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Sultan MH, Zhan Q, Wang Y, Xia Y, Jia X. Precision oncolytic viral therapy in colorectal cancer: Genetic targeting and immune modulation for personalized treatment (Review). Int J Mol Med 2025; 56:104. [PMID: 40342021 PMCID: PMC12081034 DOI: 10.3892/ijmm.2025.5545] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/13/2024] [Accepted: 04/09/2025] [Indexed: 05/11/2025] Open
Abstract
Colorectal cancer (CRC) is a leading health issue and treatments to eradicate it, such as conventional chemotherapy, are non‑selective and come with a number of complications. The present review focuses on the relatively new area of precision oncolytic viral therapy (OVT), with genetic targeting and immune modifications that offer a new future for CRC treatment. In the present review, an overview of the selection factors that are considered optimal for an oncolytic virus, mechanisms of oncolysis and immunomodulation applied to the OVT, as well as new strategies to improve the efficacy of this method are described. Additionally, cause‑and‑effect relationships are examined for OVT efficacy, mediated by the tumor microenvironment, and directions for genetic manipulation of viral specificity are explored. The possibility of synergy between OVT and immune checkpoint inhibitors and other treatment approaches are demonstrated. Incorporating the details of the present review, biomarker‑guided combination therapies in precision OVT for individualized CRC care, significant issues and future trends in this required area of medicine are highlighted. Increasingly, OVT is leaving the experimental stage and may become routine practice; it provides a new perspective on overcoming CRC and highlights the importance of further research and clinical work.
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Affiliation(s)
- Muhammad Haris Sultan
- College of Life Sciences and Medicine, Xinyuan Institute of Medicine and Biotechnology, Zhejiang Sci-Tech University, Hangzhou, Zhejiang 310018, P.R. China
- Center for Translational Medicine and Precision Medicine, Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang 325000, P.R. China
| | - Qi Zhan
- College of Life Sciences and Medicine, Xinyuan Institute of Medicine and Biotechnology, Zhejiang Sci-Tech University, Hangzhou, Zhejiang 310018, P.R. China
| | - Yigang Wang
- College of Life Sciences and Medicine, Xinyuan Institute of Medicine and Biotechnology, Zhejiang Sci-Tech University, Hangzhou, Zhejiang 310018, P.R. China
| | - Yulong Xia
- Center for Translational Medicine and Precision Medicine, Wenzhou Institute, University of Chinese Academy of Sciences, Wenzhou, Zhejiang 325000, P.R. China
| | - Xiaoyuan Jia
- College of Life Sciences and Medicine, Xinyuan Institute of Medicine and Biotechnology, Zhejiang Sci-Tech University, Hangzhou, Zhejiang 310018, P.R. China
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Guo R, Li H, Li J, Qv J, Ren G, Zhang X, Nasir S, Zhang J, Luo C, Zeshan B, Zhou Y, Xie H, Wang X. Recombinant PRV Expressing GP3 and GP5 of PRRSV Provides Effective Protection Against Coinfection With PRV and PRRSV. Transbound Emerg Dis 2025; 2025:4612568. [PMID: 40376208 PMCID: PMC12081148 DOI: 10.1155/tbed/4612568] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/21/2025] [Accepted: 04/10/2025] [Indexed: 05/18/2025]
Abstract
Porcine Reproductive and Respiratory Syndrome (PRRS) and Pseudorabies (PR) are highly contagious diseases caused by Porcine Reproductive and Respiratory Syndrome virus (PRRSV) and Pseudorabies virus (PRV). Due to the limited protective ability of currently commercialized vaccines against NADC30-like PRRSV and PRV variants, the pathological damage caused by coinfection of these two viruses has a significant impact on China's pig farming industry. In this study, six recombinant PRV stains with TK and gI/gE deletions and fused expression of GM-CSF and GP3 and GP5 proteins from NADC30-Like PRRSV were constructed by using the HDR-CRISPR/Cas9D10A system. After assessing growth characteristics and genetic stability, four strains demonstrating stable proliferation and expression of the GM-CSF, GP3, GP5 fusion protein in BHK-21 cells were selected. Evaluation of their ability to induce specific humoral and cellular immune responses in mice led to the selection of two strains with superior immunogenic effects: rPRV-ΔTK-GP3-GP5-eGFP-ΔgI/gE-mCHERRY-B and rPRV-ΔTK-eGFP-ΔgI/gE-GP3-GP5-mCHERRY-B. These strains were found to enhance NADC30-like PRRSV and PRV-specific immune responses in piglets, reduce pathological damage, and accelerate symptom resolution. In general, PRV is a promising viral vector for expressing PRRSV genes, and the data from this study provides references for new candidate vaccines against PRRSV.
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Affiliation(s)
- Ruhai Guo
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Hui Li
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Junda Li
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Jahao Qv
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Guofan Ren
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Xiao Zhang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Saba Nasir
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Jingnan Zhang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Chen Luo
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
| | - Basit Zeshan
- Faculty of Sustainable Agriculture, Universiti Malaysia Sabah, Sandakan, Sabah, Malaysia
| | - Yefei Zhou
- Department of Life Science, Nanjing Xiaozhuang University, Nanjing, Jiangsu, China
| | - Honglin Xie
- School of Animal Science and Technology, Foshan University, Foshan, Guangdong, China
| | - Xinglong Wang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi, China
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Gao Y, Zou Y, Wu C, Tao J, Nie Z, Yan J, Wang P, Huang X. Comparative evaluation of immunomodulatory cytokines for oncolytic therapy based on a high-efficient platform for oHSV1 reconstruction. Virol J 2025; 22:133. [PMID: 40325455 PMCID: PMC12054163 DOI: 10.1186/s12985-025-02758-y] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/20/2024] [Accepted: 04/22/2025] [Indexed: 05/07/2025] Open
Abstract
BACKGROUND Triple-negative breast cancer (TNBC) presents significant therapeutic challenges due to its immunosuppressive tumor microenvironment (TME). Oncolytic herpes simplex virus type 1 (oHSV1) offers dual mechanisms of tumor lysis and immune activation, yet the optimal cytokine payloads for TNBC remain undefined. METHODS We developed a CRISPR/Cas9-mediated platform for high-efficiency oHSV1 engineering, replacing the ICP47 locus with murine IFN-γ, GM-CSF, or IL-15Rα/IL-15 fusion protein (IL15Fu). Constructs were validated for cytokine secretion, MHC modulation, and cytotoxicity in 4T1 TNBC and a panel of human cancer cell lines. Antitumor efficacy and immune remodeling were evaluated in a syngeneic 4T1 model using RNA sequencing and flow cytometry. RESULTS The CRISPR platform achieved 62.5-71.4% homologous recombination efficiency, enabling rapid virus construction. In vitro, OV-IFNG exhibited upregulated MHC I/II expression and potent cytotoxicity, while OV-GMCSF attenuated oncolysis in subsets of breast cancer cell lines. In the 4T1 model, OV-IL15Fu modestly improved tumor control and extended survival without apparent toxicity, while OV-IFNG induced early mortality associated with systemic toxicity. Transcriptomic profiling revealed divergent immune modulation: OV-IL15Fu enriched T cell/NK cytotoxicity pathways, OV-IFNG amplified cytokine/chemokine signaling, and OV-GMCSF paradoxically enhanced myeloid recruitment while inhibiting MHC-II pathways. Flow cytometry confirmed functional differences in immune activation: OV-IL15Fu expanding cytotoxic lymphocytes (CD8⁺ T/NK cells), OV-IFNG preferentially promote Th1 polarization and innate immune activation, and OV-GMCSF failed to activate T cells despite myeloid infiltration. CONCLUSIONS Our findings underscore the need for rational cytokine selection in oHSV1-based immunotherapy. While IFN-γ increased immunogenic markers, its systemic toxicity and myeloid effects may limit benefit. GM-CSF exacerbated immune suppression in this context, whereas IL15Fu showed favorable immunostimulatory properties without detectable toxicity. These data support IL15Fu as a contextually promising payload for further evaluation in TNBC-targeted oncolytic virotherapy.
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Affiliation(s)
- Yingzheng Gao
- Kunming Medical University, Kunming, Yunnan, China
- Key Laboratory of The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China
| | - Yufang Zou
- Kunming Medical University, Kunming, Yunnan, China
- Key Laboratory of The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China
| | - Changjing Wu
- Kunming Medical University, Kunming, Yunnan, China
- Key Laboratory of The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China
| | - Juan Tao
- Kunming Medical University, Kunming, Yunnan, China
- Key Laboratory of The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China
| | - Zuqing Nie
- Kunming Medical University, Kunming, Yunnan, China
- Key Laboratory of The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China
| | - Jinyuan Yan
- Kunming Medical University, Kunming, Yunnan, China
- Key Laboratory of The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China
| | - Pengfei Wang
- Kunming Medical University, Kunming, Yunnan, China.
- Key Laboratory of The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China.
| | - Xinwei Huang
- Kunming Medical University, Kunming, Yunnan, China.
- Key Laboratory of The Second Affiliated Hospital of Kunming Medical University, Kunming, Yunnan, China.
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Lin Z, Yao Q, Lai K, Jiao K, Zeng X, Lei G, Zhang T, Dai H. Cas12f1 gene drives propagate efficiently in herpesviruses and induce minimal resistance. Genome Biol 2024; 25:311. [PMID: 39696608 DOI: 10.1186/s13059-024-03455-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/03/2024] [Accepted: 12/04/2024] [Indexed: 12/20/2024] Open
Abstract
BACKGROUND Synthetic CRISPR-Cas9 gene drive has been developed to control harmful species. However, resistance to Cas9 gene drive can be acquired easily when DNA repair mechanisms patch up the genetic insults introduced by Cas9 and incorporate mutations to the sgRNA target. Although many strategies to reduce the occurrence of resistance have been developed so far, they are difficult to implement and not always effective. RESULTS Here, Cas12f1, a recently developed CRISPR-Cas system with minimal potential for causing mutations within target sequences, has been explored as a potential platform for yielding low-resistance in gene drives. We construct Cas9 and Cas12f1 gene drives in a fast-replicating DNA virus, HSV1. Cas9 and Cas12f1 gene drives are able to spread among the HSV1 population with specificity towards their target sites, and their transmission among HSV1 viruses is not significantly affected by the reduced fitness incurred by the viral carriers. Cas12f1 gene drives spread similarly as Cas9 gene drives at high introduction frequency but transmit more slowly than Cas9 gene drives at low introduction frequency. However, Cas12f1 gene drives outperform Cas9 gene drives because they reach higher penetration and induce lower resistance than Cas9 gene drives in all cases. CONCLUSIONS Due to lower resistance and higher penetration, Cas12f1 gene drives could potentially supplant Cas9 gene drives for population control.
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Affiliation(s)
- Zhuangjie Lin
- Department of Immunology, School of Basic Medicine, Southern Medical University, Guangzhou, Guangdong Province, China
| | - Qiaorui Yao
- Department of Immunology, School of Basic Medicine, Southern Medical University, Guangzhou, Guangdong Province, China
| | - Keyuan Lai
- Department of Immunology, School of Basic Medicine, Southern Medical University, Guangzhou, Guangdong Province, China
| | - Kehua Jiao
- Department of Geriatric Medicine, Shanghai Health and Medical Center, Wuxi, Jiangshu Province, China
| | - Xianying Zeng
- Department of Immunology, School of Basic Medicine, Southern Medical University, Guangzhou, Guangdong Province, China
| | - Guanxiong Lei
- Affiliated Hospital of Xiangnan University, Chenzhou, Hunan Province, China
- Key Laboratory of Medical Imaging and Artificial Intelligence of Hunan Province, Chenzhou, Hunan Province, China
| | - Tongwen Zhang
- Department of Immunology, School of Basic Medicine, Southern Medical University, Guangzhou, Guangdong Province, China.
- Vaccine Biotech (Shenzhen) LTD, Shenzhen, China, & Boji Biopharmaceutical, Guangzhou, China.
| | - Hongsheng Dai
- Department of Immunology, School of Basic Medicine, Southern Medical University, Guangzhou, Guangdong Province, China.
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Sosnovtseva AO, Demidova NA, Klimova RR, Kovalev MA, Kushch AA, Starodubova ES, Latanova AA, Karpov DS. Control of HSV-1 Infection: Directions for the Development of CRISPR/Cas-Based Therapeutics and Diagnostics. Int J Mol Sci 2024; 25:12346. [PMID: 39596412 PMCID: PMC11595115 DOI: 10.3390/ijms252212346] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/05/2024] [Revised: 11/13/2024] [Accepted: 11/15/2024] [Indexed: 11/28/2024] Open
Abstract
It is estimated that nearly all individuals have been infected with herpesviruses, with herpes simplex virus type 1 (HSV-1) representing the most prevalent virus. In most cases, HSV-1 causes non-life-threatening skin damage in adults. However, in patients with compromised immune systems, it can cause serious diseases, including death. The situation is further complicated by the emergence of strains that are resistant to both traditional and novel antiviral drugs. It is, therefore, imperative that new methods of combating HSV-1 and other herpesviruses be developed without delay. CRISPR/Cas systems may prove an effective means of controlling herpesvirus infections. This review presents the current understanding of the underlying molecular mechanisms of HSV-1 infection and discusses four potential applications of CRISPR/Cas systems in the fight against HSV-1 infections. These include the search for viral and cellular genes that may serve as effective targets, the optimization of anti-HSV-1 activity of CRISPR/Cas systems in vivo, the development of CRISPR/Cas-based HSV-1 diagnostics, and the validation of HSV-1 drug resistance mutations.
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Affiliation(s)
- Anastasiia O. Sosnovtseva
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991 Moscow, Russia; (A.O.S.); (M.A.K.); (E.S.S.); (A.A.L.)
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991 Moscow, Russia
| | - Natalia A. Demidova
- N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Gamaleya Str., 18, 123098 Moscow, Russia; (N.A.D.); (R.R.K.); (A.A.K.)
| | - Regina R. Klimova
- N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Gamaleya Str., 18, 123098 Moscow, Russia; (N.A.D.); (R.R.K.); (A.A.K.)
| | - Maxim A. Kovalev
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991 Moscow, Russia; (A.O.S.); (M.A.K.); (E.S.S.); (A.A.L.)
| | - Alla A. Kushch
- N.F. Gamaleya National Research Centre for Epidemiology and Microbiology, Ministry of Health of the Russian Federation, Gamaleya Str., 18, 123098 Moscow, Russia; (N.A.D.); (R.R.K.); (A.A.K.)
| | - Elizaveta S. Starodubova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991 Moscow, Russia; (A.O.S.); (M.A.K.); (E.S.S.); (A.A.L.)
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991 Moscow, Russia
| | - Anastasia A. Latanova
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991 Moscow, Russia; (A.O.S.); (M.A.K.); (E.S.S.); (A.A.L.)
