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Marek's Disease Virus As a CRISPR/Cas9 Delivery System To Veterinary Microbiology 242 (2020) 108589 Contents lists available at ScienceDirect Veterinary Microbiology journal homepage: www.elsevier.com/locate/vetmic Marek’s disease virus as a CRISPR/Cas9 delivery system to defend against avian leukosis virus infection in chickens T Yongzhen Liu1, Zengkun Xu1, Yu Zhang, Mengmeng Yu, Suyan Wang, Yulong Gao, Changjun Liu, Yanping Zhang, Li Gao, Xiaole Qi, Hongyu Cui, Qing Pan, Kai Li*, Xiaomei Wang* Avian Immunosuppressive Diseases Division, State Key Laboratory of Veterinary Biotechnology, Harbin Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Harbin, 150069, People’s Republic of China ARTICLE INFO ABSTRACT Keywords: The CRISPR/CRISPR-associated protein 9 (Cas9) system is a powerful gene-editing tool originally discovered as Marek’s disease virus an integral mediator of bacterial adaptive immunity. Recently, this technology has been explored for its potential Avian leukosis virus utility in providing new and unique treatments for viral infection. Marek’s disease virus (MDV) and avian leu- CRISPR/Cas9 kosis virus subgroup J (ALV-J), major immunosuppressive viruses, cause significant economic losses to the Infection chicken industry. Here, we evaluated the efficacy of using MDV as a CRISPR/Cas9-delivery system to directly target and disrupt the reverse-transcribed products of the ALV-J RNA genome during its infection cycle in vitro and in vivo.Wefirst screened multiple potential guide RNA (gRNA) target sites in the ALV-J genome and identified several optimized targets capable of effectively disrupting the latently integrated viral genome and providing efficient defense against new infection by ALV-J in cells. The optimal single-gRNAs and Cas9-ex- pression cassettes were inserted into the genome of an MDV vaccine strain. The results indicated that engineered MDV stably expressing ALV-J-targeting CRISPR/Cas9 efficiently resisted ALV-J challenge in host cells. These findings demonstrated the CRISPR/Cas9 system as an effective treatment strategy against ALV-J infection. Furthermore, the results highlighted the potential of MDV as an effective delivery system for CRISPR/Cas9 in chickens. 1. Introduction treatment strategy for persistent or chronic viral infections character- ized by the persistence of a viral genome integrated into the host The CRISPR-associated protein 9 (Cas9) system was derived from genome. the integral mediator of bacterial adaptive immunity (Gasiunas et al., The design of an in vivo Cas9-associated antiviral treatment strategy 2012; Makarova et al., 2018). CRISPR/Cas9 generates single-guide RNA requires consideration of the following factors: virus type, the type of (sgRNA)-specific DNA double-strand breaks that are subsequently re- CRISPR/Cas9-delivery system, and the targeted cell type (Doerflinger paired through either non-homologous end joining or homology di- et al., 2017). Delivery of a CRISPR/Cas9 system that targets virus-in- rected repair pathways (Wiedenheft et al., 2012). The Cas9-mediated fected cells or tissues in vivo represents one of the biggest challenges to genome-editing system enables easy, fast, and economical modification its therapeutic application. Effective treatment through gene editing of endogenous genes in diverse cells and organs (Cong et al., 2013; Mali requires the CRISPR/Cas9 system to be transported to infected cells. et al., 2013; Wang et al., 2013). In the past five years, CRISPR/Cas9 Several delivery methods of therapeutic genes, including viral vectors technology has been widely used in numerous fields (Pellagatti et al., (AAV vectors, adenoviral vectors, and lentiviral vectors) and non-viral 2015), including use of Cas9-associated genome editing as a strategy for vectors (liposomes or electroporation) (Liu et al., 2017a), have been antiviral therapy against human immunodeficiency virus (HIV) (Liao developed and applied in clinical trials (Naldini, 2015). Virus-free de- et al., 2015; Yin et al., 2017), herpes simplex virus (Chen et al., 2018; livery systems are less immunogenic; however, delivery efficiency is van Diemen et al., 2016), hepatitis B virus (Bloom et al., 2018; Li et al., usually lower and not cell-type specific. Although viral vectors more 2018a), and hepatitis C virus (Moyo et al., 2018). The success of this efficiently deliver nucleic acids, size limitations on transgenic DNA are technique in antiviral therapy suggests its potential as an efficacious a primary disadvantage (Liu et al., 2017a). ⁎ Corresponding authors at: 678 Haping Road, Harbin, Heilongjiang, 150069, PR China. E-mail addresses: [email protected] (K. Li), [email protected] (X. Wang). 