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Viruses
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CRISPR/Cas in Viral Research
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CRISPR/Cas in Viral Research

A special issue of Viruses (ISSN 1999-4915). This special issue belongs to the section "Viral Immunology, Vaccines, and Antivirals".

Deadline for manuscript submissions: closed (25 February 2022) | Viewed by 52484

Special Issue Editors

Dr. Jiri Hejnar

E-Mail Website
Guest Editor
Institute of Molecular Genetics, Czech Academy of Sciences, Prague, Czech Republic
Interests: avian sarcoma/leukosis viruses; epigenetic silencing of retroviruses; receptor for retroviruses; endogenous retroviruses; syncytins; retroviral vectors; retrotransposons; transgenesis in chicken
Special Issues, Collections and Topics in MDPI journals
Prof. Dr. Ben Berkhout

E-Mail Website
Guest Editor
Laboratory of Experimental Virology, Department of Medical Microbiology, University of Amsterdam, Meibergdreef 15, 1105 AZ Amsterdam, The Netherlands
Interests: virology; antiviral approaches; virus evolution; drug resistance
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

The recent breakthroughs in gene-editing methods using clustered regularly interspaced short palindromic repeats (CRISPR) combined with Cas9 nuclease and short guide RNAs (gRNA) have opened a new era in antiviral research. Briefly, just to mention a few relevant applications, both viral genomes and virus transcripts can become targets of CRISPR/Cas9-mediated cleavage and integrated virus copies can be precisely removed using CRISPR/Cas9 tools. Editing the genes of host dependency factors, e.g., receptors for virus entry, can provide invaluable models for virus research and drug screening or even virus-resistant livestock. CRISPR/Cas9 tools may help us to better understand HIV-1 latency and functional elimination of persistent reservoirs in HIV-infected patients. Last but not least, the urgent need for robust and rapid diagnostics could be met with CRISPR-based sensors of viral nucleic acids in the near future.

For this Special Issue, we invite submissions in the form of original research articles, validated methods, resource articles, or reviews that address any aspect of CRISPR-based strategies in basic viral research and innovative applications like diagnostics and antiviral therapies. This Special Issue will cover all kinds of viruses that infect plants, animals, or humans, and we especially welcome manuscripts relevant to the current SARS-CoV-2 outbreak. In summary, our aim is to present an inspiring collection of articles covering the vibrant field of CRISPR/Cas in viral research.

Dr. Jiri Hejnar
Prof. Dr. Ben Berkhout
Guest Editors

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

  • gene editing
  • CRISPR/Cas
  • viral genomes
  • viral diagnostics
  • antiviral drug screening

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Published Papers (14 papers)