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991 Moscow, Russia
| | - Dmitry S. Karpov
- Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991 Moscow, Russia; (A.O.S.); (M.A.K.); (E.S.S.); (A.A.L.)
- Center for Precision Genome Editing and Genetic Technologies for Biomedicine, Engelhardt Institute of Molecular Biology, Russian Academy of Sciences, Vavilov Str., 32, 119991 Moscow, Russia
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Yao ZQ, Schank MB, Zhao J, El Gazzar M, Wang L, Zhang Y, Hill AC, Banik P, Pyburn JS, Moorman JP. The potential of HBV cure: an overview of CRISPR-mediated HBV gene disruption. Front Genome Ed 2024; 6:1467449. [PMID: 39444780 PMCID: PMC11496132 DOI: 10.3389/fgeed.2024.1467449] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/19/2024] [Accepted: 09/24/2024] [Indexed: 10/25/2024] Open
Abstract
Hepatitis B virus (HBV) infection is a common cause of liver disease worldwide. The current antiviral treatment using nucleotide analogues (NAs) can only suppress de novo HBV replication but cannot eliminate chronic HBV infection due to the persistence of covalently closed circular (ccc) DNA that sustains viral replication. The CRISPR/Cas9 system is a novel genome-editing tool that enables precise gene disruption and inactivation. With high efficiency and simplicity, the CRISPR/Cas9 system has been utilized in multiple studies to disrupt the HBV genome specifically, eliciting varying anti-HBV effects both in vitro and in vivo. Additionally, multi-locus gene targeting has shown enhanced antiviral activity, paving the way for combination therapy to disrupt and inactivate HBV cccDNA as well as integrated HBV DNA. Despite its promising antiviral effects, this technology faces several challenges that need to be overcome before its clinical application, i.e., off-target effects and in vivo drug delivery. As such, there is a need for improvement in CRISPR/Cas9 efficiency, specificity, versatility, and delivery. Here, we critically review the recent literature describing the tools employed in designing guide RNAs (gRNAs) targeting HBV genomes, the vehicles used for expressing and delivering CRISPR/Cas9 components, the models used for evaluating CRISPR-mediated HBV gene disruption, the methods used for assessing antiviral and off-target effects induced by CRISPR/Cas9-mediated HBV gene disruption, and the prospects of future directions and challenges in leveraging this HBV gene-editing approach, to advance the HBV treatment toward a clinical cure.
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Affiliation(s)
- Zhi Q. Yao
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
- Hepatitis (HBV/HCV/HIV) Program, James H. Quillen VA Medical Center, Department of Veterans Affairs, Johnson City, TN, United States
| | - Madison B. Schank
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Juan Zhao
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Mohamed El Gazzar
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Ling Wang
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Yi Zhang
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Addison C. Hill
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Puja Banik
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Jaeden S. Pyburn
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
| | - Jonathan P. Moorman
- Center of Excellence in Inflammation, Infectious Disease and Immunity, James H. Quillen College of Medicine, East Tennessee State University, Johnson City, TN, United States
- Department of Internal Medicine, Division of Infectious, Inflammatory and Immunologic Diseases, Quillen College of Medicine, ETSU, Johnson City, TN, United States
- Hepatitis (HBV/HCV/HIV) Program, James H. Quillen VA Medical Center, Department of Veterans Affairs, Johnson City, TN, United States
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Zhang JF, Kim SW, Shang K, Park JY, Choi YR, Jang HK, Wei B, Kang M, Cha SY. Protection of Chickens against H9N2 Avian Influenza Isolates with a Live Vector Vaccine Expressing Influenza Hemagglutinin Gene Derived from Y280 Avian Influenza Virus. Animals (Basel) 2024; 14:872. [PMID: 38539970 PMCID: PMC10967311 DOI: 10.3390/ani14060872] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/14/2024] [Revised: 03/08/2024] [Accepted: 03/10/2024] [Indexed: 11/11/2024] Open
Abstract
Since the outbreak of the H9N2/Y439 avian influenza virus in 1996, the Korean poultry industry has incurred severe economic losses. A novel possibly zoonotic H9N2 virus from the Y280-like lineage (H9N2/Y280) has been prevalent in Korea since June 2020, posing a threat to the poultry sector. Rapid mutation of influenza viruses urges the development of effective vaccines against newly generated strains. Thus, we engineered a recombinant virus rHVT/Y280 to combat H9N2/Y280. We integrated the hemagglutinin (HA) gene of the H9N2/Y280 strain into the US2 region of the herpesvirus of turkeys (HVT) Fc126 vaccine strain, utilizing CRISPR/Cas9 gene-editing technology. The successful construction of rHVT/Y280 was confirmed by polymerase chain reaction and sequencing, followed by efficacy evaluation. Four-day-old specific pathogen-free chickens received the rHVT/Y280 vaccine and were challenged with the H9N2/Y280 strain A21-MRA-003 at 3 weeks post-vaccination. In 5 days, there were no gross lesions among the vaccinated chickens. The rHVT/Y280 vaccine induced strong humoral immunity and markedly reduced virus shedding, achieving 100% inhibition of virus recovery in the cecal tonsil and significantly lowering tissue viral load. Thus, HVT vector vaccines expressing HA can be used for protecting poultry against H9N2/Y280. The induction of humoral immunity by live vaccines is vital in such cases. In summary, the recombinant virus rHVT/Y280 is a promising vaccine candidate for the protection of chickens against the H9N2/Y280.
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Affiliation(s)
- Jun-Feng Zhang
- Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea; (J.-F.Z.); (S.-W.K.); (K.S.); (J.-Y.P.); (Y.-R.C.); (H.-K.J.); (B.W.)
- College of Medical Technology and Engineering, Henan University of Science and Technology, Luoyang 471003, China
| | - Sang-Won Kim
- Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea; (J.-F.Z.); (S.-W.K.); (K.S.); (J.-Y.P.); (Y.-R.C.); (H.-K.J.); (B.W.)
| | - Ke Shang
- Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea; (J.-F.Z.); (S.-W.K.); (K.S.); (J.-Y.P.); (Y.-R.C.); (H.-K.J.); (B.W.)
- College of Animal Science and Technology, Luoyang Key Laboratory of Live Carrier Biomaterial and Animal Disease Prevention and Control, Henan University of Science and Technology, Luoyang 471000, China
| | - Jong-Yeol Park
- Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea; (J.-F.Z.); (S.-W.K.); (K.S.); (J.-Y.P.); (Y.-R.C.); (H.-K.J.); (B.W.)
| | - Yu-Ri Choi
- Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea; (J.-F.Z.); (S.-W.K.); (K.S.); (J.-Y.P.); (Y.-R.C.); (H.-K.J.); (B.W.)
| | - Hyung-Kwan Jang
- Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea; (J.-F.Z.); (S.-W.K.); (K.S.); (J.-Y.P.); (Y.-R.C.); (H.-K.J.); (B.W.)
- Bio Disease Control (BIOD) Co., Ltd., Iksan 54596, Republic of Korea
| | - Bai Wei
- Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea; (J.-F.Z.); (S.-W.K.); (K.S.); (J.-Y.P.); (Y.-R.C.); (H.-K.J.); (B.W.)
| | - Min Kang
- Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea; (J.-F.Z.); (S.-W.K.); (K.S.); (J.-Y.P.); (Y.-R.C.); (H.-K.J.); (B.W.)
- Bio Disease Control (BIOD) Co., Ltd., Iksan 54596, Republic of Korea
| | - Se-Yeoun Cha
- Department of Avian Diseases, College of Veterinary Medicine and Center for Avian Disease, Jeonbuk National University, Iksan 54596, Republic of Korea; (J.-F.Z.); (S.-W.K.); (K.S.); (J.-Y.P.); (Y.-R.C.); (H.-K.J.); (B.W.)
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8
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Wang Y. Rendezvous with Vaccinia Virus in the Post-smallpox Era: R&D Advances. Viruses 2023; 15:1742. [PMID: 37632084 PMCID: PMC10457812 DOI: 10.3390/v15081742] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/28/2023] [Revised: 08/13/2023] [Accepted: 08/14/2023] [Indexed: 08/27/2023] Open
Abstract
Smallpox was eradicated in less than 200 years after Edward Jenner's practice of cowpox variolation in 1796. The forty-three years of us living free of smallpox, beginning in 1979, never truly separated us from poxviruses. The recent outbreak of monkeypox in May 2022 might well warn us of the necessity of keeping up both the scientific research and public awareness of poxviruses. One of them in particular, the vaccinia virus (VACV), has been extensively studied as a vector given its broad host range, extraordinary thermal stability, and exceptional immunogenicity. Unceasing fundamental biological research on VACV provides us with a better understanding of its genetic elements, involvement in cellular signaling pathways, and modulation of host immune responses. This enables the rational design of safer and more efficacious next-generation vectors. To address the new technological advancement within the past decade in VACV research, this review covers the studies of viral immunomodulatory genes, modifications in commonly used vectors, novel mechanisms for rapid generation and purification of recombinant virus, and several other innovative approaches to studying its biology.
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Affiliation(s)
- Yuxiang Wang
- Vaccine Research Center, National Institutes of Health, 40 Convent Drive, Bethesda, MD 20892, USA
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9
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Wang HM, Qiao YY, Cai BY, Tan J, Na L, Wang Y, Lu H, Tang YD. Genome editing of pseudorabies virus in the CRISPR/Cas9 era: a mini-review. Front Vet Sci 2023; 10:1237186. [PMID: 37476821 PMCID: PMC10354360 DOI: 10.3389/fvets.2023.1237186] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2023] [Accepted: 06/22/2023] [Indexed: 07/22/2023] Open
Abstract
Pseudorabies virus (PRV) is an important swine virus that has a significant impact on the global swine industry. PRV is a member of the herpesvirus family, specifically the alphaherpesvirus subfamily, and has been extensively utilized as a prototype herpesvirus. Notably, recent studies have reported that PRV sporadically spills over into humans. The PRV genome is approximately 150 kb in size and is difficult to manipulate at the genomic level. The development of clustered regularly interspaced short palindromic repeat-associated protein (CRISPR/Cas9) technology has revolutionized PRV genome editing. CRISPR/Cas9 has been widely used in the construction of reporter viruses, knock-out/knock-in of genes of interest, single virus tracking and antiviral strategies. Most importantly, for vaccine development, virulence gene knockout PRV vaccine candidates can be obtained within 2 weeks using CRISPR/Cas9. In this mini-review, we provide a concise overview of the application of CRISPR/Cas9 in PRV research and mainly share our experience with methods for efficiently editing the PRV genome. Through this review, we hope to give researchers better insight into the genome editing of pseudorabies virus.
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Affiliation(s)
- Hai-Ming Wang
- Jiangsu Agri-animal Husbandry Vocational College, Taizhou, Jiangsu, China
| | - Yang-Yang Qiao
- Jiangsu Agri-animal Husbandry Vocational College, Taizhou, Jiangsu, China
| | - Bing-Yan Cai
- Jiangsu Agri-animal Husbandry Vocational College, Taizhou, Jiangsu, China
| | - Ju Tan
- Jiangsu Agri-animal Husbandry Vocational College, Taizhou, Jiangsu, China
| | - Lei Na
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China
- Jiangsu Vocational College Agriculture and Forestry, Taizhou, Jiangsu, China
| | - Yu Wang
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China
| | - Hui Lu
- Jiangsu Agri-animal Husbandry Vocational College, Taizhou, Jiangsu, China
| | - Yan-Dong Tang
- State Key Laboratory for Animal Disease Control and Prevention, Harbin Veterinary Research Institute of Chinese Academy of Agricultural Sciences, Harbin, China
- Heilongjiang Provincial Research Center for Veterinary Biomedicine, Harbin, China
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10
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Guo Y, Xu Z, Chao Y, Cao X, Jiang H, Li H, Li T, Wan Z, Shao H, Qin A, Xie Q, Ye J. An efficient double-fluorescence approach for generating fiber-2-edited recombinant serotype 4 fowl adenovirus expressing foreign gene. Front Microbiol 2023; 14:1160031. [PMID: 37065110 PMCID: PMC10102364 DOI: 10.3389/fmicb.2023.1160031] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/06/2023] [Accepted: 03/15/2023] [Indexed: 04/03/2023] Open
Abstract
Recently, the infection of serotype 4 fowl adenovirus (FAdV-4) in chicken flocks has become endemic in China, which greatly threatens the sustainable development of poultry industry. The development of recombinant FAdV-4 expressing foreign genes is an efficient strategy for controlling both FAdV-4 and other important poultry pathogens. Previous reverse genetic technique for generating the recombinant fowl adenovirus is generally inefficient. In this study, a recombinant FAdV-4 expressing enhanced green fluorescence protein (EGFP), FA4-EGFP, was used as a template virus and directly edited fiber-2 gene to develop an efficient double-fluorescence approach to generate recombinant FAdV-4 through CRISPR/Cas9 and Cre-Loxp system. Moreover, using this strategy, a recombinant virus FAdV4-HA(H9) stably expressing the HA gene of H9N2 influenza virus was generated. Chicken infection study revealed that the recombinant virus FAdV4-HA(H9) was attenuated, and could induce haemagglutination inhibition (HI) titer against H9N2 influenza virus at early time points and inhibit the viral replication in oropharynx. All these demonstrate that the novel strategy for constructing recombinant FAdV-4 expressing foreign genes developed here paves the way for rapidly developing attenuated FAdV-4-based recombinant vaccines for fighting the diseases caused by both FAdV-4 and other pathogens.
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Affiliation(s)
- Yiwen Guo
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Zhenqi Xu
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Yifei Chao
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Xudong Cao
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Huiru Jiang
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Han Li
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Tuofan Li
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Zhimin Wan
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Hongxia Shao
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Aijian Qin
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
| | - Quan Xie
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
- *Correspondence: Quan Xie,
| | - Jianqiang Ye
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, Jiangsu, China
- Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, Jiangsu, China
- Joint International Research Laboratory of Agriculture and Agri-Product Safety, The Ministry of Education of China, Yangzhou University, Yangzhou, Jiangsu, China
- Institutes of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, Jiangsu, China
- Jianqiang Ye,
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11
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Yu Y, Zhang F, Duan X, Yang C, Cui Y, Yu L. ORFV can carry TRAP gene expression via intracellular CRISPR/Cas9 gene editing technology. J Virol Methods 2023; 312:114652. [PMID: 36493528 DOI: 10.1016/j.jviromet.2022.114652] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/19/2022] [Revised: 11/09/2022] [Accepted: 11/11/2022] [Indexed: 12/12/2022]
Abstract
Orf is an acute and highly contracted human and animal infection caused by orf virus (ORFV), which mainly affects sheep, goats, and other species. Clinically, opportunistic or conditional pathogens such as Staphylococcus aureus (S. aureus) are often detected in cases of orf, which greatly increases the risk of disease progression and clinical death. It has been reported that TRAP gene products of S. aureus can broadly influence bacterial life and pathogenicity in vivo, and introduction of exogenous TRAP genes may help to inhibit the proliferation of bacteria. In order to achieve the combined control of ORFV and S. aureus, a novel approach to design a S. aureus TRAP gene vaccine using a live attenuated ORFV vector is proposed. In this study, CRISPR/Cas9 gene editing technology was used to disable vascular endothelial growth factor E of ORFV (VEGF-v) and introduced TRAP gene into this position. TRAP gene expression was detected in keratinocytes infected with recombinant virus. The construction and experimental verification of recombinant ORFV (ORFV-v/TRAP) will provide a reference for in-depth studies on the prevention and control of mixed infectious disease.