1 These authors contributed equally to this work. https://doi.org/10.1016/j.vetmic.2020.108589 Received 4 November 2019; Received in revised form 14 January 2020; Accepted 15 January 2020 0378-1135/ © 2020 Elsevier B.V. All rights reserved. Y. Liu, et al. Veterinary Microbiology 242 (2020) 108589 Major immunosuppressive viruses, such as Marek's disease virus transfected DF-1 cells relative to those in cells transfected with an EV (MDV) and avian retrovirus, cause serious economic losses to the (Fig. 2B). We then analyzed the efficiency of the CRISPR/Cas9 system poultry industry, with co-infection of MDV and avian retrovirus fre- to eradicate ALV-J proviruses at the DNA level analyzed by quantitative quently occurring in chicken flocks (Li et al., 2018b). Marek’s disease polymerase chain reaction (qPCR) during ALV-J-GFP infection in DF1 can be successfully controlled by vaccination with attenuated MDV cells. As shown in Fig. 2C, ALV-J-GFP viral titer was significantly lower strains. As with most herpesviruses, the large genome of MDV contains in DF-1 cells transfected with the CRISPR/Cas9 system relative to EV- several regions nonessential for viral replication, which allows insertion transfected cells. These results indicated that the CRISPR/Cas9 system of foreign genes and renders MDV a desirable viral vector for expressing targeting ALV-J significantly inhibited ALV-J replication and reactiva- large foreign genes. MDV establishes a persistent infection in lymphoid tion in chicken cells. tissues and can sustainably express foreign proteins (Liu et al., 2017b); however, avian retroviruses, such as avian leukosis virus (ALV), which 2.3. Generation of recombinant MDV containing the CRISPR/Cas9 system invade immune cells and integrate into the host genome to establish latent infection, represent a significant obstacle to the development of To verify whether the MDV vector can deliver the CRISPR/Cas9 efficient vaccines (Liu et al., 2016). system effective, The Cas9 and sgRNA-T8-expressing cassettes were Here, we used MDV as a CRISPR/Cas9-delivery system in order to inserted into the US2 site of the genome of an attenuated MDV vaccine promote direct targeting and disruption of reverse-transcribed products strain (814 strain), and the resulting fosmid clone (814E-Cas9/sgRNA- of the avian retrovirus RNA genome in vitro and in vivo. This study may T8) was used to replace the parental fosmid clone (814E) for re- provide a new approach for control of avian retroviral diseases in combinant viral rescue (Fig. 3A). MDV-typical plaques appeared in cells chickens. transfected with the DNA combinations (Fig. 3B), and immuno- fluorescence assays revealed that cells infected with r814-Cas9-sgLTR-8 2. Results reacted with anti-Cas9 antibodies according to the emission of a green fluorescent signal (Fig. 3B). These results indicated that the re- 2.1. Screening of the optimal target sites in the ALV-J genome combinant viruses containing the Cas9-expression cassette were suc- cessfully generated, and that recombinant MDVs expressed the Cas9 The long terminal repeat (LTR) sequence is a critical element for protein. regulating retroviral expression (Liao et al., 2015). Here, we screened 10 gRNA-targeting sites within the LTR sequence of the ALV-J genome 2.4. The antiviral efficacy of r814-Cas9-sgLTR-8 against ALV-J infection in (Fig. 1A–C). The mean fluorescence intensity (MFI) of green fluores- cells cence protein (GFP) signals associated with ALV-J-GFP was analyzed by flow cytometry. The results showed that the MFI of GFP signal in cells To determine whether r814-Cas9-sgLTR-8 was capable of pre- transfected with ALV-J LTR-sequence-specific gRNA and Cas9 were venting ALV-J infection, we first tested the antiviral activities of r814- significantly lower than those in cells transfected with empty vectors Cas9-sgLTR-8 in chicken embryo fibroblasts (CEFs) infected with r814- (EVs) or those harboring mock gRNA (Fig. 1D). We noted that the op- Cas9-sgLTR-8 or the parent MDV strain for 24 h, followed by infection timal gRNA-target site was in the R region, indicating the importance of with ALV-J and detection of ALV-J-genome copy number at 1, 2, 3, and the LTR R region to ALV replication (Fig. 1C and D). Additionally, we 4 days post-infection (dpi). As shown in Fig. 4, ALV-J-genome copy sequenced 5 DNA fragments containing the optimal gRNA-target site, number in CEFs infected with r814-Cas9-sgLTR-8 was significantly with various mutations detected among the 5 clones, and sequencing lower than that infected with parental MDV, indicating that r814-Cas9- analysis showing that disruption of GFP expression was caused by ei- sgLTR-8 provided effective antiviral activity against ALV-J infection in ther insertion or deletion (Fig. 1F). These results indicated that the CEFs. CRISPR/Cas9 system efficiently disrupted the activity of ALV-J pro- viruses through targeted destruction of proviral genomes. 2.5. The antiviral efficacy of r814-Cas9-sgLTR-8 against ALV-J challenge in chickens 2.2. Optimizing CRISPR/Cas9 components for application in chicken cells To validate the in vivo excisional efficiency of r814-Cas9-sgLTR-8, 1- The CRISPR system comprises the Cas9 protein and sgRNA, with day-old specific-pathogen-free (SPF) chickens were inoculated with their expression directly affecting the gene-editing efficiency of the r814-Cas9-sgLTR-8 or the parental virus subcutaneously, followed by CRISPR/Cas9 system (Gandhi et al., 2017).
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