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Viruses
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Applications of CRISPR Technology in Virology 2018
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Applications of CRISPR Technology in Virology 2018

A special issue of Viruses (ISSN 1999-4915).

Deadline for manuscript submissions: closed (31 October 2018) | Viewed by 120282

Special Issue Editor

Prof. Dr. Dong-Yan Jin

grade E-Mail Website
Guest Editor
School of Biomedical Sciences, The University of Hong Kong, Pokfulam 999077, Hong Kong, China
Interests: molecular virology and oncology
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Precision genome engineering by CRISPR is a game-changing technology that promises to revolutionize virology and the treatment of viral diseases. CRISPR-mediated knock-in and knock-out of viral and cellular genes have been most revealing in the study of viral pathogenesis. CRISPR-based genome-wide screens, viral delivery systems for CRISPR and other CRISPR-based enabling tools including those in live cell imaging, viral disease modeling and treatment have also come of age. The applications of CRISPR technology in virology are far-reaching. The CRISPR revolution will reach beyond the research lab. In this Special Issue of Viruses we look to assemble a timely collection of research papers and reviews focusing on applications of CRISPR technology in virology. It is hoped that it will provide an update on this emerging field and serve as a reference and guide to virologists who are and will be using CRISPR in their research. The highlights will be on cutting-edge CRISPR technologies and their applications in various fields of virology such as viral reverse genetics; the study of viral entry, viral-host interaction, viral pathogenesis and cellular response to viral infection; viral immunology; as well as design and development of vaccines and antivirals. Topics of special interest may include viral delivery systems for CRISPR; functional screening for host restriction and dependency factors using CRISPR; antiviral CRISPR; CRISPR for viral disease modeling and target discovery; CRISPR in virus-infected cells and model organisms; as well as safety issues, such as off-target CRISPR editing in virus-infected cells.

Prof. Dong-Yan Jin
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Viruses is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2600 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • Precision genome editing
  • CRISPR/Cas9 system
  • CRISPR screen
  • Reverse genetics
  • Virus-host interaction
  • Host restriction factors
  • Host dependency factors
  • Viral vectors; Vaccines
  • Antiviral agents

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Published Papers (15 papers)

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Editorial

Jump to: Research, Review

3 pages, 142 KiB  
Editorial
Special Issue: Applications of CRISPR Technology in Virology 2018
by Dong-Yan Jin
Viruses 2019, 11(9), 839; https://doi.org/10.3390/v11090839 - 10 Sep 2019
Cited by 1 | Viewed by 3183
Abstract
Precision genome engineering by CRISPR is a game-changing technology that originates from the study of virus–host interaction and promises to revolutionize virology and antiviral therapy [...] Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)