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Affiliation(s)
- YongZhong Yu
- College of Biological Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China.
| | - Fan Zhang
- College of Biological Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China
| | - Xuyang Duan
- College of Biological Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China.
| | - ChaoQun Yang
- College of Biological Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China
| | - YuDong Cui
- College of Biological Science and Technology, Heilongjiang Bayi Agricultural University, Daqing 163319, PR China
| | - Li Yu
- Division of Livestock Infectious Diseases, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, No. 427 Maduan Street, Harbin 150001, PR China
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12
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Zafar K, Khan MZ, Amin I, Mukhtar Z, Zafar M, Mansoor S. Employing template-directed CRISPR-based editing of the OsALS gene to create herbicide tolerance in Basmati rice. AOB PLANTS 2023; 15:plac059. [PMID: 36873055 PMCID: PMC9977225 DOI: 10.1093/aobpla/plac059] [Citation(s) in RCA: 8] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 04/01/2022] [Accepted: 01/06/2023] [Indexed: 06/18/2023]
Abstract
Rice (Oryza sativa) is one of the primary food crops which contributes major portion of daily calorie intake. It is used as model crop for various genome editing studies. Basmati rice was also explored for establishing non-homologous end joining-based genome editing. But it was not clear whether homology-directed repair (HDR)-based genome editing can be done in Basmati rice. The current study was designed to establish HDR-based genome editing in Basmati rice to develop herbicide tolerance. There is severe weed spread when rice is grown via direct planted rice method in various countries to save labour and water resources. Therefore, the use of herbicides is necessary to control weeds. These herbicides can also affect cultivated rice which creates the need to develop herbicide-tolerant rice. In current study, we introduced a point mutation in Acetolactate Synthase gene to convert tryptophan to leucine at position 548. For this purpose, different constructs for HDR were tested with different RNA scaffold and orientation of repair templates. Out of four different architectures, the one having repair template identical to the target DNA strand precisely edited the target site. We successfully established template-directed CRISPR-Cas9 system in Super Basmati rice by detecting desired substitutions at the target site in Acetolactate Synthase locus. Moreover, this editing of Acetolactate Synthase gene resulted in the production of herbicide tolerance in Super Basmati rice. This study suggests that such type of HDR system can be used to precisely edit other genes for crop improvement.
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Affiliation(s)
| | - Muhammad Zuhaib Khan
- Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Constituent College of Pakistan Institute of Engineering and Applied Sciences, Jhang Road, Faisalabad 37000, Pakistan
| | - Imran Amin
- Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Constituent College of Pakistan Institute of Engineering and Applied Sciences, Jhang Road, Faisalabad 37000, Pakistan
| | - Zahid Mukhtar
- Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Constituent College of Pakistan Institute of Engineering and Applied Sciences, Jhang Road, Faisalabad 37000, Pakistan
| | - Mehak Zafar
- Agricultural Biotechnology Division, National Institute for Biotechnology and Genetic Engineering (NIBGE), Constituent College of Pakistan Institute of Engineering and Applied Sciences, Jhang Road, Faisalabad 37000, Pakistan
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13
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Whelan JT, Singaravelu R, Wang F, Pelin A, Tamming LA, Pugliese G, Martin NT, Crupi MJF, Petryk J, Austin B, He X, Marius R, Duong J, Jones C, Fekete EEF, Alluqmani N, Chen A, Boulton S, Huh MS, Tang MY, Taha Z, Scut E, Diallo JS, Azad T, Lichty BD, Ilkow CS, Bell JC. CRISPR-mediated rapid arming of poxvirus vectors enables facile generation of the novel immunotherapeutic STINGPOX. Front Immunol 2023; 13:1050250. [PMID: 36713447 PMCID: PMC9880309 DOI: 10.3389/fimmu.2022.1050250] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2022] [Accepted: 12/05/2022] [Indexed: 01/15/2023] Open
Abstract
Poxvirus vectors represent versatile modalities for engineering novel vaccines and cancer immunotherapies. In addition to their oncolytic capacity and immunogenic influence, they can be readily engineered to express multiple large transgenes. However, the integration of multiple payloads into poxvirus genomes by traditional recombination-based approaches can be highly inefficient, time-consuming and cumbersome. Herein, we describe a simple, cost-effective approach to rapidly generate and purify a poxvirus vector with multiple transgenes. By utilizing a simple, modular CRISPR/Cas9 assisted-recombinant vaccinia virus engineering (CARVE) system, we demonstrate generation of a recombinant vaccinia virus expressing three distinct transgenes at three different loci in less than 1 week. We apply CARVE to rapidly generate a novel immunogenic vaccinia virus vector, which expresses a bacterial diadenylate cyclase. This novel vector, STINGPOX, produces cyclic di-AMP, a STING agonist, which drives IFN signaling critical to the anti-tumor immune response. We demonstrate that STINGPOX can drive IFN signaling in primary human cancer tissue explants. Using an immunocompetent murine colon cancer model, we demonstrate that intratumoral administration of STINGPOX in combination with checkpoint inhibitor, anti-PD1, promotes survival post-tumour challenge. These data demonstrate the utility of CRISPR/Cas9 in the rapid arming of poxvirus vectors with therapeutic payloads to create novel immunotherapies.
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Affiliation(s)
- Jack T. Whelan
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Ragunath Singaravelu
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
- Public Health Agency of Canada, Ottawa, ON, Canada
| | - Fuan Wang
- McMaster Immunology Research Centre, Department of Medicine, McMaster University, Hamilton, ON, Canada
- MG DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON, Canada
| | - Adrian Pelin
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Levi A. Tamming
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Giuseppe Pugliese
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Nikolas T. Martin
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Mathieu J. F. Crupi
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Julia Petryk
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Bradley Austin
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Xiaohong He
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Ricardo Marius
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Jessie Duong
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Carter Jones
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Emily E. F. Fekete
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Nouf Alluqmani
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Andrew Chen
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Stephen Boulton
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Michael S. Huh
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Matt Y. Tang
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Zaid Taha
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Elena Scut
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Jean-Simon Diallo
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Taha Azad
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - Brian D. Lichty
- McMaster Immunology Research Centre, Department of Medicine, McMaster University, Hamilton, ON, Canada
- MG DeGroote Institute for Infectious Disease Research, McMaster University, Hamilton, ON, Canada
| | - Carolina S. Ilkow
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
| | - John C. Bell
- Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada
- Centre for Innovation Cancer Therapeutics, Ottawa Hospital Research Institute, Ottawa, ON, Canada
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14
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Ni L, Li Y, Wu K, Deng F, Wang H, Ning YJ. Antitumor efficacy of CRISPR/Cas9-engineered ICP6 mutant herpes simplex viruses in a mouse xenograft model for lung adenocarcinoma. J Med Virol 2022; 94:6000-6015. [PMID: 35965331 DOI: 10.1002/jmv.28069] [Citation(s) in RCA: 6] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2022] [Revised: 08/03/2022] [Accepted: 08/11/2022] [Indexed: 01/06/2023]
Abstract
Oncolytic viruses (OVs), including oncolytic herpes simplex viruses (oHSVs), are promising therapeutics against cancer. Here, we report two ICP6-mutated HSVs (type I) generated by CRISPR/Cas9, rHSV1/∆RR (with ICP6 ribonucleotide reductase [RR] domain deleted) and rHSV1/∆ICP6 (with a complete deletion of ICP6), exhibiting potent antitumor efficacy against lung adenocarcinoma. Both the mutants showed strong cytotoxicity in vitro, comparable with the control viruses expressing intact ICP6, but in relatively lower titers. Moreover, these mutant viruses exhibited preferential killing ability against lung tumor cells rather than normal lung fibroblast cells. Further, unlike the control HSV-1 causing severe illness or death in the mouse model, the ICP6-mutated viruses did not induce significant pathogenicity but instead effectively reduced tumor burden in vivo and led to 100% survival of the animals, indicating notable antitumor activity and attenuated virulence. In addition, rHSV1/∆RR seemed to have even better antitumor efficacy than rHSV1/∆ICP6, albeit no statistical significance in inhibition of tumor volume. Histopathologically, rHSV1/∆RR induced massive neutrophil infiltration to the tumor microenvironment and consistently, triggered more antitumor immune and neutrophil chemotactic cytokines or higher expression levels of them (indicated by quantitative polymerase chain reaction and transcriptome analyses). These results demonstrate the anti-adenocarcinoma potential of the CRISPR/Cas9-engineered ICP6 mutant HSV1, especially the rHSV1/∆RR, which likely induces stronger innate antitumor immune response. Together, these findings may provide new valuable clues for further development of OV-based therapeutics against lung adenocarcinoma or other types of tumors.
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Affiliation(s)
- Longquan Ni
- State Key Laboratory of Virology and National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Yi Li
- Center for Cell Lineage and Development, Guangzhou Institutes of Biomedicine and Health, Chinese Academy of Sciences, Guangzhou, China
| | - Ke Wu
- State Key Laboratory of Virology and National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.,University of Chinese Academy of Sciences, Beijing, China
| | - Fei Deng
- State Key Laboratory of Virology and National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.,Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China
| | - Hualin Wang
- State Key Laboratory of Virology and National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.,Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China
| | - Yun-Jia Ning
- State Key Laboratory of Virology and National Virus Resource Center, Wuhan Institute of Virology, Chinese Academy of Sciences, Wuhan, China.,Center for Biosafety Mega-Science, Chinese Academy of Sciences, Wuhan, China
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15
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Luo C, Wang Q, Guo R, Zhang J, Zhang J, Zhang R, Ma X, Wang P, Adam FEA, Zeshan B, Yang Z, Zhou Y, Wang X. A novel Pseudorabies virus vaccine developed using HDR-CRISPR/Cas9 induces strong humoral and cellular immune response in mice. Virus Res 2022; 322:198937. [PMID: 36174845 DOI: 10.1016/j.virusres.2022.198937] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/23/2022] [Revised: 09/20/2022] [Accepted: 09/24/2022] [Indexed: 12/24/2022]
Abstract
Outbreaks of Pseudorabies (PR) by numerous highly virulent and antigenic variant Pseudorabies virus (PRV) strains have been causing severe economic losses to the pig industry in China since 2011. However, current commercial vaccines are often unable to induce thorough protective immunity. In this study, a TK/gI/gE deleted recombinant PRV expressing GM-CSF was developed by using the HDR-CRISPR/Cas9 system. Here, a four-sgRNA along with the Cas9D10A targeting system was utilized for TK/gI/gE gene deletion and GM-CSF insertion. Our study showed that the four-sgRNA targeting system appeared to have higher knock-in efficiency for PRVs editing. The replication of the recombinant PRVs were slightly lower than that of the parental strain, but they appeared to have similar properties in terms of growth curves and plaque morphology. The mice vaccinated with the recombinant PRV expressing GM-CSF via intramuscular injection showed no obvious clinical symptoms, milder pathological lesions, and were completely protected against wild-type PRV challenge. When compared to the triple gene-deleted PRV, the gB antibodies and neutralizing antibody titers were improved and the immunized mice appeared to have lower viral load and higher mRNA levels of IL-2, IL-4, IL-6, and IFN-γ in spleens. Our study offers a novel approach for recombinant PRV construction, and the triple gene-deleted PRV expressing GM-CSF could serve as a promising vaccine candidate for PR control.
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Affiliation(s)
- Chen Luo
- Department of Life Science, Nanjing Xiaozhuang University, Nanjing,Jiangsu 211171, China; College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Qianqian Wang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Ruhai Guo
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Jingnan Zhang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Jingya Zhang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Riteng Zhang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Xin Ma
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Peixin Wang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | | | - Basit Zeshan
- Faculty of Sustainable Agriculture, Universiti Malaysia Sabah, Sandakan, Sabah 90509, Malaysia
| | - Zengqi Yang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China
| | - Yefei Zhou
- Department of Life Science, Nanjing Xiaozhuang University, Nanjing,Jiangsu 211171, China.
| | - Xinglong Wang
- College of Veterinary Medicine, Northwest A&F University, Yangling, Shaanxi 712100, China.
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16
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López-Muñoz AD, Rastrojo A, Martín R, Alcami A. High-throughput engineering of cytoplasmic- and nuclear-replicating large dsDNA viruses by CRISPR/Cas9. J Gen Virol 2022; 103:001797. [PMID: 36260063 PMCID: PMC10019086 DOI: 10.1099/jgv.0.001797] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/18/2022] Open
Abstract
The application of CRISPR/Cas9 to improve genome engineering efficiency for large dsDNA viruses has been extensively described, but a robust and versatile method for high-throughput generation of marker-free recombinants for a desired locus has not yet been reported. Cytoplasmic-replicating viruses use their own repair enzymes for homologous recombination, while nuclear-replicating viruses use the host repair machinery. This is translated into a wide range of Cas9-induced homologous recombination efficiencies, depending on the virus replication compartment and viral/host repair machinery characteristics and accessibility. However, the use of Cas9 as a selection agent to target parental virus genomes robustly improves the selection of desired recombinants across large dsDNA viruses. We used ectromelia virus (ECTV) and herpes simplex virus (HSV) type 1 and 2 to optimize a CRISPR/Cas9 method that can be used versatilely for efficient genome editing and selection of both cytoplasmic- and nuclear-replicating viruses. We performed a genome-wide genetic variant analysis of mutations located at predicted off-target sequences for 20 different recombinants, showing off-target-free accuracy by deep sequencing. Our results support this optimized method as an efficient, accurate and versatile approach to enhance the two critical factors of high-throughput viral genome engineering: generation and colour-based selection of recombinants. This application of CRISPR/Cas9 reduces the time and labour for screening of desired recombinants, allowing for high-throughput generation of large collections of mutant dsDNA viruses for a desired locus, optimally in less than 2 weeks.
