SNX11 Identified as an Essential #Host Factor for #SFTS Virus #Infection by #CRISPR Knockout Screening (Virol Sin., abstract)

[Source: Virologica Sinica, full page: (LINK). Abstract, edited.]

SNX11 Identified as an Essential Host Factor for SFTS Virus Infection by CRISPR Knockout Screening

Authors: Tiezhu Liu, Jiajia Li, Yang Liu, Yuanyuan Qu, Aqian Li, Chuan Li, Quanfu Zhang,Wei Wu, Jiandong Li, Yan Liu, Dexin Li, Shiwen Wang, Mifang Liang

Research Article / First Online: 18 June 2019

 

Abstract

Severe fever with thrombocytopenia syndrome virus (SFTSV) is a highly pathogenic tick-borne bunyavirus that causes lethal infectious disease and severe fever with thrombocytopenia syndrome (SFTS) in humans. The molecular mechanisms and host cellular factors required for SFTSV infection remain uncharacterized. Using a genome-wide CRISPR-based screening strategy, we identified a host cellular protein, sorting nexin 11 (SNX11) which is involved in the intracellular endosomal trafficking pathway, as an essential cell factor for SFTSV infection. An SNX11-KO HeLa cell line was established, and SFTSV replication was significantly reduced. The glycoproteins of SFTSV were detected and remained in later endosomal compartments but were not detectable in the endoplasmic reticulum (ER) or Golgi apparatus. pH values in the endosomal compartments of the SNX11-KO cells increased compared with the pH of normal HeLa cells, and lysosomal-associated membrane protein 1 (LAMP1) expression was significantly elevated in the SNX11-KO cells. Overall, these results indicated that penetration of SFTSV from the endolysosomes into the cytoplasm of host cells was blocked in the cells lacking SNX11. Our study for the first time provides insight into the important role of the SNX11 as an essential host factor in the intracellular trafficking and penetrating process of SFTSV infection via potential regulation of viral protein sorting, membrane fusion, and other endocytic machinery.

Keywords: CRISPR – screen – Severe fever with thrombocytopenia syndrome virus (SFTSV) – Host factor – Sorting nexin 11 (SNX11)

Electronic supplementary material

The online version of this article ( https://doi.org/10.1007/s12250-019-00141-0) contains supplementary material, which is available to authorized users.

 

Notes

Acknowledgements

This work was supported by the National Key Project for Infectious Disease from the Ministry of Science and Technology (Grant No. 2018ZX10711-001).

Author Contributions

TL performed the experiments and wrote the paper; Jiajia Li, YL, and YQ performed the experiments; AL, QZ, CL, WW, YL, and Jiandong Li contributed reagents/materials/analysis tools. Jiajia Li, TL, ML, YL, Jiandong Li, and DL analyzed and discussed the data. ML and SW designed the project and edited the manuscript. All authors read and approved the final manuscript.

 

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Animal and Human Rights Statement

This article does not contain any studies with human or animal subjects performed by any of the authors.

Keywords: SFTS virus; Bunyavirus; CRISPR; Viral pathogenesis.

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Eliminating #mcr1-harbouring #plasmids in #clinical isolates using the #CRISPR/Cas9 system (J Antimicrob Chemother., abstract)

[Source: Journal of Antimicrobial Chemotherapy, full page: (LINK). Abstract, edited.]

Eliminating mcr-1-harbouring plasmids in clinical isolates using the CRISPR/Cas9 system

Pengxia Wang, Dongmei He, Baiyuan Li, Yunxue Guo, Weiquan Wang, Xiongjian Luo, Xuanyu Zhao, Xiaoxue Wang

Journal of Antimicrobial Chemotherapy, dkz246, https://doi.org/10.1093/jac/dkz246

Published: 15 June 2019

 

Abstract

Objectives

To eliminate mcr-1-harbouring plasmids and MDR plasmids in clinical Escherichia coli isolates.

Methods

Plasmid pMBLcas9 expressing Cas9 was constructed and used to clone target single-guide RNAs (sgRNAs) for plasmid curing. The recombinant plasmid pMBLcas9-sgRNA was transferred by conjugation into two clinical E. coliisolates. The curing efficiency of different sgRNAs targeting conserved genes was tested. The elimination of targeted plasmids and the generation of transposase-mediated recombination of p14EC033a variants were characterized by PCR and DNA sequencing.