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Research

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19 pages, 2461 KiB  
Article
In Silico Prediction and Selection of Target Sequences in the SARS-CoV-2 RNA Genome for an Antiviral Attack
by Mouraya Hussein, Zaria Andrade dos Ramos, Ben Berkhout and Elena Herrera-Carrillo
Viruses 2022, 14(2), 385; https://doi.org/10.3390/v14020385 - 14 Feb 2022
Cited by 3 | Viewed by 2869
Abstract
The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. [...] Read more.
The SARS-CoV-2 pandemic has urged the development of protective vaccines and the search for specific antiviral drugs. The modern molecular biology tools provides alternative methods, such as CRISPR-Cas and RNA interference, that can be adapted as antiviral approaches, and contribute to this search. The unique CRISPR-Cas13d system, with the small crRNA guide molecule, mediates a sequence-specific attack on RNA, and can be developed as an anti-coronavirus strategy. We analyzed the SARS-CoV-2 genome to localize the hypothetically best crRNA-annealing sites of 23 nucleotides based on our extensive expertise with sequence-specific antiviral strategies. We considered target sites of which the sequence is well-conserved among SARS-CoV-2 isolates. As we should prepare for a potential future outbreak of related viruses, we screened for targets that are conserved between SARS-CoV-2 and SARS-CoV. To further broaden the search, we screened for targets that are conserved between SARS-CoV-2 and the more distantly related MERS-CoV, as well as the four other human coronaviruses (OC43, 229E, NL63, HKU1). Finally, we performed a search for pan-corona target sequences that are conserved among all these coronaviruses, including the new Omicron variant, that are able to replicate in humans. This survey may contribute to the design of effective, safe, and escape-proof antiviral strategies to prepare for future pandemics. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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14 pages, 1768 KiB  
Article
Knock-Out of Retrovirus Receptor Gene Tva in the Chicken Confers Resistance to Avian Leukosis Virus Subgroups A and K and Affects Cobalamin (Vitamin B12)-Dependent Level of Methylmalonic Acid
by Anna Koslová, Pavel Trefil, Jitka Mucksová, Veronika Krchlíková, Jiří Plachý, Jakub Krijt, Markéta Reinišová, Dana Kučerová, Josef Geryk, Jiří Kalina, Filip Šenigl, Daniel Elleder, Viktor Kožich and Jiří Hejnar
Viruses 2021, 13(12), 2504; https://doi.org/10.3390/v13122504 - 14 Dec 2021
Cited by 13 | Viewed by 3063
Abstract
The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the [...] Read more.
The chicken Tva cell surface protein, a member of the low-density lipoprotein receptor family, has been identified as an entry receptor for avian leukosis virus of classic subgroup A and newly emerging subgroup K. Because both viruses represent an important concern for the poultry industry, we introduced a frame-shifting deletion into the chicken tva locus with the aim of knocking-out Tva expression and creating a virus-resistant chicken line. The tva knock-out was prepared by CRISPR/Cas9 gene editing in chicken primordial germ cells and orthotopic transplantation of edited cells into the testes of sterilized recipient roosters. The resulting tva −/− chickens tested fully resistant to avian leukosis virus subgroups A and K, both in in vitro and in vivo assays, in contrast to their susceptible tva +/+ and tva +/− siblings. We also found a specific disorder of the cobalamin/vitamin B12 metabolism in the tva knock-out chickens, which is in accordance with the recently recognized physiological function of Tva as a receptor for cobalamin in complex with transcobalamin transporter. Last but not least, we bring a new example of the de novo resistance created by CRISPR/Cas9 editing of pathogen dependence genes in farm animals and, furthermore, a new example of gene editing in chicken. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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13 pages, 1467 KiB  
Article
Transient CRISPR-Cas Treatment Can Prevent Reactivation of HIV-1 Replication in a Latently Infected T-Cell Line
by Ye Liu, Rienk E. Jeeninga, Bep Klaver, Ben Berkhout and Atze T. Das
Viruses 2021, 13(12), 2461; https://doi.org/10.3390/v13122461 - 08 Dec 2021
Cited by 7 | Viewed by 3092
Abstract