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Affiliation(s)
- Alberto Domingo López-Muñoz
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain.,Present address: Cellular Biology Section, Laboratory of Viral Diseases, NIAID, NIH, Bethesda, Maryland, USA
| | - Alberto Rastrojo
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain.,Present address: Genetic Unit, Department of Biology, Universidad Autónoma de Madrid, Madrid, Spain
| | - Rocío Martín
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain
| | - Antonio Alcami
- Centro de Biología Molecular Severo Ochoa, Consejo Superior de Investigaciones Científicas and Universidad Autónoma de Madrid, Madrid, Spain
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17
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Advances in CRISPR/Cas9. BIOMED RESEARCH INTERNATIONAL 2022; 2022:9978571. [PMID: 36193328 PMCID: PMC9525763 DOI: 10.1155/2022/9978571] [Citation(s) in RCA: 9] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 06/02/2022] [Revised: 08/09/2022] [Accepted: 08/22/2022] [Indexed: 11/30/2022]
Abstract
CRISPR/Cas9 technology has become the most examined gene editing technology in recent years due to its simple design, yet low cost, high efficiency, and simple operation, which can also achieve simultaneous editing of multiple loci. It can also be carried out without using plasmids, saving lots of troubles caused by plasmids. CRISPR/Cas9 has shown great potential in the study of genes or genomic functions in microorganisms, plants, animals, and human beings. In this review, we will examine the history, structure, and basic mechanisms of the CRISPR/Cas9 system, describe its great value in precision medicine and sgRNA library screening, and dig its great potential in a new field: DNA information storage.
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18
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Najafi S, Tan SC, Aghamiri S, Raee P, Ebrahimi Z, Jahromi ZK, Rahmati Y, Sadri Nahand J, Piroozmand A, Jajarmi V, Mirzaei H. Therapeutic potentials of CRISPR-Cas genome editing technology in human viral infections. Biomed Pharmacother 2022; 148:112743. [PMID: 35228065 PMCID: PMC8872819 DOI: 10.1016/j.biopha.2022.112743] [Citation(s) in RCA: 31] [Impact Index Per Article: 10.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/13/2022] [Revised: 02/16/2022] [Accepted: 02/18/2022] [Indexed: 11/25/2022] Open
Abstract
Viral infections are a common cause of morbidity worldwide. The emergence of Coronavirus Disease 2019 (COVID-19) has led to more attention to viral infections and finding novel therapeutics. The CRISPR-Cas9 system has been recently proposed as a potential therapeutic tool for the treatment of viral diseases. Here, we review the research progress in the use of CRISPR-Cas technology for treating viral infections, as well as the strategies for improving the delivery of this gene-editing tool in vivo. Key challenges that hinder the widespread clinical application of CRISPR-Cas9 technology are also discussed, and several possible directions for future research are proposed.
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Affiliation(s)
- Sajad Najafi
- Student Research Committee, Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Shing Cheng Tan
- UKM Medical Molecular Biology Institute, Universiti Kebangsaan Malaysia, Kuala Lumpur, Malaysia
| | - Shahin Aghamiri
- Student Research Committee, Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Pourya Raee
- Department of Biology and Anatomical Sciences, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Zahra Ebrahimi
- Student Research Committee, Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Zahra Kargar Jahromi
- Central Research Laboratory, Jahrom University of Medical Sciences, Jahrom, Iran
| | - Yazdan Rahmati
- Department of Medical Genetics and Molecular Biology, Faculty of Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Javid Sadri Nahand
- Infectious and Tropical Diseases Research Center, Tabriz University of Medical Sciences, Tabriz, Iran
| | - Ahmad Piroozmand
- Autoimmune Diseases Research Center, Kashan University of Medical Sciences, Kashan, Iran
| | - Vahid Jajarmi
- Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran,Correspondence to: Department of Medical Biotechnology, School of Advanced Technologies in Medicine, Shahid Beheshti University of Medical Sciences, Tehran 19395-4818, Iran
| | - Hamed Mirzaei
- Student Research Committee, Kashan University of Medical Sciences, Kashan, Iran,Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran,Corresponding author at: Research Center for Biochemistry and Nutrition in Metabolic Diseases, Institute for Basic Sciences, Kashan University of Medical Sciences, Kashan, Iran
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19
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Liu CY, Jin M, Guo H, Zhao HZ, Hou LN, Yang Y, Wen YJ, Wang FX. Concurrent Gene Insertion, Deletion, and Inversion during the Construction of a Novel Attenuated BoHV-1 Using CRISPR/Cas9 Genome Editing. Vet Sci 2022; 9:vetsci9040166. [PMID: 35448664 PMCID: PMC9029512 DOI: 10.3390/vetsci9040166] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/28/2022] [Revised: 03/12/2022] [Accepted: 03/28/2022] [Indexed: 02/01/2023] Open
Abstract
Bovine herpesvirus type I (BoHV-1) is an important pathogen that causes respiratory disease in bovines. The disease is prevalent worldwide, causing huge economic losses to the cattle industry. Gene-deficient vaccines with immunological markers to distinguish them from wild-type infections have become a mainstream in vaccine research and development. In order to knock out the gE gene BoHV-1, we employed the CRISPR/Cas9 system. Interesting phenomena were observed at the single guide RNA (sgRNA) splicing site, including gene insertion, gene deletion, and the inversion of 5′ and 3′ ends of the sgRNA splicing site. In addition to the deletion of the gE gene, the US9 gene, and the non-coding regions of gE and US9, it was found that the US4 sequence, US6 sequence, and part of the US7 sequence were inserted into the EGFP sgRNA splicing site and the 3′ end of the EGFP sequence was deleted. Similar to the BoHV-1 parent, the BoHV-1 mutants induced high neutralizing antibodies titer levels in mice. In summary, we developed a series of recombinant gE-deletion BoHV-1 samples using the CRISPR/Cas9 gene editing system. The mutant viruses with EGFP+ or EGFP− will lay the foundation for research on BoHV-1 and vaccine development in the future.
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Affiliation(s)
- Chun-Yu Liu
- College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China; (C.-Y.L.); (M.J.); (H.G.); (H.-Z.Z.); (L.-N.H.)
| | - Ming Jin
- College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China; (C.-Y.L.); (M.J.); (H.G.); (H.-Z.Z.); (L.-N.H.)
| | - Hao Guo
- College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China; (C.-Y.L.); (M.J.); (H.G.); (H.-Z.Z.); (L.-N.H.)
| | - Hong-Zhe Zhao
- College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China; (C.-Y.L.); (M.J.); (H.G.); (H.-Z.Z.); (L.-N.H.)
| | - Li-Na Hou
- College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China; (C.-Y.L.); (M.J.); (H.G.); (H.-Z.Z.); (L.-N.H.)
| | - Yang Yang
- School of Life Sciences, Inner Mongolia University, Hohhot 010018, China;
| | - Yong-Jun Wen
- College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China; (C.-Y.L.); (M.J.); (H.G.); (H.-Z.Z.); (L.-N.H.)
- Correspondence: (Y.-J.W.); (F.-X.W.)
| | - Feng-Xue Wang
- College of Veterinary Medicine, Inner Mongolia Agricultural University, Hohhot 010018, China; (C.-Y.L.); (M.J.); (H.G.); (H.-Z.Z.); (L.-N.H.)
- Correspondence: (Y.-J.W.); (F.-X.W.)
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20
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Targeted deletion of glycoprotein B gene by CRISPR/Cas9 nuclease inhibits Gallid herpesvirus type 3 in dually-infected Marek's disease virus-transformed lymphoblastoid cell line MSB-1. J Virol 2022; 96:e0202721. [PMID: 35107377 PMCID: PMC8941929 DOI: 10.1128/jvi.02027-21] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
Marek’s disease virus (MDV) is a member of the genus Mardivirus in the subfamily Alphaherpesvirinae. There are three different serotypes of MDV designated as MDV-1 (Gallid herpesvirus type 2), MDV-2 (Gallid herpesvirus type 3), and MDV-3 (Meleagrid herpesvirus 1, herpesvirus of turkeys, HVT). MDV-1 is the only serotype that induces Marek’s disease (MD), a lymphoproliferative disorder resulting in aggressive T-cell lymphomas and paralytic symptoms. In the lymphomas and lymphoblastoid cell lines (LCL) derived from them, MDV establishes latent infection with limited viral gene expression. The latent viral genome in LCL can be activated by co-cultivation with chicken embryo fibroblast (CEF) monolayers. MSB-1, one of the first MDV-transformed LCL established from the splenic lymphoma, is distinct in harboring both the oncogenic MDV-1 and non-oncogenic MDV-2 viruses. Following the successful application of CRISPR/Cas9 editing approach for precise knockdown of the MDV-1 genes in LCL, we describe here the targeted deletion of MDV-2 glycoprotein B (gB) in MSB-1 cells. Due to the essential nature of gB for infectivity, the production of MDV-2 plaques on CEF was completely abolished in the MDV-2-gB-deleted MSB-1 cells. Our study has demonstrated that the CRISPR/Cas9 system can be used for targeted inactivation of the co-infecting MDV-2 without affecting the MDV-1 in the MSB-1 cell line. Successful inactivation of MDV-2 demonstrated here also points toward the possibility of using targeted gene editing as an antiviral strategy against pathogenic MDV-1 and other viruses infecting chickens. IMPORTANCE Marek’s disease (MD) is a lymphoproliferative disease of chickens characterized by rapid-onset lymphomas in multiple organs and by infiltration into peripheral nerves, causing paralysis. Lymphoblastoid cell lines (LCL) derived from MD lymphomas have served as valuable resources to improve understanding of distinct aspects of virus-host interactions in transformed cells including transformation, latency, and reactivation. MDV-transformed LCL MSB-1, derived from spleen lymphoma induced by the BC-1 strain of MDV, has a unique feature of harboring an additional non-pathogenic MDV-2 strain HPRS-24. By targeted deletion of essential gene glycoprotein B from the MDV-2 genome within the MSB-1 cells, we demonstrated the total inhibition of MDV-2 virus replication on co-cultivated CEF, with no effect on MDV-1 replication. The identified viral genes critical for reactivation/inhibition of viruses will be useful as targets for development of de novo disease resistance in chickens to avian pathogens.
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21
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Li Y, Wei Y, Li G, Huang S, Xu J, Ding Q, Hong J. Targeting NECTIN-1 Based on CRISPR/Cas9 System Attenuated the Herpes Simplex Virus Infection in Human Corneal Epithelial Cells In Vitro. Transl Vis Sci Technol 2022; 11:8. [PMID: 35119473 PMCID: PMC8819356 DOI: 10.1167/tvst.11.2.8] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/06/2021] [Accepted: 01/05/2022] [Indexed: 11/24/2022] Open
Abstract
PURPOSE Viral keratitis caused by herpes simplex virus 1 (HSV-1) is a lifelong recurring disease and an unignored cause of blindness worldwide. Current antiviral therapy cannot eliminate the transcriptionally silent HSV-1 in latently infected patients. With the explosive applications of the clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated nuclease (Cas) 9 gene-editing system in recent years, we aim to develop a CRISPR/Cas9 system targeting down the major HSV receptor, NECTIN-1 on human corneal epithelial cells (HCECs), to provide a novel strategy for herpes simplex keratitis (HSK) treatment. METHODS The selected single guide RNAs (sgRNAs) targeting human nectin cell adhesion molecule 1 (NECTIN-1), together with Cas-9, were assembled into lentivirus. HCECs were infected with Lenti-Cas9-gRNAs to establish NECTIN-1 knockdown cells. Following HSV-green fluorescent protein (GFP) infection, cell survival and virus infection were determined by fluorescence microscopy and flow cytometry. Relative HSV DNA amount was also compared through quantitative reverse transcriptase-polymerase chain reaction. RESULTS Lentivirus packaged with the CRISPR/Cas9 system and the two selected sgRNAs both successfully edited down the protein levels of NECTIN-1 of HCECs. After HSV-GFP infection, the infection rate of HCECs in knockdown groups dramatically decreased, especially in the NECTIN-1 knockdown group 1. In addition, the relative HSV DNA amount of both knockdown groups was only 30% when compared with the control group. CONCLUSIONS We successfully knocked down the NECTIN-1 expression in vitro by the CRISPR/Cas9 system, which alleviated the HSV infection in HCECs. TRANSLATIONAL RELEVANCE This study offered a promising target for the cure of HSK.
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Affiliation(s)
- Yue Li
- Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, Shanghai, China
- NHC Key Laboratory of Myopia, Fudan University, Shanghai, China
- Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China
| | - Yuda Wei
- CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Gang Li
- Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, Shanghai, China
- NHC Key Laboratory of Myopia, Fudan University, Shanghai, China
- Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China
| | - Shiqian Huang
- CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Jianjiang Xu
- Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, Shanghai, China
- NHC Key Laboratory of Myopia, Fudan University, Shanghai, China
- Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China
| | - Qiurong Ding
- CAS Key Laboratory of Nutrition and Metabolism, Institute for Nutritional Sciences, Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences, Shanghai, China
| | - Jiaxu Hong
- Eye Institute and Department of Ophthalmology, Eye & ENT Hospital, Fudan University, Shanghai, China
- NHC Key Laboratory of Myopia, Fudan University, Shanghai, China
- Shanghai Key Laboratory of Visual Impairment and Restoration, Shanghai, China
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22
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Wang L, Chen Y, Liu X, Li Z, Dai X. The Application of CRISPR/Cas9 Technology for Cancer Immunotherapy: Current Status and Problems. Front Oncol 2022; 11:704999. [PMID: 35111663 PMCID: PMC8801488 DOI: 10.3389/fonc.2021.704999] [Citation(s) in RCA: 13] [Impact Index Per Article: 4.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/04/2021] [Accepted: 12/27/2021] [Indexed: 12/19/2022] Open
Abstract
Cancer is one of the main causes of disease-related deaths in the world. Although cancer treatment strategies have been improved in recent years, the survival time of cancer patients is still far from satisfied. Cancer immunotherapy, such as Oncolytic virotherapy, Immune checkpoints inhibition, Chimeric antigen receptor T (CAR-T) cell therapy, Chimeric antigen receptor natural killer (CAR-NK) cell therapy and macrophages genomic modification, has emerged as an effective therapeutic strategy for different kinds of cancer. However, many patients do not respond to the cancer immunotherapy which warrants further investigation to optimize this strategy. The clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR/Cas9), as a versatile genome engineering tool, has become popular in the biology research field and it was also applied to optimize tumor immunotherapy. Moreover, CRISPR-based high-throughput screening can be used in the study of immunomodulatory drug resistance mechanism. In this review, we summarized the development as well as the application of CRISPR/Cas9 technology in the cancer immunotherapy and discussed the potential problems that may be caused by this combination.