Research

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14 pages, 1544 KiB  
Article
The Impact of HIV-1 Genetic Diversity on CRISPR-Cas9 Antiviral Activity and Viral Escape
by Gilles Darcis, Caroline S. Binda, Bep Klaver, Elena Herrera-Carrillo, Ben Berkhout and Atze T. Das
Viruses 2019, 11(3), 255; https://doi.org/10.3390/v11030255 - 13 Mar 2019
Cited by 30 | Viewed by 4638
Abstract
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA (dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs [...] Read more.
The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas9 system is widely explored for sequence-specific attack on HIV-1 proviral DNA. We recently identified dual-guide RNA (dual-gRNA) combinations that can block HIV-1 replication permanently in infected cell cultures and prevent viral escape. Although the gRNAs were designed to target highly conserved viral sequences, their efficacy may be challenged by high genetic variation in the HIV-1 genome. We therefore evaluated the breadth of these dual-gRNA combinations against distinct HIV-1 isolates, including several subtypes. Replication of nearly all virus isolates could be prevented by at least one gRNA combination, which caused inactivation of the proviral genomes and the gradual loss of replication-competent virus over time. The dual-gRNA efficacy was not affected by most single nucleotide (nt) mismatches between gRNA and the viral target. However, 1-nt mismatches at the Cas9 cleavage site and two mismatches anywhere in the viral target sequence significantly reduced the inhibitory effect. Accordingly, sequence analysis of viruses upon breakthrough replication revealed the acquisition of escape mutations in perfectly matching and most 1-nt mismatching targets, but not in targets with a mismatch at the Cas9 cleavage site or with two mismatches. These results demonstrate that combinatorial CRISPR-Cas9 treatment can cure T cells infected by distinct HIV-1 isolates, but even minor sequence variation in conserved viral target sites can affect the efficacy of this strategy. Successful cure attempts against isolates with divergent target sequences may therefore require adaptation of the gRNAs. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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9 pages, 1435 KiB  
Article
Engineering RNA Virus Interference via the CRISPR/Cas13 Machinery in Arabidopsis
by Rashid Aman, Ahmed Mahas, Haroon Butt, Zahir Ali, Fatimah Aljedaani and Magdy Mahfouz
Viruses 2018, 10(12), 732; https://doi.org/10.3390/v10120732 - 19 Dec 2018
Cited by 74 | Viewed by 8609
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome [...] Read more.
Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are key immune mechanisms helping prokaryotic species fend off RNA and DNA viruses. CRISPR/Cas9 has broad applications in basic research and biotechnology and has been widely used across eukaryotic species for genome engineering and functional analysis of genes. The recently developed CRISPR/Cas13 systems target RNA rather than DNA and thus offer new potential for transcriptome engineering and combatting RNA viruses. Here, we used CRISPR/LshCas13a to stably engineer Arabidopsis thaliana for interference against the RNA genome of Turnip mosaic virus (TuMV). Our data demonstrate that CRISPR RNAs (crRNAs) guiding Cas13a to the sequences encoding helper component proteinase silencing suppressor (HC-Pro) or GFP target 2 (GFP-T2) provide better interference compared to crRNAs targeting other regions of the TuMV RNA genome. This work demonstrates the exciting potential of CRISPR/Cas13 to be used as an antiviral strategy to obstruct RNA viruses, and encourages the search for more robust and effective Cas13 variants or CRISPR systems that can target RNA. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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17 pages, 4148 KiB  
Article
Anti-CRISPR-Based and CRISPR-Based Genome Editing of Sulfolobus islandicus Rod-Shaped Virus 2
by David Mayo-Muñoz, Fei He, Jacob Bruun Jørgensen, Poul Kári Madsen, Yuvaraj Bhoobalan-Chitty and Xu Peng
Viruses 2018, 10(12), 695; https://doi.org/10.3390/v10120695 - 08 Dec 2018
Cited by 20 | Viewed by 6409
Abstract
Genetic engineering of viruses has generally been challenging. This is also true for archaeal rod-shaped viruses, which carry linear double-stranded DNA genomes with hairpin ends. In this paper, we describe two different genome editing approaches to mutate the Sulfolobus islandicus rod-shaped virus 2 [...] Read more.
Genetic engineering of viruses has generally been challenging. This is also true for archaeal rod-shaped viruses, which carry linear double-stranded DNA genomes with hairpin ends. In this paper, we describe two different genome editing approaches to mutate the Sulfolobus islandicus rod-shaped virus 2 (SIRV2) using the archaeon Sulfolobus islandicus LAL14/1 and its derivatives as hosts. The anti-CRISPR (Acr) gene acrID1, which inhibits CRISPR-Cas subtype I-D immunity, was first used as a selection marker to knock out genes from SIRV2M, an acrID1-null mutant of SIRV2. Moreover, we harnessed the endogenous CRISPR-Cas systems of the host to knock out the accessory genes consecutively, which resulted in a genome comprised solely of core genes of the 11 SIRV members. Furthermore, infection of this series of knockout mutants in the CRISPR-null host of LAL14/1 (Δarrays) confirmed the non-essentiality of the deleted genes and all except the last deletion mutant propagated as efficiently as the WT SIRV2. This suggested that the last gene deleted, SIRV2 gp37, is important for the efficient viral propagation. The generated viral mutants will be useful for future functional studies including searching for new Acrs and the approaches described in this case are applicable to other viruses. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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11 pages, 3613 KiB  
Article
CRISPRStudio: A User-Friendly Software for Rapid CRISPR Array Visualization
by Moïra B. Dion, Simon J. Labrie, Shiraz A. Shah and Sylvain Moineau