Results

In this study, four native plasmids in isolate 14EC033 and two native plasmids in isolate 14EC007 were successfully eliminated in a step-by-step manner using pMBLcas9. Moreover, two native plasmids in 14EC007 were simultaneously eliminated by tandemly cloning multiple sgRNAs in pMBLcas9, sensitizing 14EC007 to polymyxin and carbenicillin. In 14EC033 with two mcr-1-harbouring plasmids, IncI2 plasmid p14EC033a and IncX4 plasmid p14EC033b, a single mcr-1 sgRNA mediated the loss of p14EC033b and generated a mutant p14EC033a in which the mcr-1 gene was deleted. An insertion element, IS5, located upstream of mcr-1 in p14EC033a was responsible for transposase-mediated recombination, resulting in mcr-1 gene deletion instead of plasmid curing.

Conclusions

CRISPR/Cas9 can be used to efficiently sensitize clinical isolates to antibiotics in vitro. For isolates with multiple plasmids, the CRISPR/Cas9 approach can either remove each plasmid in a stepwise manner or simultaneously remove multiple plasmids in one step. Moreover, this approach can be used to delete multiple gene copies by using only one sgRNA. However, caution must be exercised to avoid unwanted recombination events during genetic manipulation.

Topic: plasmids – crispr

Issue Section: ORIGINAL RESEARCH

Keywords: Antibiotics; Drugs Resistance; MCR1; Colistin; CRISPR.

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A #CRISPR #activation screen identifies #genes protecting from #Zika virus #infection (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

A CRISPR activation screen identifies genes protecting from Zika virus infection

Anna Dukhovny, Kevin Lamkiewicz, Qian Chen, Markus Fricke, Nabila Jabrane-Ferrat, Manja Marz, Jae U. Jung, Ella H. Sklan

DOI: 10.1128/JVI.00211-19

 

ABSTRACT

Zika virus (ZIKV) is an arthropod borne emerging pathogen causing febrile illness. ZIKV is associated Guillain-Barré syndrome and other neurological complications. Infection during pregnancy is associated with pregnancy complications and developmental and neurological abnormalities collectively defined as congenital Zika syndrome. There is still no vaccine or specific treatment for ZIKV infection. To identify host factors that can rescue cells from ZIKV infection we used a genome scale CRISPR activation screen. Our highly ranking hits included a short list of interferon stimulated genes (ISGs) previously reported to have antiviral activity. Validation of the screen results highlighted IFNL2 and IFI6 as genes providing high levels of protection from ZIKV. Activation of these genes had an effect on an early stage in viral infection. In addition, infected cells expressing sgRNAs for both of these genes displayed lower levels of cell death compared to controls. Furthermore, the identified genes were significantly induced in ZIKV infected placenta explants. Thus, these results highlight a set of ISGs directly relevant for rescuing cells from ZIKV infection or its associated cell death and substantiates CRISPR activation screens as a tool to identify host factors impeding pathogen infection.

 

IMPORTANCE

Zika virus (ZIKV) is an emerging vector-borne pathogen causing a febrile disease. ZIKV infection might also trigger Guillain-Barré syndrome, neuropathy and myelitis. Vertical transmission of ZIKV can cause fetus demise, still birth or severe congenital abnormalities and neurological complications. There is no vaccine or specific antiviral treatment against ZIKV. We used a genome wide CRISPR activation screen, where genes are activated from their native promoters to identify host cell factors that protect cells from ZIKV infection or associated cell death. The results provide better understanding of key host factors that protect cells from ZIKV infection and might assist in identifying novel antiviral targets.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Zika Virus; CRISPR; Genetics.

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The #application of #CRISPR/Cas9-based genome editing in studying the #mechanism of #pandrug #resistance in #Klebsiella pneumoniae (Antimicrob Agents Chemother., abstract)

[Source: Antimicrobial Agents and Chemotherapy, full page: (LINK). Abstract, edited.]

