#Host-specific #restriction of #avian #influenza virus caused by differential dynamics of ANP32 family members (J Infect Dis., abstract)

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

Host-specific restriction of avian influenza virus caused by differential dynamics of ANP32 family members

Young Hyun Park, Kelly Chungu, Su Bin Lee, Seung Je Woo, Ho Yeon Cho, Hong Jo Lee, Deivendran Rengaraj, Ji-Ho Lee, Chang-Seon Song, Jeong Mook Lim, Jae Yong Han

The Journal of Infectious Diseases, jiz506, https://doi.org/10.1093/infdis/jiz506

Published: 03 October 2019

 

Abstract

Influenza viruses must utilize host factors to complete their lifecycle. Species-specific differences in host factors between birds and mammals mean that avian influenza viruses (AIVs) replicate well in avian hosts but not in human hosts. Acidic nuclear phosphoprotein 32 family member A (ANP32A) has been identified as the host restriction factor for the viral polymerase (vPol) activity of AIVs. ANP32A belongs to the conserved ANP32 family, the functional roles of which during viral replication remain unclear. Here, we targeted chicken ANP32A using CRISPR/Cas9-mediated genome editing to examine the functional roles of ANP32A and other members of the ANP32 family. We found that each chicken and human ANP32 family member had different effects on vPol activity, suggesting that species-specific vPol activity of AIVs could be caused by the differential functions and overall competency of ANP32 family members.

ANP32 family, cellular host factor, host restriction, influenza virus

Topic: aves – chickens – avian influenza – influenza a virus, avian – mammals – orthomyxoviridae – phosphoproteins – virus replication – viruses – host factor – crispr – genome editing

Issue Section: Major Article

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This article is published and distributed under the terms of the Oxford University Press, Standard Journals Publication Model (https://academic.oup.com/journals/pages/open_access/funder_policies/chorus/standard_publication_model)

Keywords: Influenza A; Genetics; CRISPR-Cas9; Animal models.

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Conjugative #delivery of #CRISPR-Cas9 for the selective #depletion of #antibiotic-resistant #enterococci (Antimicrob Agents Chemother., abstract)

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

Conjugative delivery of CRISPR-Cas9 for the selective depletion of antibiotic-resistant enterococci

Marinelle Rodrigues, Sara W. McBride, Karthik Hullahalli, Kelli L. Palmer, Breck A. Duerkop

DOI: 10.1128/AAC.01454-19

 

ABSTRACT

The innovation of new therapies to combat multidrug-resistant (MDR) bacteria is being outpaced by the continued rise of MDR bacterial infections. Of particular concern are hospital-acquired infections (HAIs) recalcitrant to antibiotic therapies. The Gram-positive intestinal pathobiont Enterococcus faecalis is associated with HAIs and some strains are MDR. Therefore, novel strategies to control E. faecalis populations are needed. We previously characterized an E. faecalis Type II CRISPR-Cas system and demonstrated its utility in the sequence-specific removal of antibiotic resistance determinants. Here we present work describing the adaption of this CRISPR-Cas system into a constitutively expressed module encoded on a pheromone-responsive conjugative plasmid that efficiently transfers to E. faecalis for the selective removal of antibiotic resistance genes. Using in vitro competition assays, we show that these CRISPR-Cas-encoding delivery plasmids, or CRISPR-Cas antimicrobials, can reduce the occurrence of antibiotic resistance in enterococcal populations in a sequence-specific manner. Furthermore, we demonstrate that deployment of CRISPR-Cas antimicrobials in the murine intestine reduces the occurrence of antibiotic-resistant E. faecalis by several orders of magnitude. Finally, we show that E. faecalis donor strains harboring CRISPR-Cas antimicrobials are immune to uptake of antibiotic resistance determinants in vivo. Our results demonstrate that conjugative delivery of CRISPR-Cas antimicrobials may be adaptable for future deployment from probiotic bacteria for exact targeting of defined MDR bacteria or for precision engineering of polymicrobial communities in the mammalian intestine.

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

Keywords: Antibiotics; Drugs Resistance; Enterococcus faecalis; CRISPR; Animal models.

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#CRISPR-Edited #StemCells in a #Patient with #HIV and Acute Lymphocytic #Leukemia (N Engl J Med., abstract)

[Source: The New England Journal of Medicine, full page: (LINK). Summary, edited.]

CRISPR-Edited Stem Cells in a Patient with HIV and Acute Lymphocytic Leukemia

Lei Xu, M.D., Ph.D., Jun Wang, M.D., Ph.D., Yulin Liu, B.S., Liangfu Xie, B.S., Bin Su, Ph.D., Danlei Mou, M.D., Ph.D., Longteng Wang, B.S., Tingting Liu, M.D., Xiaobao Wang, B.S., Bin Zhang, M.D., Ph.D., Long Zhao, Ph.D., Liangding Hu, M.D., et al.