Viruses 2018, 10(11), 602; https://doi.org/10.3390/v10110602 - 01 Nov 2018
Cited by 31 | Viewed by 5755
Abstract
The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain [...] Read more.
The CRISPR-Cas system biologically serves as an adaptive defense mechanism against phages. However, there is growing interest in exploiting the hypervariable nature of the CRISPR locus, often of viral origin, for microbial typing and tracking. Moreover, the spacer content of any given strain provides a phage resistance profile. Large-scale CRISPR typing studies require an efficient method for showcasing CRISPR array similarities across multiple isolates. Historically, CRISPR arrays found in microbes have been represented by colored shapes based on nucleotide sequence identity and, while this approach is now routinely used, only scarce computational resources are available to automate the process, making it very time-consuming for large datasets. To alleviate this tedious task, we introduce CRISPRStudio, a command-line tool developed to accelerate CRISPR analysis and standardize the preparation of CRISPR array figures. It first compares nucleotide spacer sequences present in a dataset and then clusters them based on sequence similarity to assign a meaningful representative color. CRISPRStudio offers versatility to suit different biological contexts by including options such as automatic sorting of CRISPR loci and highlighting of shared spacers, while remaining fast and user-friendly. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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10 pages, 4167 KiB  
Article
CRISPR/Cas9 Mutagenesis of UL21 in Multiple Strains of Herpes Simplex Virus Reveals Differential Requirements for pUL21 in Viral Replication
by Renée L. Finnen and Bruce W. Banfield
Viruses 2018, 10(5), 258; https://doi.org/10.3390/v10050258 - 15 May 2018
Cited by 21 | Viewed by 4530
Abstract
Studies from multiple laboratories using different strains or species of herpes simplex virus (HSV) with deletions in UL21 have yielded conflicting results regarding the necessity of pUL21 in HSV infection. To resolve this discrepancy, we utilized CRISPR/Cas9 mutagenesis to isolate pUL21 deficient viruses [...] Read more.
Studies from multiple laboratories using different strains or species of herpes simplex virus (HSV) with deletions in UL21 have yielded conflicting results regarding the necessity of pUL21 in HSV infection. To resolve this discrepancy, we utilized CRISPR/Cas9 mutagenesis to isolate pUL21 deficient viruses in multiple HSV backgrounds, and performed a side-by-side comparison of the cell-to-cell spread and replication phenotypes of these viruses. These analyses confirmed previous studies implicating the involvement of pUL21 in cell-to-cell spread of HSV. Cell-to-cell spread of HSV-2 was more greatly affected by the lack of pUL21 than HSV-1, and strain-specific differences in the requirement for pUL21 in cell-to-cell spread were also noted. HSV-2 strain 186 lacking pUL21 was particularly crippled in both cell-to-cell spread and viral replication in non-complementing cells, in comparison to other HSV strains lacking pUL21, suggesting that the strict requirement for pUL21 by strain 186 may not be representative of the HSV-2 species as a whole. This work highlights CRISPR/Cas9 technology as a useful tool for rapidly constructing deletion mutants of alphaherpesviruses, regardless of background strain, and should find great utility whenever strain-specific differences need to be investigated. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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16 pages, 3755 KiB  
Article
Characterization of an N-Terminal Non-Core Domain of RAG1 Gene Disrupted Syrian Hamster Model Generated by CRISPR Cas9
by Jinxin Miao, Baoling Ying, Rong Li, Ann E. Tollefson, Jacqueline F. Spencer, William S. M. Wold, Seok-Hwan Song, Il-Keun Kong, Karoly Toth, Yaohe Wang and Zhongde Wang
Viruses 2018, 10(5), 243; https://doi.org/10.3390/v10050243 - 06 May 2018
Cited by 24 | Viewed by 5404
Abstract
The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion [...] Read more.
The accumulating evidence demonstrates that Syrian hamsters have advantages as models for various diseases. To develop a Syrian hamster (Mesocricetus auratus) model of human immunodeficiency caused by RAG1 gene mutations, we employed the CRISPR/Cas9 system and introduced an 86-nucleotide frameshift deletion in the hamster RAG1 gene encoding part of the N-terminal non-core domain of RAG1. Histological and immunohistochemical analyses demonstrated that these hamsters (referred herein as RAG1-86nt hamsters) had atrophic spleen and thymus, and developed significantly less white pulp and were almost completely devoid of splenic lymphoid follicles. The RAG1-nt86 hamsters had barely detectable CD3+ and CD4+ T cells. The expression of B and T lymphocyte-specific genes (CD3γ and CD4 for T cell-specific) and (CD22 and FCMR for B cell-specific) was dramatically reduced, whereas the expression of macrophage-specific (CD68) and natural killer (NK) cell-specific (CD94 and KLRG1) marker genes was increased in the spleen of RAG1-nt86 hamsters compared to wildtype hamsters. Interestingly, despite the impaired development of B and T lymphocytes, the RAG1-86nt hamsters still developed neutralizing antibodies against human adenovirus type C6 (HAdV-C6) upon intranasal infection and were capable of clearing the infectious viruses, albeit with slower kinetics. Therefore, the RAG1-86nt hamster reported herein (similar to the hypomorphic RAG1 mutations in humans that cause Omenn syndrome), may provide a useful model for studying the pathogenesis of the specific RAG1-mutation-induced human immunodeficiency, the host immune response to adenovirus infection and other pathogens as well as for evaluation of cell and gene therapies for treatment of this subset of RAG1 mutation patients. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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Review