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Affiliation(s)
- Luyao Wang
- Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, First Hospital, Jilin University, Changchun, China
- National-Local Joint Engineering Laboratory of Animal Models for Human Disease, First Hospital, Jilin University, Changchun, China
| | - Yurong Chen
- Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, First Hospital, Jilin University, Changchun, China
- National-Local Joint Engineering Laboratory of Animal Models for Human Disease, First Hospital, Jilin University, Changchun, China
| | - Xinrui Liu
- Neurosurgery Department, First Hospital, Jilin University, Changchun, China
| | - Ziyi Li
- Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, First Hospital, Jilin University, Changchun, China
- National-Local Joint Engineering Laboratory of Animal Models for Human Disease, First Hospital, Jilin University, Changchun, China
| | - Xiangpeng Dai
- Key Laboratory of Organ Regeneration and Transplantation of Ministry of Education, First Hospital, Jilin University, Changchun, China
- National-Local Joint Engineering Laboratory of Animal Models for Human Disease, First Hospital, Jilin University, Changchun, China
- *Correspondence: Xiangpeng Dai,
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23
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Oh HS, Diaz FM, Zhou C, Carpenter N, Knipe DM. CRISPR-Cas9 Expressed in Stably Transduced Cell Lines Promotes Recombination and Selects for Herpes Simplex Virus Recombinants. CURRENT RESEARCH IN VIROLOGICAL SCIENCE 2022; 3:100023. [PMID: 36330462 PMCID: PMC9629518 DOI: 10.1016/j.crviro.2022.100023] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 05/12/2023]
Abstract
Recombinant herpes simplex virus strains can be constructed by several methods, including homologous recombination, bacterial artificial chromosome manipulation, and yeast genetic methods. Homologous recombination may have the advantage of introducing fewer genetic alterations in the viral genome, but the low level of recombinants can make this method more time consuming if there is no screen or selection. In this study we used complementing cell lines that express Cas9 and guide RNAs targeting the parental virus to rapidly generate recombinant viruses. Analysis of the progeny viruses indicated that CRISPR-Cas9 both promoted recombination to increase recombinant viruses and selected against parental viruses in the transfection progeny viruses. This approach can also be used to enrich for recombinants made by any of the current methods.
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Affiliation(s)
- Hyung Suk Oh
- Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA
| | - Fernando M. Diaz
- Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA
| | - Changhong Zhou
- Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA
| | - Nicholas Carpenter
- Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA
| | - David M. Knipe
- Department of Microbiology, Blavatnik Institute, Harvard Medical School, Boston, MA
- To whom correspondence should be directed:
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24
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Söllner JH, Mettenleiter TC, Petersen B. Genome Editing Strategies to Protect Livestock from Viral Infections. Viruses 2021; 13:1996. [PMID: 34696426 PMCID: PMC8539128 DOI: 10.3390/v13101996] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/27/2021] [Revised: 09/27/2021] [Accepted: 09/29/2021] [Indexed: 12/26/2022] Open
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 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.
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Affiliation(s)
- Jenny-Helena Söllner
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, 31535 Neustadt am Rübenberge, Germany;
| | | | - Björn Petersen
- Institute of Farm Animal Genetics, Friedrich-Loeffler-Institut, 31535 Neustadt am Rübenberge, Germany;
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25
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Tang N, Zhang Y, Shen Z, Yao Y, Nair V. Application of CRISPR-Cas9 Editing for Virus Engineering and the Development of Recombinant Viral Vaccines. CRISPR J 2021; 4:477-490. [PMID: 34406035 DOI: 10.1089/crispr.2021.0017] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/26/2022] Open
Abstract
CRISPR-Cas technology, discovered originally as a bacterial defense system, has been extensively repurposed as a powerful tool for genome editing for multiple applications in biology. In the field of virology, CRISPR-Cas9 technology has been widely applied on genetic recombination and engineering of genomes of various viruses to ask some fundamental questions about virus-host interactions. Its high efficiency, specificity, versatility, and low cost have also provided great inspiration and hope in the field of vaccinology to solve a series of bottleneck problems in the development of recombinant viral vaccines. This review highlights the applications of CRISPR editing in the technological advances compared to the traditional approaches used for the construction of recombinant viral vaccines and vectors, the main factors affecting their application, and the challenges that need to be overcome for further streamlining their effective usage in the prevention and control of diseases. Factors affecting efficiency, target specificity, and fidelity of CRISPR-Cas editing in the context of viral genome editing and development of recombinant vaccines are also discussed.
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Affiliation(s)
- Na Tang
- Shandong Binzhou Animal Science and Veterinary Medicine Academy and UK-China Centre of Excellence for Research on Avian Diseases, Binzhou, P.R. China; University of Oxford, Oxford, United Kingdom
| | - Yaoyao Zhang
- The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash road, Guildford, Surrey, United Kingdom; University of Oxford, Oxford, United Kingdom
| | - Zhiqiang Shen
- Shandong Binzhou Animal Science and Veterinary Medicine Academy and UK-China Centre of Excellence for Research on Avian Diseases, Binzhou, P.R. China; University of Oxford, Oxford, United Kingdom
| | - Yongxiu Yao
- The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash road, Guildford, Surrey, United Kingdom; University of Oxford, Oxford, United Kingdom
| | - Venugopal Nair
- The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash road, Guildford, Surrey, United Kingdom; University of Oxford, Oxford, United Kingdom.,The Jenner Institute Laboratories, University of Oxford, Oxford, United Kingdom; and University of Oxford, Oxford, United Kingdom.,Department of Zoology, University of Oxford, Oxford, United Kingdom
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Fu PF, Cheng X, Su BQ, Duan LF, Wang CR, Niu XR, Wang J, Yang GY, Chu BB. CRISPR/Cas9-based generation of a recombinant double-reporter pseudorabies virus and its characterization in vitro and in vivo. Vet Res 2021; 52:95. [PMID: 34174954 PMCID: PMC8233574 DOI: 10.1186/s13567-021-00964-4] [Citation(s) in RCA: 4] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/19/2021] [Accepted: 05/27/2021] [Indexed: 11/24/2022] Open
Abstract
Pseudorabies, caused by pseudorabies virus (PRV) variants, has broken out among commercial PRV vaccine-immunized swine herds and resulted in major economic losses to the pig industry in China since late 2011. However, the mechanism of virulence enhancement of variant PRV is currently unclear. Here, a recombinant PRV (rPRV HN1201-EGFP-Luc) with stable expression of enhanced green fluorescent protein (EGFP) and firefly luciferase as a double reporter virus was constructed on the basis of the PRV variant HN1201 through CRISPR/Cas9 gene-editing technology coupled with two sgRNAs. The biological characteristics of the recombinant virus and its lethality to mice were similar to those of the parental strain and displayed a stable viral titre and luciferase activity through 20 passages. Moreover, bioluminescence signals were detected in mice at 12 h after rPRV HN1201-EGFP-Luc infection. Using the double reporter PRV, we also found that 25-hydroxycholesterol had a significant inhibitory effect on PRV both in vivo and in vitro. These results suggested that the double reporter PRV based on PRV variant HN1201 should be an excellent tool for basic virology studies and evaluating antiviral agents.
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Affiliation(s)
- Peng-Fei Fu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China
| | - Xuan Cheng
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China
| | - Bing-Qian Su
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China
| | - Li-Fang Duan
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China
| | - Cong-Rong Wang
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China
| | - Xin-Rui Niu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China
| | - Jiang Wang
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China
| | - Guo-Yu Yang
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China.
| | - Bei-Bei Chu
- College of Veterinary Medicine, Henan Agricultural University, Zhengzhou, 450046, Henan, China.
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27
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Huang X, Li X, Yang L, Wang P, Yan J, Nie Z, Gao Y, Li Z, Wen J, Cao X. Construction and optimization of herpes simplex virus vectors for central nervous system gene delivery based on CRISPR/Cas9-mediated genome editing. Curr Gene Ther 2021; 22:66-77. [PMID: 34148538 DOI: 10.2174/1566523219666210618154326] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Revised: 04/13/2021] [Accepted: 04/18/2021] [Indexed: 11/22/2022]
Abstract
AIMS We aim to define parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors and optimize the expression cassettes to achieve robust and sustained expression in CNS. BACKGROUND Engineered, attenuated Herpes simplex virus (HSV) vectors are promising vehicles for gene delivery to the peripheral and central nervous systems. The virus latent promoter (LAP) is commonly used to drive exogenous gene expression; however, parameters affecting the safety and long-term transgene expression of attenuated HSV-1 vectors have not been fully understood. OBJECTIVE This study aimed to construct attenuated HSV-1 vectors using the CRISPR-Cas9 system and examine the influence of transgene cassette construction and insertion site on transgene expression and vector safety. METHOD In this study, we used a CRISPR-Cas9 system to accurately and efficiently edit attenuated HSV-1 strain 1716, and constructed two series of recombinant virus LMR and LMRx with different sets of gene cassettes insertion in Exon1(LAP2) and 2.0 kb intron downstream of LAP, respectively. The transgene expression and viral gene transcriptional kinetics were compared in in-vitro cell lines. The reporter gene expression and safety profiles of each vector were further evaluated in the mouse hippocampus gene transduction model. RESULT The in-vitro cell line analysis indicated that the insertion of a gene expression cassette would disrupt virus gene transcription. Mouse hippocampus transducing analysis suggested that complete expression cassette insertion at 2.0 kb intron could achieve robust and longtime gene expression than the other constructs. Recombinants with gene expression cassettes lacked Poly (A), which induced significant neuronal inflammation due to persistent viral antigen expression and microglia activation. CONCLUSION Our results indicated that the integrity of LAT transcripts was not necessary for the establishment of long-term latent expression. Exogenous strong promoters (like cBh promoter) could remain active during latency when placed in Exon1 or 2.0 Kb Intron of LAT locus, although their transcriptional activity declined with time. Consistent with previous research, the foreign gene expression would last much longer when the gene cassette was located downstream of Exon1, which suggested a role of LAP2 in maintaining promoter activity during latency. Besides, over-transcription of the downstream part of LAT may induce continuous activation of the attenuated vectors, suggesting an important role of LAT in maintaining viral reactivation potential.
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Affiliation(s)
- Xinwei Huang
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
| | - Xiuqing Li
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
| | - Lijuan Yang
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
| | - Pengfei Wang
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
| | - Jingyuan Yan
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
| | - Zuqing Nie
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
| | - Yingzheng Gao
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
| | - Zhiwei Li
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
| | - Jie Wen
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
| | - Xia Cao
- Key Laboratory of The Second Affiliated Hospital of Kuming Medical College, Kunming, 650101, China
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Methods for the Manipulation of Herpesvirus Genome and the Application to Marek's Disease Virus Research. Microorganisms 2021; 9:microorganisms9061260. [PMID: 34200544 PMCID: PMC8228275 DOI: 10.3390/microorganisms9061260] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/30/2021] [Revised: 05/29/2021] [Accepted: 06/08/2021] [Indexed: 11/30/2022] Open
Abstract
Herpesviruses are a group of double-strand DNA viruses that infect a wide range of hosts, including humans and animals. In the past decades, numerous methods have been developed to manipulate herpesviruses genomes, from the introduction of random mutations to specific genome editing. The development of genome manipulation methods has largely advanced the study of viral genes function, contributing not only to the understanding of herpesvirus biology and pathogenesis, but also the generation of novel vaccines and therapies to control and treat diseases. In this review, we summarize the major methods of herpesvirus genome manipulation with emphasis in their application to Marek’s disease virus research.
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29
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Hassanzadeh P. The significance of bioengineered nanoplatforms against SARS-CoV-2: From detection to genome editing. Life Sci 2021; 274:119289. [PMID: 33676931 PMCID: PMC7930743 DOI: 10.1016/j.lfs.2021.119289] [Citation(s) in RCA: 5] [Impact Index Per Article: 1.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/06/2020] [Revised: 02/12/2021] [Accepted: 02/20/2021] [Indexed: 12/19/2022]
Abstract
COVID-19 outbreak can impose serious negative impacts on the infrastructures of societies including the healthcare systems. Despite the increasing research efforts, false positive or negative results that may be associated with serologic or even RT-PCR tests, inappropriate or variable immune response, and high rates of mutations in coronavirus may negatively affect virus detection process and effectiveness of the vaccines or drugs in development. Nanotechnology-based research attempts via developing state-of-the-art techniques such as nanomechatronics ones and advanced materials including the sensors for detecting the pathogen loads at very low concentrations or site-specific delivery of therapeutics, and real-time protections against the pandemic outbreaks by nanorobots can provide outstanding biomedical breakthroughs. Considering the unique characteristics of pathogens particularly the newly-emerged ones and avoiding the exaggerated optimism or simplistic views on the prophylactic and therapeutic approaches including the one-size-fits-all ones or presenting multiple medications that may be associated with synergistic toxicities rather than enhanced efficiencies might pave the way towards the development of more appropriate treatment strategies with reduced safety concerns. This paper highlights the significance of nanoplatforms against the viral disorders and their capabilities of genome editing that may facilitate taking more appropriate measures against SARS-CoV-2.
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Affiliation(s)
- Parichehr Hassanzadeh
- Nanotechnology Research Center, Faculty of Pharmacy, Tehran University of Medical Sciences, Tehran 13169-43551, Iran.
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30
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Shi J, Zheng S, Wu X, Peng Z, Li C, Wang S, Xin C, Xu S, Li J. Efficient influence of ssDNA virus PCV2 replication by CRISPR/Cas9 targeting of the viral genome. Mol Immunol 2021; 133:63-66. [PMID: 33631556 DOI: 10.1016/j.molimm.2021.01.024] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/12/2020] [Revised: 11/23/2020] [Accepted: 01/19/2021] [Indexed: 11/20/2022]
Abstract
Porcine circovirus type 2 (PCV2), a ubiquitous pathogen that primary cause of postweaning multisystemic wasting syndrome (PMWS), had caused significant morbidity and mortality in swine populations with huge economic losses in the worldwide swine industry. Currently, looking for effective antiviral drugs for PCV2 infection remains an important works. In our study, CRISPR/Cas9 system was used to further detected the key sites of PCV2 replication. We designed 8 single guide RNAs (sgRNA) by targeting essential genes across the genome of PCV2. Western-blot(WB), Cell counting kit-8 for high-throughput sgRNA screening were applied to detect PCV2 replication levels. The results showed that Oc8, O13, O134, NQT and NPS sgRNAs can edit the PCV2 genome efficiently and inhibit PCV2 replication in PK-15 cell; H3 sgRNA cannot edit the PCV2 genome successfully; NAT sgRNA can edit the PCV2 genome efficiently to improve the PCV2 replication in PK-15 cell; O26 sgRNA can edit the PCV2 genome successfully but it is not known yet of its effect on PCV2 replication, besides the Cas9 expression had no effect on cell viability. These data suggest that CRISPR/Cas9 system targeting PCV2 essential genes may serve as a novel therapeutic agent against PCV2 infection in the future.