Novel therapeutic strategies aiming at the permanent inactivation of the HIV-1 reservoir in infected individuals are currently being explored, including approaches based on CRISPR-Cas gene editing. Extinction of all infectious HIV provirus in infected T-cell cultures was previously achieved when cells were transduced [...] Read more.
Novel therapeutic strategies aiming at the permanent inactivation of the HIV-1 reservoir in infected individuals are currently being explored, including approaches based on CRISPR-Cas gene editing. Extinction of all infectious HIV provirus in infected T-cell cultures was previously achieved when cells were transduced with lentiviral vectors for the stable expression of CRISPR-Cas9 or Cas12a systems targeting HIV DNA. Because lentiviral transduction and long-term CRISPR-Cas activity are less suitable for in vivo application of this antiviral strategy, we investigated whether HIV can also be completely inactivated by transient CRISPR-Cas activity. Latently infected SupT1 T-cells were repeatedly transfected with different Cas9 and Cas12a mRNA/protein sources in combination with dual gRNAs/crRNAs targeting highly conserved viral sequences. Upon repeated Cas9 protein treatment, viral replication could no longer be reactivated. We demonstrate that this was due to complete mutational inactivation of the proviral DNA, mostly through mutations at the target sites, but also through excision or inversion of the viral DNA fragment between the two target sites. These results demonstrate that repeated transient CRISPR-Cas treatment of a latently infected T-cell culture can lead to the permanent inactivation of HIV replication, indicating that transient CRISPR-Cas delivery methods can be considered for in vivo application. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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20 pages, 2326 KiB  
Article
A CRISPR Activation Screen Identifies an Atypical Rho GTPase That Enhances Zika Viral Entry
by Anh Phuong Luu, Zhenlan Yao, Sangeetha Ramachandran, Stephanie A. Azzopardi, Linde A. Miles, William M. Schneider, H.-Heinrich Hoffmann, Leonia Bozzacco, Gustavo Garcia, Jr., Danyang Gong, Robert Damoiseaux, Hengli Tang, Kouki Morizono, Charles M. Rudin, Ren Sun, Vaithilingaraja Arumugaswami, John T. Poirier, Margaret R. MacDonald, Charles M. Rice and Melody M. H. Li
Viruses 2021, 13(11), 2113; https://doi.org/10.3390/v13112113 - 20 Oct 2021
Cited by 8 | Viewed by 3811
Abstract
Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically [...] Read more.
Zika virus (ZIKV) is a re-emerging flavivirus that has caused large-scale epidemics. Infection during pregnancy can lead to neurologic developmental abnormalities in children. There is no approved vaccine or therapy for ZIKV. To uncover cellular pathways required for ZIKV that can be therapeutically targeted, we transcriptionally upregulated all known human coding genes with an engineered CRISPR–Cas9 activation complex in human fibroblasts deficient in interferon (IFN) signaling. We identified Ras homolog family member V (RhoV) and WW domain-containing transcription regulator 1 (WWTR1) as proviral factors, and found them to play important roles during early ZIKV infection in A549 cells. We then focused on RhoV, a Rho GTPase with atypical terminal sequences and membrane association, and validated its proviral effects on ZIKV infection and virion production in SNB-19 cells. We found that RhoV promotes infection of some flaviviruses and acts at the step of viral entry. Furthermore, RhoV proviral effects depend on the complete GTPase cycle. By depleting Rho GTPases and related proteins, we identified RhoB and Pak1 as additional proviral factors. Taken together, these results highlight the positive role of RhoV in ZIKV infection and confirm CRISPR activation as a relevant method to identify novel host–pathogen interactions. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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15 pages, 1205 KiB  
Article
Comparison of CRISPR–Cas9 Tools for Transcriptional Repression and Gene Disruption in the BEVS
by Mark R. Bruder, Sadru-Dean Walji and Marc G. Aucoin
Viruses 2021, 13(10), 1925; https://doi.org/10.3390/v13101925 - 24 Sep 2021
Cited by 7 | Viewed by 2414
Abstract
The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR–Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional [...] Read more.