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17 pages, 637 KiB  
Review
Modeling Host-Virus Interactions in Viral Infectious Diseases Using Stem-Cell-Derived Systems and CRISPR/Cas9 Technology
by Jihoon Kim, Bon-Kyoung Koo and Ki-Jun Yoon
Viruses 2019, 11(2), 124; https://doi.org/10.3390/v11020124 - 30 Jan 2019
Cited by 18 | Viewed by 7080
Abstract
Pathologies induced by viral infections have undergone extensive study, with traditional model systems such as two-dimensional (2D) cell cultures and in vivo mouse models contributing greatly to our understanding of host-virus interactions. However, the technical limitations inherent in these systems have constrained efforts [...] Read more.
Pathologies induced by viral infections have undergone extensive study, with traditional model systems such as two-dimensional (2D) cell cultures and in vivo mouse models contributing greatly to our understanding of host-virus interactions. However, the technical limitations inherent in these systems have constrained efforts to more fully understand such interactions, leading to a search for alternative in vitro systems that accurately recreate in vivo physiology in order to advance the study of viral pathogenesis. Over the last decade, there have been significant technological advances that have allowed researchers to more accurately model the host environment when modeling viral pathogenesis in vitro, including induced pluripotent stem cells (iPSCs), adult stem-cell-derived organoid culture systems and CRISPR/Cas9-mediated genome editing. Such technological breakthroughs have ushered in a new era in the field of viral pathogenesis, where previously challenging questions have begun to be tackled. These include genome-wide analysis of host-virus crosstalk, identification of host factors critical for viral pathogenesis, and the study of viral pathogens that previously lacked a suitable platform, e.g., noroviruses, rotaviruses, enteroviruses, adenoviruses, and Zika virus. In this review, we will discuss recent advances in the study of viral pathogenesis and host-virus crosstalk arising from the use of iPSC, organoid, and CRISPR/Cas9 technologies. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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12 pages, 219 KiB  
Review
Viral Delivery Systems for CRISPR
by Christine L. Xu, Merry Z. C. Ruan, Vinit B. Mahajan and Stephen H. Tsang
Viruses 2019, 11(1), 28; https://doi.org/10.3390/v11010028 - 04 Jan 2019
Cited by 164 | Viewed by 10868
Abstract
The frontiers of precision medicine have been revolutionized by the development of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 as an editing tool. CRISPR/Cas9 has been used to develop animal models, understand disease mechanisms, and validate treatment targets. In addition, it is regarded as [...] Read more.
The frontiers of precision medicine have been revolutionized by the development of Clustered Regularly-Interspaced Short Palindromic Repeats (CRISPR)/Cas9 as an editing tool. CRISPR/Cas9 has been used to develop animal models, understand disease mechanisms, and validate treatment targets. In addition, it is regarded as an effective tool for genome surgery when combined with viral delivery vectors. In this article, we will explore the various viral mechanisms for delivering CRISPR/Cas9 into tissues and cells, as well as the benefits and drawbacks of each method. We will also review the history and recent development of CRISPR and viral vectors and discuss their applications as a powerful tool in furthering our exploration of disease mechanisms and therapies. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
11 pages, 790 KiB  
Review
Phage Genetic Engineering Using CRISPR–Cas Systems
by Asma Hatoum-Aslan
Viruses 2018, 10(6), 335; https://doi.org/10.3390/v10060335 - 19 Jun 2018
Cited by 50 | Viewed by 9774
Abstract
Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR–Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural [...] Read more.