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Affiliation(s)
- Jianli Shi
- Division of Swine Diseases, Shandong Provincial Key Laboratory of Animal Disease Control & Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan, 250100, China
| | - Shuxuan Zheng
- University Medical Center Utrecht, Department of Medical Microbiology, the Netherlands
| | - Xiaoyan Wu
- Division of Swine Diseases, Shandong Provincial Key Laboratory of Animal Disease Control & Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan, 250100, China; Shandong Normal University, Jinan, 250014, China
| | - Zhe Peng
- Division of Swine Diseases, Shandong Provincial Key Laboratory of Animal Disease Control & Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan, 250100, China
| | - Chen Li
- Division of Swine Diseases, Shandong Provincial Key Laboratory of Animal Disease Control & Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan, 250100, China
| | - Shuo Wang
- Division of Swine Diseases, Shandong Provincial Key Laboratory of Animal Disease Control & Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan, 250100, China; Shandong Normal University, Jinan, 250014, China
| | - Changxun Xin
- Division of Swine Diseases, Shandong Provincial Key Laboratory of Animal Disease Control & Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan, 250100, China; Qingdao Agricultural University, Qingdao, 266000, China
| | - Shaojian Xu
- Division of Swine Diseases, Shandong Provincial Key Laboratory of Animal Disease Control & Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan, 250100, China
| | - Jun Li
- Division of Swine Diseases, Shandong Provincial Key Laboratory of Animal Disease Control & Breeding, Institute of Animal Science and Veterinary Medicine Shandong Academy of Agricultural Sciences, Jinan, 250100, China; Qingdao Agricultural University, Qingdao, 266000, China; Shandong Normal University, Jinan, 250014, China.
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31
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Latest Advances of Virology Research Using CRISPR/Cas9-Based Gene-Editing Technology and Its Application to Vaccine Development. Viruses 2021; 13:v13050779. [PMID: 33924851 PMCID: PMC8146441 DOI: 10.3390/v13050779] [Citation(s) in RCA: 35] [Impact Index Per Article: 8.8] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2021] [Revised: 04/26/2021] [Accepted: 04/27/2021] [Indexed: 02/07/2023] Open
Abstract
In recent years, the CRISPR/Cas9-based gene-editing techniques have been well developed and applied widely in several aspects of research in the biological sciences, in many species, including humans, animals, plants, and even in viruses. Modification of the viral genome is crucial for revealing gene function, virus pathogenesis, gene therapy, genetic engineering, and vaccine development. Herein, we have provided a brief review of the different technologies for the modification of the viral genomes. Particularly, we have focused on the recently developed CRISPR/Cas9-based gene-editing system, detailing its origin, functional principles, and touching on its latest achievements in virology research and applications in vaccine development, especially in large DNA viruses of humans and animals. Future prospects of CRISPR/Cas9-based gene-editing technology in virology research, including the potential shortcomings, are also discussed.
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32
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Xie Q, Cao S, Zhang W, Wang W, Li L, Kan Q, Fu H, Geng T, Li T, Wan Z, Gao W, Shao H, Qin A, Ye J. A novel fiber-2-edited live attenuated vaccine candidate against the highly pathogenic serotype 4 fowl adenovirus. Vet Res 2021; 52:35. [PMID: 33640033 PMCID: PMC7912893 DOI: 10.1186/s13567-021-00907-z] [Citation(s) in RCA: 24] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/17/2020] [Accepted: 01/21/2021] [Indexed: 02/06/2023] Open
Abstract
Recently, the outbreaks of hydropericardium-hepatitis syndrome (HHS) caused by the highly pathogenic fowl adenovirus serotype 4 (FAdV-4) have resulted in huge economic losses to the poultry industry globally. Although several inactivated or subunit vaccines have been developed against FAdV-4, live-attenuated vaccines for FAdV-4 are rarely reported. In this study, a recombinant virus FA4-EGFP expressing EGFP-Fiber-2 fusion protein was generated by the CRISPR/Cas9 technique. Although FA4-EGFP shows slightly lower replication ability than the wild type (WT) FAdV-4, FA4-EGFP was significantly attenuated in vivo compared with the WT FAdV-4. Chickens infected with FA4-EGFP did not show any clinical signs, and all survived to 14 day post-infection (dpi), whereas those infected with FAdV-4 showed severe clinical signs with HHS and all died at 4 dpi. Besides, the inoculation of FA4-EGFP in chickens provided efficient protection against lethal challenge with FAdV-4. Compared with an inactivated vaccine, FA4-EGFP induced neutralizing antibodies with higher titers earlier. All these data not only provide a live-attenuated vaccine candidate against the highly pathogenic FAdV-4 but also give a potential insertion site for developing FAdV-4-based vaccine vectors for delivering foreign antigens.
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Affiliation(s)
- Quan Xie
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Shiya Cao
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Wei Zhang
- Sinopharm Yangzhou VAC Biological Engineering Co.Ltd, Yangzhou, 225127, Jiangsu, China
| | - Weikang Wang
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Luyuan Li
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Qiuqi Kan
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Hui Fu
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Tuoyu Geng
- College of Animal Science and Technology, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Tuofan Li
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Zhimin Wan
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Wei Gao
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Hongxia Shao
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Aijian Qin
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China.,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China.,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China
| | - Jianqiang Ye
- Key Laboratory of Jiangsu Preventive Veterinary Medicine, Key Laboratory for Avian Preventive Medicine, Ministry of Education, College of Veterinary Medicine, Yangzhou University, Yangzhou, 225009, Jiangsu, China. .,Jiangsu Co-Innovation Center for Prevention and Control of Important Animal Infectious Diseases and Zoonoses, Yangzhou, 225009, Jiangsu, China. .,Joint International Research Laboratory of Agriculture and Agri-Product Safety, the Ministry of Education of China, Yangzhou University, Yangzhou, 225009, Jiangsu, China. .,Institute of Agricultural Science and Technology Development, Yangzhou University, Yangzhou, 225009, Jiangsu, China.
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33
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Kushch AA, Kisteneva LB, Klimova RR, Cheshik SG. [The role of herpesviruses in development of diseases of the urogenital tract and infertility in women]. Vopr Virusol 2021; 65:317-325. [PMID: 33533228 DOI: 10.36233/0507-4088-2020-65-6-2] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/07/2021] [Accepted: 01/07/2021] [Indexed: 11/05/2022]
Abstract
This review presents the data on the spreading of all known human herpesviruses (НHVs) in female urogenital tract. According to the WHO almost 500 million people worldwide suffer from genital infection caused by НHVs. НHVs were detected in various inflammatory diseases of female upper and lower genital tract (vaginitis and cervicitis), in extrauterine pregnancy (in fallopian tubes), in infertility (cervical channel, endometrium and ovaries). Herpes simplex virus 1 (HSV‑1) was identified for the first time in oocytes after failed in vitro fertilization (IVF). НHVs produce negative effect on the entire reproductive process from conception to childbirth. It was established that HSV, cytomegalovirus (CMV) and human herpesvirus 6 (HHV-6) markedly increase the risk of spontaneous abortion, preterm birth and stillbirth. Intrauterine НHV infection is a major cause of congenital malformations. Data on humoral and cell immunity in genital herpesvirus infections (НHVI) are also reviewed. Intravaginal HSV‑2 infection changes cell composition of vaginal mucosa, i.e., together with cells mobilized from the blood, protective role is performed by resident memory T‑cells (TRM), natural killer cells (NK‑cells) and regulatory T‑cells (Treg) whose function consists in maintaining the balance of the activities of lymphocytes. Constant НHVI spreading is largely explained by transition of primary infection to potentially reactivating latent form, since latent virus is unavailable to immune recognition and medicines. The genome editing system CRISPR/Cas9 can recognize and modify not only active but also latent viruses. The promising pilot results with the use of this system offer the possibility of developing innovative technologies for НHV elimination and НHVI eradication.
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Affiliation(s)
- A A Kushch
- Ivanovsky Institute of Virology of FSBI «National Research Centre of Epidemiology and Microbiology named after honorary academician N.F. Gamaleya» of the Ministry of Health of Russia
| | - L B Kisteneva
- Ivanovsky Institute of Virology of FSBI «National Research Centre of Epidemiology and Microbiology named after honorary academician N.F. Gamaleya» of the Ministry of Health of Russia
| | - R R Klimova
- Ivanovsky Institute of Virology of FSBI «National Research Centre of Epidemiology and Microbiology named after honorary academician N.F. Gamaleya» of the Ministry of Health of Russia
| | - S G Cheshik
- Ivanovsky Institute of Virology of FSBI «National Research Centre of Epidemiology and Microbiology named after honorary academician N.F. Gamaleya» of the Ministry of Health of Russia
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Matía A, Lorenzo MM, Blasco R. Tools for the targeted genetic modification of poxvirus genomes. Curr Opin Virol 2020; 44:183-190. [PMID: 33242829 DOI: 10.1016/j.coviro.2020.10.006] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.2] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2020] [Revised: 10/27/2020] [Accepted: 10/27/2020] [Indexed: 12/14/2022]
Abstract
The potential of viruses as biotechnology platforms is becoming more appealing due to technological advances in synthetic biology techniques and to the increasing accessibility of means to manipulate virus genomes. Among viral systems, poxviruses, and their prototype member Vaccinia Virus, are one of the outstanding choices for different biotechnological and medical applications based on heterologous gene expression, recombinant vaccines or oncolytic viruses. The refinement of genetic engineering methods on Vaccinia Virus over the last decades have contributed to facilitate the manipulation of the genomes of poxviruses, and may aid in the improvement of virus variants designed for different goals through reverse genetic approaches. Targeted genetic changes are usually performed by homologous recombination with the viral genome. In addition to the classic approach, recent methodological advances that may assist new strategies for the mutation or edition of poxvirus genomes are reviewed.
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Affiliation(s)
- Alejandro Matía
- Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (I.N.I.A.), Ctra. La Coruña km 7.5, E-28040 Madrid, Spain
| | - María M Lorenzo
- Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (I.N.I.A.), Ctra. La Coruña km 7.5, E-28040 Madrid, Spain
| | - Rafael Blasco
- Departamento de Biotecnología, Instituto Nacional de Investigación y Tecnología Agraria y Alimentaria (I.N.I.A.), Ctra. La Coruña km 7.5, E-28040 Madrid, Spain.
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Vilela J, Rohaim MA, Munir M. Application of CRISPR/Cas9 in Understanding Avian Viruses and Developing Poultry Vaccines. Front Cell Infect Microbiol 2020; 10:581504. [PMID: 33330126 PMCID: PMC7732654 DOI: 10.3389/fcimb.2020.581504] [Citation(s) in RCA: 8] [Impact Index Per Article: 1.6] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/08/2020] [Accepted: 09/25/2020] [Indexed: 12/15/2022] Open
Abstract
Clustered regularly interspaced short palindromic repeats associated protein nuclease 9 (CRISPR-Cas9) technology offers novel approaches to precisely, cost-effectively, and user-friendly edit genomes for a wide array of applications and across multiple disciplines. This methodology can be leveraged to underpin host-virus interactions, elucidate viral gene functions, and to develop recombinant vaccines. The successful utilization of CRISPR/Cas9 in editing viral genomes has paved the way of developing novel and multiplex viral vectored poultry vaccines. Furthermore, CRISPR/Cas9 can be exploited to rectify major limitations of conventional approaches including reversion to virulent form, recombination with field viruses and transgene, and genome instability. This review provides comprehensive analysis of the potential of CRISPR/Cas9 genome editing technique in understanding avian virus-host interactions and developing novel poultry vaccines. Finally, we discuss the simplest and practical aspects of genome editing approaches in generating multivalent recombinant poultry vaccines that conform simultaneous protection against major avian diseases.
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Affiliation(s)
- Julianne Vilela
- Division of Biomedical and Life Sciences, The Lancaster University, Lancaster, United Kingdom
| | - Mohammed A Rohaim
- Division of Biomedical and Life Sciences, The Lancaster University, Lancaster, United Kingdom
| | - Muhammad Munir
- Division of Biomedical and Life Sciences, The Lancaster University, Lancaster, United Kingdom
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A highly efficient recombinant canarypox virus-based vaccine against canine distemper virus constructed using the CRISPR/Cas9 gene editing method. Vet Microbiol 2020; 251:108920. [PMID: 33197867 DOI: 10.1016/j.vetmic.2020.108920] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/08/2020] [Accepted: 11/01/2020] [Indexed: 12/11/2022]
Abstract
Canine distemper virus (CDV) is the causative agent of canine distemper (CD), which is one of the most important infectious diseases affecting wild and domestic carnivores. Vaccination represents an effective approach to prevent CDV infection among domestic carnivores. Canarypox-vectored recombinant CD vaccines (such as Recombitek CDV, PureVax Ferret Distemper, and Merial) with the CDV hemagglutinin (H) and fusion (F) genes can induce a potent immune response in dogs and ferrets. However, the vaccine's effectiveness varies with the species. In the current study, we developed a highly efficient recombinant canarypox virus termed as "ALVAC-CDV-M-F-H/C5-" that contained CDV virus-like particles (VLPs) by using the CRISPR/Cas9 gene editing method, which enabled concurrent expression of the matrix (M), H, and F genes. The recombinant strain provided faster seroconversion than the parent strain among minks as well as provided higher rates of antibody positivity than the parent strain among foxes and minks even before the administration of a second booster vaccination. We demonstrated, for the first time, that the CRISPR/Cas9 system can be applied for the rapid and efficient modification of the ALVAC-CDV-F-H genome and also that a high-dose new recombinant strain that produces CDV VLPs may present good outcomes in the prevention of CD among foxes and minks.
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Rapid poxvirus engineering using CRISPR/Cas9 as a selection tool. Commun Biol 2020; 3:643. [PMID: 33144673 PMCID: PMC7641209 DOI: 10.1038/s42003-020-01374-6] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/09/2020] [Accepted: 10/01/2020] [Indexed: 02/08/2023] Open
Abstract
In standard uses of CRISPR/Cas9 technology, the cutting of genomes and their efficient repair are considered to go hand-in-hand to achieve desired genetic changes. This includes the current approach for engineering genomes of large dsDNA viruses. However, for poxviruses we show that Cas9-guide RNA complexes cut viral genomes soon after their entry into cells, but repair of these breaks is inefficient. As a result, Cas9 targeting makes only modest, if any, improvements to basal rates of homologous recombination between repair constructs and poxvirus genomes. Instead, Cas9 cleavage leads to inhibition of poxvirus DNA replication thereby suppressing virus spread in culture. This unexpected outcome allows Cas9 to be used as a powerful tool for selecting conventionally generated poxvirus recombinants, which are otherwise impossible to separate from a large background of parental virus without the use of marker genes. This application of CRISPR/Cas9 greatly speeds up the generation of poxvirus-based vaccines, making this platform considerably more attractive in the context of personalised cancer vaccines and emerging disease outbreaks. Gowripalan, Smith et al. use CRISPR/Cas9 technology to rapidly select recombinant poxviruses without using selectable marker genes. They find that Cas9 cleavage inhibits poxvirus DNA replication, suppressing virus spread in culture. This application makes poxviruses more attractive vector platforms for fighting cancer and emerging disease outbreaks.