The generation of knock-out viruses using recombineering of bacmids has greatly accelerated scrutiny of baculovirus genes for a variety of applications. However, the CRISPR–Cas9 system is a powerful tool that simplifies sequence-specific genome editing and effective transcriptional regulation of genes compared to traditional recombineering and RNAi approaches. Here, the effectiveness of the CRISPR–Cas9 system for gene disruption and transcriptional repression in the BEVS was compared. Cell lines constitutively expressing the cas9 or dcas9 gene were developed, and recombinant baculoviruses delivering the sgRNA were evaluated for disruption or repression of a reporter green fluorescent protein gene. Finally, endogenous AcMNPV genes were targeted for disruption or downregulation to affect gene expression and baculovirus replication. This study provides a proof-of-concept that CRISPR–Cas9 technology may be an effective tool for efficient scrutiny of baculovirus genes through targeted gene disruption and transcriptional repression. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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16 pages, 2271 KiB  
Article
Efficient Inhibition of HIV Using CRISPR/Cas13d Nuclease System
by Hoang Nguyen, Hannah Wilson, Sahana Jayakumar, Viraj Kulkarni and Smita Kulkarni
Viruses 2021, 13(9), 1850; https://doi.org/10.3390/v13091850 - 16 Sep 2021
Cited by 15 | Viewed by 4305
Abstract
Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13-mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit [...] Read more.
Recently discovered Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas13 proteins are programmable RNA-guided ribonucleases that target single-stranded RNA (ssRNA). CRISPR/Cas13-mediated RNA targeting has emerged as a powerful tool for detecting and eliminating RNA viruses. Here, we demonstrate the effectiveness of CRISPR/Cas13d to inhibit HIV-1 replication. We designed guide RNAs (gRNAs) targeting highly conserved regions of HIV-1. RfxCas13d (CasRx) in combination with HIV-specific gRNAs efficiently inhibited HIV-1 replication in cell line models. Furthermore, simultaneous targeting of four distinct, non-overlapping sites in the HIV-1 transcript resulted in robust inhibition of HIV-1 replication. We also show the effective HIV-1 inhibition in primary CD4+ T-cells and suppression of HIV-1 reactivated from latently infected cells using the CRISPR/Cas13d system. Our study demonstrates the utility of the CRISPR/Cas13d nuclease system to target acute and latent HIV infection and provides an alternative treatment modality against HIV. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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20 pages, 2594 KiB  
Article
A Genome-Wide CRISPR/Cas9 Screen Reveals the Requirement of Host Sphingomyelin Synthase 1 for Infection with Pseudorabies Virus Mutant gD–Pass
by Julia E. Hölper, Finn Grey, John Kenneth Baillie, Tim Regan, Nicholas J. Parkinson, Dirk Höper, Thiprampai Thamamongood, Martin Schwemmle, Katrin Pannhorst, Lisa Wendt, Thomas C. Mettenleiter and Barbara G. Klupp
Viruses 2021, 13(8), 1574; https://doi.org/10.3390/v13081574 - 09 Aug 2021
Cited by 9 | Viewed by 3722
Abstract
Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an [...] Read more.
Herpesviruses are large DNA viruses, which encode up to 300 different proteins including enzymes enabling efficient replication. Nevertheless, they depend on a multitude of host cell proteins for successful propagation. To uncover cellular host factors important for replication of pseudorabies virus (PrV), an alphaherpesvirus of swine, we performed an unbiased genome-wide CRISPR/Cas9 forward screen. To this end, a porcine CRISPR-knockout sgRNA library (SsCRISPRko.v1) targeting 20,598 genes was generated and used to transduce porcine kidney cells. Cells were then infected with either wildtype PrV (PrV-Ka) or a PrV mutant (PrV-gD–Pass) lacking the receptor-binding protein gD, which regained infectivity after serial passaging in cell culture. While no cells survived infection with PrV-Ka, resistant cell colonies were observed after infection with PrV-gD–Pass. In these cells, sphingomyelin synthase 1 (SMS1) was identified as the top hit candidate. Infection efficiency was reduced by up to 90% for PrV-gD–Pass in rabbit RK13-sgms1KO cells compared to wildtype cells accompanied by lower viral progeny titers. Exogenous expression of SMS1 partly reverted the entry defect of PrV-gD–Pass. In contrast, infectivity of PrV-Ka was reduced by 50% on the knockout cells, which could not be restored by exogenous expression of SMS1. These data suggest that SMS1 plays a pivotal role for PrV infection, when the gD-mediated entry pathway is blocked. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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22 pages, 2806 KiB  