Since their discovery over a decade ago, the class of prokaryotic immune systems known as CRISPR–Cas have afforded a suite of genetic tools that have revolutionized research in model organisms spanning all domains of life. CRISPR-mediated tools have also emerged for the natural targets of CRISPR–Cas immunity, the viruses that specifically infect bacteria, or phages. Despite their status as the most abundant biological entities on the planet, the majority of phage genes have unassigned functions. This reality underscores the need for robust genetic tools to study them. Recent reports have demonstrated that CRISPR–Cas systems, specifically the three major types (I, II, and III), can be harnessed to genetically engineer phages that infect diverse hosts. Here, the mechanisms of each of these systems, specific strategies used, and phage editing efficacies will be reviewed. Due to the relatively wide distribution of CRISPR–Cas systems across bacteria and archaea, it is anticipated that these immune systems will provide generally applicable tools that will advance the mechanistic understanding of prokaryotic viruses and accelerate the development of novel technologies based on these ubiquitous organisms. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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12 pages, 570 KiB  
Review
Potential Application of the CRISPR/Cas9 System against Herpesvirus Infections
by Yuan-Chuan Chen, Jingxue Sheng, Phong Trang and Fenyong Liu
Viruses 2018, 10(6), 291; https://doi.org/10.3390/v10060291 - 29 May 2018
Cited by 55 | Viewed by 9401
Abstract
The CRISPR/Cas9 system has been applied in the genome editing and disruption of latent infections for herpesviruses such as the herpes simplex virus, Epstein–Barr virus, cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus. CRISPR/Cas9-directed mutagenesis can introduce similar types of mutations to the viral genome as [...] Read more.
The CRISPR/Cas9 system has been applied in the genome editing and disruption of latent infections for herpesviruses such as the herpes simplex virus, Epstein–Barr virus, cytomegalovirus, and Kaposi’s sarcoma-associated herpesvirus. CRISPR/Cas9-directed mutagenesis can introduce similar types of mutations to the viral genome as can bacterial artificial chromosome recombination engineering, which maintains and reconstitutes the viral genome successfully. The cleavage mediated by CRISPR/Cas9 enables the manipulation of disease-associated viral strains with unprecedented efficiency and precision. Additionally, current therapies for herpesvirus productive and latent infections are limited in efficacy and cannot eradicate viruses. CRISPR/Cas9 is potentially adapted for antiviral treatment by specifically targeting viral genomes during latent infections. This review, which focuses on recently published progress, suggests that the CRISPR/Cas9 system is not only a useful tool for basic virology research, but also a promising strategy for the control and prevention of herpesvirus latent infections. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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13 pages, 5961 KiB  
Review
Rapid CRISPR/Cas9-Mediated Cloning of Full-Length Epstein-Barr Virus Genomes from Latently Infected Cells
by Misako Yajima, Kazufumi Ikuta and Teru Kanda
Viruses 2018, 10(4), 171; https://doi.org/10.3390/v10040171 - 03 Apr 2018
Cited by 7 | Viewed by 8218
Abstract
Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of [...] Read more.
Herpesviruses have relatively large DNA genomes of more than 150 kb that are difficult to clone and sequence. Bacterial artificial chromosome (BAC) cloning of herpesvirus genomes is a powerful technique that greatly facilitates whole viral genome sequencing as well as functional characterization of reconstituted viruses. We describe recently invented technologies for rapid BAC cloning of herpesvirus genomes using CRISPR/Cas9-mediated homology-directed repair. We focus on recent BAC cloning techniques of Epstein-Barr virus (EBV) genomes and discuss the possible advantages of a CRISPR/Cas9-mediated strategy comparatively with precedent EBV-BAC cloning strategies. We also describe the design decisions of this technology as well as possible pitfalls and points to be improved in the future. The obtained EBV-BAC clones are subjected to long-read sequencing analysis to determine complete EBV genome sequence including repetitive regions. Rapid cloning and sequence determination of various EBV strains will greatly contribute to the understanding of their global geographical distribution. This technology can also be used to clone disease-associated EBV strains and test the hypothesis that they have special features that distinguish them from strains that infect asymptomatically. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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Graphical abstract

