensis MR-1 using a broad host-range pBBR1-based plasmid

Suzuki

Kouzuma

Watanabe

2020

J. Gen. Appl. Microbiol.

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“…24 CRISPR-Cas system is a bacterial adaptive immune system protecting itself against invaders and has been used in several bacteria such as E. coli, 25 Staphylococcus aureus and Shewanella algae. 26,27 This system provided an effective strategy for sequence-specific curing of the plasmid.…”

Section: Discussionmentioning

confidence: 99%

Reversal of mcr-1-Mediated Colistin Resistance in Escherichia coli by CRISPR-Cas9 System

Wan¹,

Cui²,

Zhi-gang³

et al. 2020

IDR

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The plasmid-borne mobilized colistin resistance gene (mcr-1) was discovered in 2015. Subsequently, the rapid horizontal transfer of mcr-1 gene to diverse bacterial species poses a serious threat to public health, which urgently needs the introduction of novel antimicrobial strategies. Therefore, the purpose of this study is to sensitize bacteria to colistin and reduce the propagation of mcr-1 gene by curing mcr-1-harboring plasmid in Escherichia coli (E. coli) using the CRISPR-Cas9 system. Methods: Two sgRNAs specific to mcr-1 gene were designed and cloned into plasmid pCas9. The recombinant plasmid pCas9-mcr was transformed into E. coli carrying pUC19mcr-1 or pHNSHP45, separately. The elimination efficiency in strains was evaluated by PCR and quantitative real-time PCR (qPCR). The antimicrobial susceptibility test was performed using the broth microdilution method. Results: In this study, we constructed the high copy number plasmid pUC19-mcr-1 and recombinant plasmid pCas9-m1 or pCas9-m2, which contain 20 nt or 30 nt sgRNA sequences targeted to mcr-1, respectively. PCR and qPCR results showed that mcr-1-harboring plasmids could be efficiently eliminated, and there was no significant correlation between sgRNA lengths and curing efficiency. However, when comparing restructured high copy number plasmid (pUC19-mcr-1) to natural resistance plasmid (pHNSHP45) in eliminating efficiency, we found that the content of plasmid backbone had an influence on efficiency. Furthermore, the conjugation assays verified that the engineered CRISPR-Cas9 system in bacteria or in bacteria genome can protect the recipient from plasmid-borne mcr-1 transfer via conjugation. Additionally, sequence analysis showed that three different types of defects in CRISPR-Cas9 system lead to escape mutants. Conclusion: We presented a method that only one plasmid-mediated CRISPR-Cas9 system can be used to efficiently resensitize E. coli to colistin. Moreover, this system provided a great potentiality to counteract the propagation of mcr-1 among bacterial pathogens.

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“…are believed to be susceptible to most antibiotics used in human medicine, such as third-generation cephalosporins, gentamicin and ciprofloxacin [ 7 ]. However, recent studies have shown an increasing resistance of new isolates [ 1 , 13 , 14 ]. This resistance, in addition to the increasing number of infections, underscores the importance of research focusing on these bacteria and how to prevent new infections.…”

Section: Introductionmentioning

confidence: 99%

First isolation and whole-genome sequencing of a Shewanella algae strain from a swine farm in Brazil

et al. 2020

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Background Infections caused by Shewanella spp. have been increasingly reported worldwide. The advances in genomic sciences have enabled better understanding about the taxonomy and epidemiology of this agent. However, the scarcity of DNA sequencing data is still an obstacle for understanding the genus and its association with infections in humans and animals. Results In this study, we report the first isolation and whole-genome sequencing of a Shewanella algae strain from a swine farm in Brazil using the boot sock method, as well as the resistance profile of this strain to antimicrobials. The isolate was first identified as Shewanella putrefaciens, but after whole-genome sequencing it showed greater similarity with Shewanella algae. The strain showed resistance to 46.7% of the antimicrobials tested, and 26 resistance genes were identified in the genome. Conclusions This report supports research made with Shewanella spp. and gives a step forward for understanding its taxonomy and epidemiology. It also highlights the risk of emerging pathogens with high resistance to antimicrobial formulas that are important to public health.

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Reversal of carbapenem-resistance in Shewanella algae by CRISPR/Cas9 genome editing

Abstract: Graphical abstract

Cited by 38 publications

References 49 publications

CRISPR/Cas9-mediated genome editing of <i>Shewanella oneidensis</i> MR-1 using a broad host-range pBBR1-based plasmid

CRISPR/Cas9-mediated genome editing of <i>Shewanella oneidensis</i> MR-1 using a broad host-range pBBR1-based plasmid

<p>Reversal of <em>mcr-1</em>-Mediated Colistin Resistance in <em>Escherichia coli</em> by CRISPR-Cas9 System</p>

First isolation and whole-genome sequencing of a Shewanella algae strain from a swine farm in Brazil

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