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38
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Zhu ZJ, Teng M, Li HZ, Zheng LP, Liu JL, Chai SJ, Yao YX, Nair V, Zhang GP, Luo J. Marek's Disease Virus ( Gallid alphaherpesvirus 2)-Encoded miR-M2-5p Simultaneously Promotes Cell Proliferation and Suppresses Apoptosis Through RBM24 and MYOD1-Mediated Signaling Pathways. Front Microbiol 2020; 11:596422. [PMID: 33224130 PMCID: PMC7669912 DOI: 10.3389/fmicb.2020.596422] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.4] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/19/2020] [Accepted: 10/08/2020] [Indexed: 11/13/2022] Open
Abstract
MicroRNAs (miRNAs) have been demonstrated for their involvement in virus biology and pathogenesis, including functioning as key determinants of virally-induced cancers. As an important oncogenic α-herpesvirus affecting poultry health, Marek’s disease virus serotype 1 [Gallid alphaherpesvirus 2 (GaHV-2)] induces rapid-onset T-cell lymphomatous disease commonly referred to as Marek’s disease (MD), an excellent biological model for the study of virally-induced cancer in the natural hosts. Previously, we have demonstrated that GaHV-2-encoded miRNAs (especially those within the Meq-cluster) have the potential to act as critical regulators of multiple processes such as virus replication, latency, pathogenesis, and/or oncogenesis. In addition to miR-M4-5p (miR-155 homolog) and miR-M3-5p, we have recently found that miR-M2-5p possibly participate in inducing MD lymphomagenesis. Here, we report the identification of two tumor suppressors, the RNA-binding protein 24 (RBM24) and myogenic differentiation 1 (MYOD1), being two biological targets for miR-M2-5p. Our experiments revealed that as a critical miRNA, miR-M2-5p promotes cell proliferation via regulating the RBM24-mediated p63 overexpression and MYOD1-mediated IGF2 signaling and suppresses apoptosis by targeting the MYOD1-mediated Caspase-3 signaling pathway. Our data present a new strategy of a single viral miRNA exerting dual role to potentially participate in the virally-induced T-cell lymphomagenesis by simultaneously promoting the cell proliferation and suppressing apoptosis.
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Affiliation(s)
- Zhi-Jian Zhu
- Research Center of Avian Diseases, College of Veterinary Medicine, Sichuan Agricultural University, Chengdu, China.,Key Laboratory of Animal Immunology, Ministry of Agriculture and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China.,UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou, China
| | - Man Teng
- Key Laboratory of Animal Immunology, Ministry of Agriculture and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China.,UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou, China
| | - Hui-Zhen Li
- Key Laboratory of Animal Immunology, Ministry of Agriculture and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China.,UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou, China.,College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, China
| | - Lu-Ping Zheng
- Key Laboratory of Animal Immunology, Ministry of Agriculture and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China.,UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou, China
| | - Jin-Ling Liu
- Key Laboratory of Animal Immunology, Ministry of Agriculture and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China.,UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou, China
| | - Shu-Jun Chai
- Key Laboratory of Animal Immunology, Ministry of Agriculture and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China.,UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou, China
| | - Yong-Xiu Yao
- The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Guildford, United Kingdom
| | - Venugopal Nair
- The Pirbright Institute and UK-China Centre of Excellence for Research on Avian Diseases, Guildford, United Kingdom
| | - Gai-Ping Zhang
- College of Animal Science and Veterinary Medicine, Henan Agricultural University, Zhengzhou, China.,Jiangsu Co-Innovation Center for the Prevention and Control of Important Animal Infectious Disease and Zoonoses, Yangzhou University, Yangzhou, China
| | - Jun Luo
- Key Laboratory of Animal Immunology, Ministry of Agriculture and Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou, China.,UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou, China.,Key Laboratory of Animal Disease and Public Safety, College of Animal Science and Technology, Henan University of Science and Technology, Luoyang, China
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Azangou-Khyavy M, Ghasemi M, Khanali J, Boroomand-Saboor M, Jamalkhah M, Soleimani M, Kiani J. CRISPR/Cas: From Tumor Gene Editing to T Cell-Based Immunotherapy of Cancer. Front Immunol 2020; 11:2062. [PMID: 33117331 PMCID: PMC7553049 DOI: 10.3389/fimmu.2020.02062] [Citation(s) in RCA: 46] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/05/2020] [Accepted: 07/29/2020] [Indexed: 12/26/2022] Open
Abstract
The clustered regularly interspaced short palindromic repeats system has demonstrated considerable advantages over other nuclease-based genome editing tools due to its high accuracy, efficiency, and strong specificity. Given that cancer is caused by an excessive accumulation of mutations that lead to the activation of oncogenes and inactivation of tumor suppressor genes, the CRISPR/Cas9 system is a therapy of choice for tumor genome editing and treatment. In defining its superior use, we have reviewed the novel applications of the CRISPR genome editing tool in discovering, sorting, and prioritizing targets for subsequent interventions, and passing different hurdles of cancer treatment such as epigenetic alterations and drug resistance. Moreover, we have reviewed the breakthroughs precipitated by the CRISPR system in the field of cancer immunotherapy, such as identification of immune system-tumor interplay, production of universal Chimeric Antigen Receptor T cells, inhibition of immune checkpoint inhibitors, and Oncolytic Virotherapy. The existing challenges and limitations, as well as the prospects of CRISPR based systems, are also discussed.
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Affiliation(s)
| | - Mobina Ghasemi
- Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Javad Khanali
- Student Research Committee, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | | | - Monire Jamalkhah
- Department of Biotechnology, College of Science, University of Tehran, Tehran, Iran
| | - Masoud Soleimani
- Hematology Department, Faculty of Medical Sciences, Tarbiat Modares University, Tehran, Iran
| | - Jafar Kiani
- Oncopathology Research Center, Iran University of Medical Sciences, Tehran, Iran
- Department of Molecular Medicine, Faculty of Advanced Technologies in Medicine, Iran University of Medical Sciences, Tehran, Iran
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40
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Efficient Editing of an Adenoviral Vector Genome with CRISPR/Cas9. Indian J Microbiol 2020; 61:91-95. [PMID: 33505098 DOI: 10.1007/s12088-020-00905-3] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/03/2020] [Accepted: 08/26/2020] [Indexed: 10/23/2022] Open
Abstract
Immunotherapy based on genetic modification of T cells has played an important role in the treatment of tumors and viral infections. Moreover, adenoviral vectors engineered with improved safety due to their inability to integrate into the host genome have been key in the clinical application of T cell therapy. However, the commonly used adenoviral vector Ad5 exhibits low efficiency of infection of human T cells and the details of the intracellular trafficking pathway of adenoviral vectors in human primary T cells remains unclear. Resolution of these issues will depend on successful modification of the adenoviral vector. To this end, here we describe the successful establishment of a simple and efficient method for editing adenoviral vectors in vitro using the CRISPR-Cas9 gene editing system to target the adenoviral fiber gene.
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41
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Gong Y, Bi Y, Li Z, Li Y, Yao Y, Long Q, Pu T, Chen C, Liu T, Dong S, Cun W. High-efficiency nonhomologous insertion of a foreign gene into the herpes simplex virus genome. J Gen Virol 2020; 101:982-996. [PMID: 32602833 PMCID: PMC7654747 DOI: 10.1099/jgv.0.001451] [Citation(s) in RCA: 7] [Impact Index Per Article: 1.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/09/2020] [Accepted: 05/26/2020] [Indexed: 12/14/2022] Open
Abstract
Efficient, accurate and convenient foreign-gene insertion strategies are crucial for the high-throughput and rapid construction of large DNA viral vectors, but relatively inefficient and labour-intensive methods have limited the application of recombinant viruses. In this study, we applied the nonhomologous insertion (NHI) strategy, which is based on the nonhomologous end joining (NHEJ) repair pathway. Compared to the currently used homologous recombination (HR) strategy, we obtained a higher efficiency of foreign-gene insertion into the herpes simplex virus (HSV) genome that reached 45 % after optimization. By using NHI, we rapidly constructed recombinant reporter viruses using a small amount of clinical viruses, and the recombinant virus was stable for at least ten consecutive passages. The fidelity of NHI ranged from 70-100% and was related to the sequence background of the insertion site according to the sequencing results. Finally, we depict the dynamic process by which the foreign-gene donor plasmid and viral genome are rapidly cleaved by Cas9, as revealed by quantitative pulse analysis. Furthermore, the NHI strategy exerted selection pressure on the wild-type and reverse-integrated viral genomes to efficiently integrate the foreign gene in a predetermined direction. Our results indicate that the use of a rationally designed NHI strategy can allow rapid and efficient foreign gene knock-in into the HSV genome and provide useful guidance for gene insertion into large DNA viral genomes using NHI.
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Affiliation(s)
- Yue Gong
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
| | - Yanwei Bi
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
| | - Zhihua Li
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
| | - Yuzhong Li
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
| | - Yueting Yao
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
| | - Qiong Long
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
| | - Tao Pu
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
| | - Chen Chen
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
| | - Tongyun Liu
- The First Affiliated Hospital of Kunming Medical University, Kunming 650118, Yunnan, PR China
| | - Shaozhong Dong
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
| | - Wei Cun
- Institute of Medical Biology, Chinese Academy of Medical Sciences & Peking Union Medical College, Kunming 650118, Yunnan, PR China
- Yunnan Key Laboratory of Vaccine Research and Development of Severe Infectious Disease, Kunming, 650118, Yunnan, PR China
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Zhao Y, Wang LQ, Zheng HH, Yang YR, Liu F, Zheng LL, Jin Y, Chen HY. Construction and immunogenicity of a gE/gI/TK-deleted PRV based on porcine pseudorabies virus variant. Mol Cell Probes 2020; 53:101605. [PMID: 32464159 DOI: 10.1016/j.mcp.2020.101605] [Citation(s) in RCA: 41] [Impact Index Per Article: 8.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/18/2020] [Revised: 05/08/2020] [Accepted: 05/20/2020] [Indexed: 01/10/2023]
Abstract
Pseudorabies (PR) caused by re-emerging pseudorabies virus (PRV) variant has outbroken among PRV vaccine-immunized swine herds on many Chinese pig farms, with severe socioeconomic consequences since late 2011. Here, a gE/gI/TK-deleted recombinant virus (rPRV NY-gE-/gI-/TK-) was constructed based on PRV NY strain from 2012 through homologous DNA recombination and gene-editing technology termed clustered regularly interspaced palindromic repeats (CRISPR)/associated (Cas9) system. The rPRV NY-gE-/gI-/TK- strain showed similar growth kinetics to the parental PRV NY strain in vitro, and was safe for mice. Sixty mice were injected subcutaneously (s.c.) twice with 106.0 TCID50 of rPRV NY-gE-/gI-/TK- and DMEM, respectively, with two-week interval. The levels of PRV gB antibodies and neutralizing antibodies against PRV NY in mice immunized with rPRV NY-gE-/gI-/TK- were higher than those in the DMEM control group. The number of T lymphocyte subclasses CD3+, CD4+ and CD8+ in rPRV NY-gE-/gI-/TK--immunized mice was higher than that in DMEM-injected mice. After challenge with 106.0 TCID50 PRV NY at 42 dpi, all rPRV NY-gE-/gI-/TK--immunized mice survived without exhibiting any pathological lesions in different tissues and intranuclear eosinophilic inclusions of the brain, and the viral genomic copy numbers in various organs of mice were obviously lower than DMEM group. These results showed the rPRV NY-gE-/gI-/TK- could be a promising next-generation vaccine to control now epidemic PR in China.
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Affiliation(s)
- Yu Zhao
- Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Nongye Road 63#, Zhengzhou, 450002, Henan Province, People's Republic of China
| | - Lin-Qing Wang
- Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Nongye Road 63#, Zhengzhou, 450002, Henan Province, People's Republic of China; Department of Life Science, Zhengzhou Normal University, Zhengzhou, 450044, Henan Province, People's Republic of China
| | - Hui-Hua Zheng
- Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Nongye Road 63#, Zhengzhou, 450002, Henan Province, People's Republic of China
| | - Yu-Rong Yang
- Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Nongye Road 63#, Zhengzhou, 450002, Henan Province, People's Republic of China
| | - Fang Liu
- Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Nongye Road 63#, Zhengzhou, 450002, Henan Province, People's Republic of China
| | - Lan-Lan Zheng
- Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Nongye Road 63#, Zhengzhou, 450002, Henan Province, People's Republic of China
| | - Yue Jin
- Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Nongye Road 63#, Zhengzhou, 450002, Henan Province, People's Republic of China.
| | - Hong-Ying Chen
- Zhengzhou Key Laboratory for Pig Disease Prevention and Control, College of Animal Science and Veterinary Medicine, Henan Agricultural University, Nongye Road 63#, Zhengzhou, 450002, Henan Province, People's Republic of China.
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Luo J, Teng M, Zai X, Tang N, Zhang Y, Mandviwala A, Reddy VRAP, Baigent S, Yao Y, Nair V. Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System. Viruses 2020; 12:E466. [PMID: 32325942 PMCID: PMC7232411 DOI: 10.3390/v12040466] [Citation(s) in RCA: 12] [Impact Index Per Article: 2.4] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/25/2020] [Revised: 04/16/2020] [Accepted: 04/17/2020] [Indexed: 01/06/2023] Open
Abstract
The virus-encoded microRNAs (miRNAs) have been demonstrated to have important regulatory roles in herpesvirus biology, including virus replication, latency, pathogenesis and/or tumorigenesis. As an emerging efficient tool for gene editing, the clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system has been successfully applied in manipulating the genomes of large DNA viruses. Herein, utilizing the CRISPR/Cas9 system with a double-guide RNAs transfection/virus infection strategy, we have established a new platform for mutagenesis of viral miRNAs encoded by the Marek's disease virus serotype 1 (MDV-1), an oncogenic alphaherpesvirus that can induce rapid-onset T-cell lymphomas in chickens. A series of miRNA-knocked out (miR-KO) mutants with deletions of the Meq- or the mid-clustered miRNAs, namely RB-1B∆Meq-miRs, RB-1B∆M9-M2, RB-1B∆M4, RB-1B∆M9 and RB-1B∆M11, were generated from vvMDV strain RB-1B virus. Interestingly, mutagenesis of the targeted miRNAs showed changes in the in vitro virus growth kinetics, which is consistent with that of the in vivo proliferation curves of our previously reported GX0101 mutants produced by the bacterial artificial chromosome (BAC) clone and Rec E/T homologous recombination techniques. Our data demonstrate that the CRISPR/Cas9-based gene editing is a simple, efficient and relatively nondisruptive approach for manipulating the small non-coding genes from the genome of herpesvirus and will undoubtedly contribute significantly to the future progress in herpesvirus biology.