22 pages, 1845 KiB  
Review
CRISPR–Cas9 Genetic Analysis of Virus–Host Interactions
by Makda Gebre, Jason L. Nomburg and Benjamin E. Gewurz
Viruses 2018, 10(2), 55; https://doi.org/10.3390/v10020055 - 30 Jan 2018
Cited by 19 | Viewed by 12689
Abstract
Clustered regularly interspaced short palindromic repeats (CRISPR) has greatly expanded the ability to genetically probe virus–host interactions. CRISPR systems enable focused or systematic, genomewide studies of nearly all aspects of a virus lifecycle. Combined with its relative ease of use and high reproducibility, [...] Read more.
Clustered regularly interspaced short palindromic repeats (CRISPR) has greatly expanded the ability to genetically probe virus–host interactions. CRISPR systems enable focused or systematic, genomewide studies of nearly all aspects of a virus lifecycle. Combined with its relative ease of use and high reproducibility, CRISPR is becoming an essential tool in studies of the host factors important for viral pathogenesis. Here, we review the use of CRISPR–Cas9 for the loss-of-function analysis of host dependency factors. We focus on the use of CRISPR-pooled screens for the systematic identification of host dependency factors, particularly in Epstein–Barr virus-transformed B cells. We also discuss the use of CRISPR interference (CRISPRi) and gain-of-function CRISPR activation (CRISPRa) approaches to probe virus–host interactions. Finally, we comment on the future directions enabled by combinatorial CRISPR screens. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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27 pages, 2127 KiB  
Review
CRISPR/Cas9—Advancing Orthopoxvirus Genome Editing for Vaccine and Vector Development
by Arinze Okoli, Malachy I. Okeke, Morten Tryland and Ugo Moens
Viruses 2018, 10(1), 50; https://doi.org/10.3390/v10010050 - 22 Jan 2018
Cited by 25 | Viewed by 11788
Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription [...] Read more.
The clustered regularly interspaced short palindromic repeat (CRISPR)/associated protein 9 (Cas9) technology is revolutionizing genome editing approaches. Its high efficiency, specificity, versatility, flexibility, simplicity and low cost have made the CRISPR/Cas9 system preferable to other guided site-specific nuclease-based systems such as TALENs (Transcription Activator-like Effector Nucleases) and ZFNs (Zinc Finger Nucleases) in genome editing of viruses. CRISPR/Cas9 is presently being applied in constructing viral mutants, preventing virus infections, eradicating proviral DNA, and inhibiting viral replication in infected cells. The successful adaptation of CRISPR/Cas9 to editing the genome of Vaccinia virus paves the way for its application in editing other vaccine/vector-relevant orthopoxvirus (OPXV) strains. Thus, CRISPR/Cas9 can be used to resolve some of the major hindrances to the development of OPXV-based recombinant vaccines and vectors, including sub-optimal immunogenicity; transgene and genome instability; reversion of attenuation; potential of spread of transgenes to wildtype strains and close contacts, which are important biosafety and risk assessment considerations. In this article, we review the published literature on the application of CRISPR/Cas9 in virus genome editing and discuss the potentials of CRISPR/Cas9 in advancing OPXV-based recombinant vaccines and vectors. We also discuss the application of CRISPR/Cas9 in combating viruses of clinical relevance, the limitations of CRISPR/Cas9 and the current strategies to overcome them. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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Graphical abstract

28 pages, 714 KiB  
Review
CRISPR-Cas Targeting of Host Genes as an Antiviral Strategy
by Shuliang Chen, Xiao Yu and Deyin Guo
Viruses 2018, 10(1), 40; https://doi.org/10.3390/v10010040 - 16 Jan 2018
Cited by 32 | Viewed by 9830
Abstract
Currently, a new gene editing tool—the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) system—is becoming a promising approach for genetic manipulation at the genomic level. This simple method, originating from the adaptive immune defense system in prokaryotes, has been developed and [...] Read more.
Currently, a new gene editing tool—the Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) associated (Cas) system—is becoming a promising approach for genetic manipulation at the genomic level. This simple method, originating from the adaptive immune defense system in prokaryotes, has been developed and applied to antiviral research in humans. Based on the characteristics of virus-host interactions and the basic rules of nucleic acid cleavage or gene activation of the CRISPR-Cas system, it can be used to target both the virus genome and host factors to clear viral reservoirs and prohibit virus infection or replication. Here, we summarize recent progress of the CRISPR-Cas technology in editing host genes as an antiviral strategy. Full article
(This article belongs to the Special Issue Applications of CRISPR Technology in Virology 2018)
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