Article
An MHV-68 Mutator Phenotype Mutant Virus, Confirmed by CRISPR/Cas9-Mediated Gene Editing of the Viral DNA Polymerase Gene, Shows Reduced Viral Fitness
by Erika Trompet, Arturo Temblador, Sarah Gillemot, Dimitrios Topalis, Robert Snoeck and Graciela Andrei
Viruses 2021, 13(6), 985; https://doi.org/10.3390/v13060985 - 26 May 2021
Cited by 1 | Viewed by 2954
Abstract
Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. [...] Read more.
Drug resistance studies on human γ-herpesviruses are hampered by the absence of an in vitro system that allows efficient lytic viral replication. Therefore, we employed murine γ-herpesvirus-68 (MHV-68) that efficiently replicates in vitro as a model to study the antiviral resistance of γ-herpesviruses. In this study, we investigated the mechanism of resistance to nucleoside (ganciclovir (GCV)), nucleotide (cidofovir (CDV), HPMP-5azaC, HPMPO-DAPy) and pyrophosphate (foscarnet (PFA)) analogues and the impact of these drug resistance mutations on viral fitness. Viral fitness was determined by dual infection competition assays, where MHV-68 drug-resistant viral clones competed with the wild-type virus in the absence and presence of antivirals. Using next-generation sequencing, the composition of the viral populations was determined at the time of infection and after 5 days of growth. Antiviral drug resistance selection resulted in clones harboring mutations in the viral DNA polymerase (DP), denoted Y383SGCV, Q827RHPMP-5azaC, G302WPFA, K442TPFA, G302W+K442TPFA, C297WHPMPO-DAPy and C981YCDV. Without antiviral pressure, viral clones Q827RHPMP-5azaC, G302WPFA, K442TPFA and G302W+K442TPFA grew equal to the wild-type virus. However, in the presence of antivirals, these mutants had a growth advantage over the wild-type virus that was moderately to very strongly correlated with antiviral resistance. The Y383SGCV mutant was more fit than the wild-type virus with and without antivirals, except in the presence of brivudin. The C297W and C981Y changes were associated with a mutator phenotype and had a severely impaired viral fitness in the absence and presence of antivirals. The mutator phenotype caused by C297W in MHV-68 DP was validated by using a CRISPR/Cas9 genome editing approach. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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14 pages, 2710 KiB  
Article
A Crucial Role of ACBD3 Required for Coxsackievirus Infection in Animal Model Developed by AAV-Mediated CRISPR Genome Editing Technique
by Hye Jin Shin, Keun Bon Ku, Soojin Kim, Heon Seok Kim, Yeon-Soo Kim, Bum-Tae Kim, Seong-Jun Kim and Chonsaeng Kim
Viruses 2021, 13(2), 237; https://doi.org/10.3390/v13020237 - 03 Feb 2021
Cited by 3 | Viewed by 2194
Abstract
Genetic screens using CRISPR/Cas9 have been exploited to discover host–virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for [...] Read more.
Genetic screens using CRISPR/Cas9 have been exploited to discover host–virus interactions. These screens have identified viral dependencies on host proteins during their life cycle and potential antiviral strategies. The acyl-CoA binding domain containing 3 (ACBD3) was identified as an essential host factor for the Coxsackievirus B3 (CVB3) infection. Other groups have also investigated the role of ACBD3 as a host factor for diverse enteroviruses in cultured cells. However, it has not been tested if ACBD3 is required in the animal model of CVB3 infection. Owing to embryonic lethality, conventional knockout mice were not available for in vivo study. As an alternative approach, we used adeno-associated virus (AAV)-mediated CRISPR genome editing to generate mice that lacked ACBD3 within the pancreas, the major target organ for CVB3. Delivery of sgRNAs using self-complementary (sc) AAV8 efficiently induced a loss-of-function mutation in the pancreas of the Cas9 knock-in mice. Loss of ACBD3 in the pancreas resulted in a 100-fold reduction in the CVB3 titer within the pancreas and a noticeable reduction in viral protein expression. These results indicate a crucial function of ACBD3 in CVB3 infection in vivo. AAV-mediated CRISPR genome editing may be applicable to many in vivo studies on the virus–host interaction and identify a novel target for antiviral therapeutics. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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Review