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Affiliation(s)
- Jun Luo
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
- Key Laboratory of Animal Immunology, Ministry of Agriculture & Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
- UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
| | - Man Teng
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
- Key Laboratory of Animal Immunology, Ministry of Agriculture & Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
- UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
| | - Xusheng Zai
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
- Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University, Yangzhou 225009, China
| | - Na Tang
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
- Binzhou Animal Science and Veterinary Medicine Academy & UK-China Centre of Excellence for Research on Avian Diseases, Binzhou 256600, China
| | - Yaoyao Zhang
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
- College of Animal Science and Technology, Guangxi University, Nanning 530004, China
| | - Ahmedali Mandviwala
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
| | - Vishwanatha R. A. P. Reddy
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
| | - Susan Baigent
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
| | - Yongxiu Yao
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
| | - Venugopal Nair
- The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK; (M.T.); (X.Z.); (N.T.); (Y.Z.); (A.M.); (V.R.A.P.R.); (S.B.); (Y.Y.)
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Ren J, Wang H, Zhou L, Ge X, Guo X, Han J, Yang H. Glycoproteins C and D of PRV Strain HB1201 Contribute Individually to the Escape From Bartha-K61 Vaccine-Induced Immunity. Front Microbiol 2020; 11:323. [PMID: 32210933 PMCID: PMC7076175 DOI: 10.3389/fmicb.2020.00323] [Citation(s) in RCA: 15] [Impact Index Per Article: 3.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/11/2019] [Accepted: 02/14/2020] [Indexed: 12/21/2022] Open
Abstract
The newly emerged pseudorabies virus (PRV) novel variants can escape from the immunity induced by the classical vaccine Bartha-K61. Here we investigated the underlying mechanisms by constructing chimeric mutants between epidemic strain HB1201 and the Bartha-K61 vaccine. Our analyses focused on three viral envelope glycoproteins, namely gB, gC, and gD, as they exhibit remarkable genetic variations and are also involved in induction of protective immunity. The corresponding genes were swapped reciprocally either individually or in combination by using CRISPR/Cas9 technology and homologous recombination. The rescued chimeric viruses exhibited differential sensitivity to neutralizing antibodies in vitro, and gC was found to be the major contributor to inefficient neutralization against HB1201 by anti-Bartha-K61 serum. When tested in the 4-week-piglet model, substitution with HB1201 gC enabled Bartha-K61 to induce a protective immunity against HB1201 at a high challenge dose of 107 TCID50. Interestingly, despite a relatively lower cross-neutralization ability, the gD exchange also enabled Bartha-K61 to protect piglets from lethal challenge. In both cases, clinical signs and microscopic lesions were eased, and so was the viral tissue load with the exception of brain. A better protection could be achieved when both gC and gD were swapped in terms of reducing viral load in brain and virus-induced microscopic lesions. Thus, our studies not only revealed individual roles of gC and gD variations in the immune escape and also suggested a synergistic effect of both proteins on induction of protective immunity. These findings have important implications in novel vaccine development for PRV control in China.
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Affiliation(s)
- Jianle Ren
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, and College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Haibao Wang
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, and College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Lei Zhou
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, and College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Xinna Ge
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, and College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Xin Guo
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, and College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Jun Han
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, and College of Veterinary Medicine, China Agricultural University, Beijing, China
| | - Hanchun Yang
- Key Laboratory of Animal Epidemiology of the Ministry of Agriculture and Rural Affairs, and College of Veterinary Medicine, China Agricultural University, Beijing, China
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Generation of A Triple Insert Live Avian Herpesvirus Vectored Vaccine Using CRISPR/Cas9-Based Gene Editing. Vaccines (Basel) 2020; 8:vaccines8010097. [PMID: 32098149 PMCID: PMC7157232 DOI: 10.3390/vaccines8010097] [Citation(s) in RCA: 36] [Impact Index Per Article: 7.2] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/27/2020] [Revised: 02/18/2020] [Accepted: 02/19/2020] [Indexed: 01/10/2023] Open
Abstract
Herpesvirus of turkeys (HVT), used originally as a vaccine against Marek’s disease (MD), has recently been shown to be a highly effective viral vector for generation of recombinant vaccines that deliver protective antigens of other avian pathogens. Until the recent launch of commercial HVT-vectored dual insert vaccines, most of the HVT-vectored vaccines in the market carry a single foreign gene and are usually developed with slow and less efficient conventional recombination methods. There is immense value in developing multivalent HVT-vectored vaccines capable of inducing simultaneous protection against multiple avian pathogens, particularly to overcome the interference between individual recombinant HVT vaccines. Here we demonstrate the use of a previously developed CRISPR/Cas9 gene editing protocol for the insertion of ILTV gD-gI and the H9N2 AIV hemagglutinin expression cassettes into the distinct locations of the recombinant HVT-IBDV VP2 viral genome, to generate the triple insert HVT-VP2-gDgI-HA recombinant vaccine. The insertion, protein expression, and stability of each insert were then evaluated by PCR, immunostaining and Western blot analyses. The successful generation of the first triple insert recombinant HVT vaccine with the potential for the simultaneous protection against three major avian viral diseases in addition to MD is a major innovation in vaccination-based control of major poultry diseases.
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Yang YB, Tang YD, Hu Y, Yu F, Xiong JY, Sun MX, Lyu C, Peng JM, Tian ZJ, Cai XH, An TQ. Single Virus Tracking with Quantum Dots Packaged into Enveloped Viruses Using CRISPR. NANO LETTERS 2020; 20:1417-1427. [PMID: 31930919 DOI: 10.1021/acs.nanolett.9b05103] [Citation(s) in RCA: 25] [Impact Index Per Article: 5.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/10/2023]
Abstract
Labeling viruses with high-photoluminescence quantum dots (QDs) for single virus tracking provides a visual tool to aid our understanding of viral infection mechanisms. However, efficiently labeling internal viral components without modifying the viral envelope and capsid remains a challenge, and existing strategies are not applicable to most viruses. Here, we have devised a strategy using the clustered regularly interspaced short palindromic repeats (CRISPR) imaging system to label the nucleic acids of Pseudorabies virus (PRV) with QDs. In this strategy, QDs were conjugated to viral nucleic acids with the help of nuclease-deactivated Cas9/gRNA complexes in the nuclei of living cells and then packaged into PRV during virion assembly. The processes of PRV-QD adsorption, cytoplasmic transport along microtubules, and nuclear entry were monitored in real time in both Vero and HeLa cells, demonstrating the utility and efficiency of the strategy in the study of viral infection.
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Affiliation(s)
- Yong-Bo Yang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Yan-Dong Tang
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Yue Hu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Fang Yu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Jun-Yao Xiong
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Ming-Xia Sun
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Chuang Lyu
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Jin-Mei Peng
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Zhi-Jun Tian
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Xue-Hui Cai
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
| | - Tong-Qing An
- State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute , Chinese Academy of Agricultural Sciences , Harbin 150069 , China
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Atasoy MO, Rohaim MA, Munir M. Simultaneous Deletion of Virulence Factors and Insertion of Antigens into the Infectious Laryngotracheitis Virus Using NHEJ-CRISPR/Cas9 and Cre-Lox System for Construction of a Stable Vaccine Vector. Vaccines (Basel) 2019; 7:vaccines7040207. [PMID: 31817447 PMCID: PMC6963826 DOI: 10.3390/vaccines7040207] [Citation(s) in RCA: 19] [Impact Index Per Article: 3.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2019] [Revised: 12/01/2019] [Accepted: 12/02/2019] [Indexed: 12/13/2022] Open
Abstract
Infectious laryngotracheitis virus (ILTV) is a promising vaccine vector due to its heterologous gene accommodation capabilities, low pathogenicity, and potential to induce cellular and humoral arms of immunity. Owing to these characteristics, different gene-deletion versions of ILTVs have been successfully deployed as a vector platform for the development of recombinant vaccines against multiple avian viruses using conventional recombination methods, which are tedious, time-demanding, and error-prone. Here, we applied a versatile, and customisable clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 accompanied with Cre–Lox system to simultaneously delete virulence factors and to insert foreign genes in the ILTV genome. Using this pipeline, we successfully deleted thymidine kinase (TK) and unique short 4 (US4) genes and inserted fusion (F) gene of the Newcastle disease virus without adversely affecting ILTV replication and expression of the F protein. Taken together, the proposed approach offers novel tools to attenuate (by deletion of virulence factor) and to generate multivalent (by insertion of heterologous genes) vaccine vectors to protect chickens against pathogens of poultry and public health importance.
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Chang P, Ameen F, Sealy JE, Sadeyen JR, Bhat S, Li Y, Iqbal M. Application of HDR-CRISPR/Cas9 and Erythrocyte Binding for Rapid Generation of Recombinant Turkey Herpesvirus-Vectored Avian Influenza Virus Vaccines. Vaccines (Basel) 2019; 7:vaccines7040192. [PMID: 31766655 PMCID: PMC6963405 DOI: 10.3390/vaccines7040192] [Citation(s) in RCA: 16] [Impact Index Per Article: 2.7] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/30/2019] [Revised: 11/12/2019] [Accepted: 11/20/2019] [Indexed: 11/16/2022] Open
Abstract
Avian influenza viruses (AIVs) are highly contagious and have caused huge economical loss to the poultry industry. AIV vaccines remain one of the most effective methods of controlling this disease. Turkey herpesvirus (HVT) is a commonly used live attenuated vaccine against Marek’s disease; it has also been used as a viral vector for recombinant AIV vaccine development. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a gene editing tool which, in vaccinology, has facilitated the development of recombinant DNA viral-vectored vaccines. Here, we utilize homology-directed repair (HDR) for the generation of a HVT–H7N9 HA bivalent vaccine; a H7N9 HA expression cassette was inserted into the intergenic region between UL45 and UL46 of HVT. To optimize the selection efficiency of our bivalent vaccine, we combined CRISPR/Cas9 with erythrocyte binding to rapidly generate recombinant HVT–H7HA candidate vaccines.
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Affiliation(s)
- Pengxiang Chang
- Avian Influenza Group, The Pirbright Institute, Pirbright, Woking GU24 0NF, UK; (P.C.); (J.E.S.); (J.-R.S.); (S.B.)
| | - Faisal Ameen
- Department of Microbiology, University of Veterinary and Animal Sciences Lahore 54000, Pakistan;
| | - Joshua E. Sealy
- Avian Influenza Group, The Pirbright Institute, Pirbright, Woking GU24 0NF, UK; (P.C.); (J.E.S.); (J.-R.S.); (S.B.)
| | - Jean-Remy Sadeyen
- Avian Influenza Group, The Pirbright Institute, Pirbright, Woking GU24 0NF, UK; (P.C.); (J.E.S.); (J.-R.S.); (S.B.)
| | - Sushant Bhat
- Avian Influenza Group, The Pirbright Institute, Pirbright, Woking GU24 0NF, UK; (P.C.); (J.E.S.); (J.-R.S.); (S.B.)
| | - Yongqing Li
- Institute of Animal Husbandry and Veterinary Medicine, Beijing Academy of Agricultural and Forestry Sciences, Beijing 100097, China;
- Sino–UK Joint Laboratory for the Prevention & Control of Infectious Diseases in Livestock and Poultry, Institute of Animal Husbandry and Veterinary Medicine, Beijing 100097, China
| | - Munir Iqbal
- Avian Influenza Group, The Pirbright Institute, Pirbright, Woking GU24 0NF, UK; (P.C.); (J.E.S.); (J.-R.S.); (S.B.)
- Sino–UK Joint Laboratory for the Prevention & Control of Infectious Diseases in Livestock and Poultry, Institute of Animal Husbandry and Veterinary Medicine, Beijing 100097, China
- Correspondence: ; Tel.: +44-1483-231441
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Generation and validation of recombinant herpes simplex type 1 viruses (HSV-1) using CRISPR/Cas9 genetic disruption. Methods Enzymol 2019; 635:167-184. [PMID: 32122544 DOI: 10.1016/bs.mie.2019.08.011] [Citation(s) in RCA: 3] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/06/2023]
Abstract
Herpes simplex virus type 1 (HSV-1) is a large DNA virus that has been popular for oncolytic virus development in pre-clinical research and clinical trials. An oncolytic HSV-1 encoding granulocyte-macrophage colony stimulating factor (GM-CSF), designated talimogene laherparepvec (T-VEC) was approved for the treatment of patients with advanced melanoma in 2015. There are numerous advantages of HSV-1 for oncolytic development, including the ease of recombinant engineering, presence of non-essential genes allowing attenuation of pathogenicity and space for foreign transgene expression. In addition, most recombinants retain sensitivity to acyclovir providing an additional safety feature. In this chapter, we will focus on the key methods for the development of oncolytic HSV-1 vectors and some of the commonly utilized laboratory protocols used to characterize and assess the structure and oncolytic activity of recombinant HSV-1 viruses.
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50
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Koganti R, Yadavalli T, Shukla D. Current and Emerging Therapies for Ocular Herpes Simplex Virus Type-1 Infections. Microorganisms 2019; 7:microorganisms7100429. [PMID: 31658632 PMCID: PMC6843252 DOI: 10.3390/microorganisms7100429] [Citation(s) in RCA: 55] [Impact Index Per Article: 9.2] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2019] [Revised: 10/02/2019] [Accepted: 10/03/2019] [Indexed: 12/19/2022] Open
Abstract
Herpes simplex virus type-1 (HSV-1) is a neurotropic, double-stranded DNA virus that can cause a wide variety of diseases, including many ocular pathologies. It is one of the leading causes of infectious blindness in the United States. Because of its ubiquitous nature and its potential to cause serious ocular maladies, there is a significant need for more effective antiviral therapies against ocular HSV-1. In this review, we discuss the lifecycle of HSV-1 as it pertains to corneal infections and the clinically approved as well as emerging treatments to combat HSV-1 infections. We also highlight some newly identified host targets for the antiviral drug development.
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Affiliation(s)
- Raghuram Koganti
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor St, Chicago, IL 60612, USA.
| | - Tejabhiram Yadavalli
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor St, Chicago, IL 60612, USA.
| | - Deepak Shukla
- Department of Ophthalmology and Visual Sciences, University of Illinois at Chicago, 1855 W. Taylor St, Chicago, IL 60612, USA.
- Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612, USA.
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