 

Summary

The safety of CRISPR (clustered regularly interspaced short palindromic repeats)–based genome editing in the context of human gene therapy is largely unknown. CCR5 is a reasonable but not absolutely protective target for a cure of human immunodeficiency virus type 1 (HIV-1) infection, because CCR5-null blood cells are largely resistant to HIV-1 entry. We transplanted CRISPR-edited CCR5-ablated hematopoietic stem and progenitor cells (HSPCs) into a patient with HIV-1 infection and acute lymphoblastic leukemia. The acute lymphoblastic leukemia was in complete remission with full donor chimerism, and donor cells carrying the ablated CCR5 persisted for more than 19 months without gene editing–related adverse events. The percentage of CD4+ cells with CCR5 ablation increased by a small degree during a period of antiretroviral-therapy interruption. Although we achieved successful transplantation and long-term engraftment of CRISPR-edited HSPCs, the percentage of CCR5 disruption in lymphocytes was only approximately 5%, which indicates the need for further research into this approach. (Funded by the Beijing Municipal Science and Technology Commission and others; ClinicalTrials.gov number, NCT03164135.)

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Supported by grants from the Beijing Municipal Science and Technology Commission (D171100000517004), the National Key Research and Development Program of China (2017YFA0103000), the Science and Technology Planning Project of Beijing City  (Z181100001818004), and the National 13th Five-Year Grand Program on Key Infectious Disease Control (2017ZX10202102-005-003 and 2017ZX10202101-004-001).

Disclosure forms provided by the authors are available with the full text of this article at NEJM.org.

Dr. L. Xu, Dr. J. Wang, Mr. Y. Liu, and Mr. L. Xie and Drs. H. Wu, H. Deng, and H. Chen contributed equally to this article.

This article was published on September 11, 2019, at NEJM.org.

A data sharing statement provided by the authors is available with the full text of this article at NEJM.org.

We thank Zeyu Chen, Shicheng Sun, Chengyan Wang, Weifeng Lai, and Shunyi Liao for assistance with the experiment design and with revising an earlier version of the manuscript; Edouard Stanley and Tung-Tien Sun for assistance with revising an earlier version of the manuscript; Yang Gao, Yang Liu, Huangfan Xie, Hanwei Li, Jingyi Wei, Kailong Xia, Cui Zhang, Qiang Shi, Guoliang Hu, Xiaoyue Gao, and Xuanling Shi for technical assistance; and Jing Zhang, Min Jiang, Yongfeng Su, Na Liu, Xiao Lou, Yizhi Wang, Ying Sun, and Yafei Wang, of the 307 Hospital of the People’s Liberation Army, for contributions to the care of the patient.

Author Affiliations

From the Department of Hematopoietic Stem Cell Transplantation (L. Xu, J.W., T.L., B.Z., L.H., H.N., Y.Z., H.C.) and the Cell and Gene Therapy Center (B.Z., L.Z., L.H., H.C.), 307 Hospital of the People’s Liberation Army, the Fifth Medical Center of the People’s Liberation Army General Hospital, the Department of Cell Biology, School of Basic Medical Sciences, Peking University Stem Cell Research Center, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, and the Ministry of Education (MOE) Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking–Tsinghua Center for Life Sciences (Y.L., L. Xie, X.W., J.X., H.D.), and the School of Life Sciences, Center for Statistical Science and Center for Bioinformatics (L.W., C.L.), Peking University, and the Center for Infectious Diseases, Beijing Youan Hospital, Capital Medical University, Beijing Key Laboratory for HIV–AIDS Research (B.S., D.M., L.L., X.L., T.Z., H.W.), Beijing, and the Institute of Human Virology, Zhongshan School of Medicine, Sun Yat-sen University, Guangzhou (K.D.) — all in China.
Address reprint requests to Dr. H. Deng at the Department of Cell Biology, School of Basic Medical Sciences, Peking University Stem Cell Research Center, State Key Laboratory of Natural and Biomimetic Drugs, Peking University Health Science Center, and the MOE Key Laboratory of Cell Proliferation and Differentiation, College of Life Sciences, Peking–Tsinghua Center for Life Sciences, Peking University, No. 5, Yiheyuan Rd., Beijing 100871, China, or at hongkui_deng@pku.edu.cn.

Hu Chen, M.D., Ph.D., is deceased.

Keywords: HIV/AIDS; Leukemia; Crispr.

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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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