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14 pages, 503 KiB  
Review
Application of the CRISPR/Cas9 System to Study Regulation Pathways of the Cellular Immune Response to Influenza Virus
by Daria Prokhorova, Natalya Zhukova (Eschenko), Anna Lemza, Mariia Sergeeva, Rinat Amirkhanov and Grigory Stepanov
Viruses 2022, 14(2), 437; https://doi.org/10.3390/v14020437 - 21 Feb 2022
Cited by 3 | Viewed by 3270
Abstract
Influenza A virus (IAV) causes a respiratory infection that affects millions of people of different age groups and can lead to acute respiratory distress syndrome. Currently, host genes, receptors, and other cellular components critical for IAV replication are actively studied. One of the [...] Read more.
Influenza A virus (IAV) causes a respiratory infection that affects millions of people of different age groups and can lead to acute respiratory distress syndrome. Currently, host genes, receptors, and other cellular components critical for IAV replication are actively studied. One of the most convenient and accessible genome-editing tools to facilitate these studies is the CRISPR/Cas9 system. This tool allows for regulating the expression of both viral and host cell genes to enhance or impair viral entry and replication. This review considers the effect of the genome editing system on specific target genes in cells (human and chicken) in terms of subsequent changes in the influenza virus life cycle and the efficiency of virus particle production. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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15 pages, 1478 KiB  
Review
Recent Progress and Future Prospective in HBV Cure by CRISPR/Cas
by Yu-Chan Yang and Hung-Chih Yang
Viruses 2022, 14(1), 4; https://doi.org/10.3390/v14010004 - 21 Dec 2021
Cited by 18 | Viewed by 4637
Abstract
Hepatitis B virus (HBV) infection remains an important issue of global public health. Although current antiviral therapy has dramatically reduced the mortality and morbidity of chronic hepatitis B (CHB), it fails to cure it. Rebound viremia often occurs after stopping antiviral therapy. Persistent [...] Read more.
Hepatitis B virus (HBV) infection remains an important issue of global public health. Although current antiviral therapy has dramatically reduced the mortality and morbidity of chronic hepatitis B (CHB), it fails to cure it. Rebound viremia often occurs after stopping antiviral therapy. Persistent HBV covalently closed circular DNA (cccDNA) and integrated DNA under antiviral therapy form the major barrier to eradication of HBV infection. CRISPR-mediated genome editing has emerged as a promising therapeutic approach to specifically destroy persistent HBV genomes, both cccDNA and integrated DNA, for HBV cure. However, the cleavage of integrated HBV DNA by CRISPR-Cas9 will cause double-strand break (DSB) of host genome, raising a serious safety concern about genome instability and carcinogenesis. The newly developed CRISPR-derived base editors (BEs), which fuse a catalytically disabled nuclease with a nucleobase deaminase enzyme, can be used to permanently inactivate HBV genome by introducing irreversible point mutations for generation of premature stop codons without DSBs of host genome. Although promising, CRISPR-mediated base editing still faces daunting challenges before its clinical application, including the base-editing efficacy, the off-target effect, the difficulty in finding conserved target HBV sequences, and in vivo delivery efficiency. Several strategies have been adopted to optimize the efficiency and specificity of CRISPR-BEs and to improve in vivo delivery efficacy through novel viral and non-viral delivery approaches. Particularly, the non-viral delivery of Cas9 mRNA and ribonucleoprotein by lipid nanoparticles exhibits attractive potential for liver-targeted delivery in clinical. Along with all progress above, the CRISPR-mediated gene therapy will ultimately achieve HBV cure. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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18 pages, 1144 KiB  
Review
CRISPR Screening: Molecular Tools for Studying Virus–Host Interactions
by Vladimir Chulanov, Anastasiya Kostyusheva, Sergey Brezgin, Natalia Ponomareva, Vladimir Gegechkori, Elena Volchkova, Nikolay Pimenov and Dmitry Kostyushev