The application of CRISPR/Cas9-based genome editing in studying the mechanism of pandrug resistance in Klebsiella pneumoniae

Qiaoling Sun, Yu Wang, Ning Dong, Lingwei Shen, Hongwei Zhou, Yan-yan Hu, Danxia Gu, Sheng Chen, Rong Zhang, Quanjiang Ji

DOI: 10.1128/AAC.00113-19

 

ABSTRACT

In this study, a CRISPR/Cas9-mediated genome editing method was used to study the functions of genes mgrB, tetA and ramR in mediating colistin and tigecycline resistance in carbapenem-resistant Klebsiella pneumoniae. Inactivation of the tetA, ramR, or mgrB genes by CRISPR/Cas9 affected bacterial susceptibility to tigecycline or colistin, respectively. This study proved that the CRISPR/Cas9-based genome editing method could be effectively applied to K. pneumoniae and should be further utilized for genetic characterization.

Copyright © 2019 American Society for Microbiology. All Rights Reserved.

Keywords: Antibiotics; Drugs Resistance; Colistin; Tigecycline; CRISPR.

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Efficient #genome engineering of a virulent #Klebsiella #bacteriophage using #CRISPR-Cas9 (J Virol., abstract)

[Source: Journal of Virology, full page: (LINK). Abstract, edited.]

Efficient genome engineering of a virulent Klebsiella bacteriophage using CRISPR-Cas9

Juntao Shen1,  Jinjie Zhou1, Guo-Qiang Chen2 and Zhi-Long Xiu1⇑

Author Affiliations: 1 School of Life Science and Biotechnology, Dalian University of Technology, Dalian, PR China; 2 School of Life Sciences, Tsinghua University, Beijing, PR China

 

ABSTRACT

Klebsiella pneumoniae is one of the most common nosocomial opportunistic pathogens usually with multiple drug-resistance. Phage therapy, a potential new therapeutics to replace or supplement antibiotics, has attracted much attention. However, very few Klebsiella phages have been well-characterized as the lack of efficient genome editing tools. Here, Cas9 from Streptococcus pyogenes and a single guide RNA (sgRNA) were used to modify a virulent Klebsiella bacteriophage phiKpS2. We firstly evaluated the distribution of sgRNA activity in phages and proved that it’s largely inconsistent with the predicted activity from current models trained on eukaryotic cell datasets. A simple CRISPR-based phage genome editing procedure was developed based on the discovery that homologous arms as short as 30-60 bp was sufficient to introduce point mutation, gene deletion and swap. We also demonstrated that weak sgRNAs could be used for precise phage genome editing but failed to select random recombinants, possibly because inefficient cleavage can be tolerated through continuous repair by homologous recombination with the uncut genomes. Small frameshift deletion was proved to be an efficient way to evaluate the essentiality of phage genes. By using the above strategies, a putative promoter and nine genes of phiKpS2 were successfully deleted. Interestingly, the holin gene can be deleted with little effect on phiKpS2 infection, but the reason is not yet clear. This study established an efficient, time-saving, and cost-effective procedure for phage genome editing, which is expected to significantly promote the development of bacteriophage therapy.

 

IMPORTANCE

In the present study, we have addressed an efficient, time-saving and cost-effective CRISPR-based phage genome editing of Klebsiella phage, which has the potential to significantly expand our knowledge of phage-host interactions and to promote the applications of phage therapy. The distribution of sgRNA activity was first evaluated in phages. Short homologous arms were proved enough to introduce point mutation, small frameshift deletion, gene deletion and swap into phages, and weak sgRNAs were proved useful for precise phage genome editing but failed to select random recombinants, which all make the CRISPR-based phage genome editing easier to use.

 

FOOTNOTES

Corresponding author: Prof. Dr. Zhi-Long Xiu, School of Life Science and Biotechnology, Dalian University of Technology, 2 Linggong Road, Dalian, 116024, PR China., E-mail: zhlxiu@dlut.edu.cn, Tel: +86-0411-84706369

Copyright © 2018 American Society for Microbiology. All Rights Reserved.

Keywords: Antibiotics; Drugs Resistance; Klebsiella pneumoniae; Bacteriophages; CRISPR-Cas9.

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