Viruses 2021, 13(11), 2258; https://doi.org/10.3390/v13112258 - 11 Nov 2021
Cited by 7 | Viewed by 6382
Abstract
CRISPR/Cas is a powerful tool for studying the role of genes in viral infections. The invention of CRISPR screening technologies has made it possible to untangle complex interactions between the host and viral agents. Moreover, whole-genome and pathway-specific CRISPR screens have facilitated identification [...] Read more.
CRISPR/Cas is a powerful tool for studying the role of genes in viral infections. The invention of CRISPR screening technologies has made it possible to untangle complex interactions between the host and viral agents. Moreover, whole-genome and pathway-specific CRISPR screens have facilitated identification of novel drug candidates for treating viral infections. In this review, we highlight recent developments in the fields of CRISPR/Cas with a focus on the use of CRISPR screens for studying viral infections and identifying new candidate genes to aid development of antivirals. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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23 pages, 28527 KiB  
Review
CRISPR Tackles Emerging Viral Pathogens
by Emily N. Kirby, Byron Shue, Paul Q. Thomas and Michael R. Beard
Viruses 2021, 13(11), 2157; https://doi.org/10.3390/v13112157 - 26 Oct 2021
Cited by 4 | Viewed by 4104
Abstract
Understanding the dynamic relationship between viral pathogens and cellular host factors is critical to furthering our knowledge of viral replication, disease mechanisms and development of anti-viral therapeutics. CRISPR genome editing technology has enhanced this understanding, by allowing identification of pro-viral and anti-viral cellular [...] Read more.
Understanding the dynamic relationship between viral pathogens and cellular host factors is critical to furthering our knowledge of viral replication, disease mechanisms and development of anti-viral therapeutics. CRISPR genome editing technology has enhanced this understanding, by allowing identification of pro-viral and anti-viral cellular host factors for a wide range of viruses, most recently the cause of the COVID-19 pandemic, SARS-CoV-2. This review will discuss how CRISPR knockout and CRISPR activation genome-wide screening methods are a robust tool to investigate the viral life cycle and how other class 2 CRISPR systems are being repurposed for diagnostics. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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20 pages, 1717 KiB  
Review
Genome Editing Strategies to Protect Livestock from Viral Infections
by Jenny-Helena Söllner, Thomas C. Mettenleiter and Björn Petersen
Viruses 2021, 13(10), 1996; https://doi.org/10.3390/v13101996 - 04 Oct 2021
Cited by 5 | Viewed by 3107
Abstract
The livestock industry is constantly threatened by viral disease outbreaks, including infections with zoonotic potential. While preventive vaccination is frequently applied, disease control and eradication also depend on strict biosecurity measures. Clustered regularly interspaced palindromic repeats (CRISPR) and associated proteins (Cas) have been [...] Read more.
The livestock industry is constantly threatened by viral disease outbreaks, including infections with zoonotic potential. While preventive vaccination is frequently applied, disease control and eradication also depend on strict biosecurity measures. Clustered regularly interspaced palindromic repeats (CRISPR) and associated proteins (Cas) have been repurposed as genome editors to induce targeted double-strand breaks at almost any location in the genome. Thus, CRISPR/Cas genome editors can also be utilized to generate disease-resistant or resilient livestock, develop vaccines, and further understand virus–host interactions. Genes of interest in animals and viruses can be targeted to understand their functions during infection. Furthermore, transgenic animals expressing CRISPR/Cas can be generated to target the viral genome upon infection. Genetically modified livestock can thereby reduce disease outbreaks and decrease zoonotic threats. Full article
(This article belongs to the Special Issue CRISPR/Cas in Viral Research)
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