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Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System

文献类型： 外文期刊

作者： Luo, Jun 1 ; Teng, Man 1 ; Zai, Xusheng 1 ; Tang, Na 1 ; Zhang, Yaoyao 1 ; Mandviwala, Ahmedali 1 ; Reddy, Vishwanatha 1 ;

作者机构： 1.Pirbright, Pirbright Inst, Ash Rd, Guildford GU24 0NF, Surrey, England

2.Pirbright, UK China Ctr Excellence Res Avian Dis, Ash Rd, Guildford GU24 0NF, Surrey, England

3.Henan Acad Agr Sci, Key Lab Anim Immunol, Minist Agr, Zhengzhou 450002, Peoples R China

4.Henan Acad Agr Sci, Henan Prov Key Lab Anim Immunol, Zhengzhou 450002, Peoples R China

5.Henan Acad Agr Sci, UK China Ctr Excellence Res Avian Dis, Zhengzhou 450002, Peoples R China

6.Yangzhou Univ, Minist Educ, Key Lab Avian Prevent Med, Yangzhou 225009, Jiangsu, Peoples R China

7.Binzhou Anim Sci & Vet Med Acad, Binzhou 256600, Peoples R China

8.UK China Ctr Excellence Res Avian Dis, Binzhou 256600, Peoples R China

9.Guangxi Univ, Coll Anim Sci & Technol, Nanning 530004, Peoples R China

关键词： CRISPR; herpesvirus; Marek's disease virus; miRNA; gene editing

期刊名称：VIRUSES-BASEL （ 影响因子：5.048； 五年影响因子：5.127 ）

ISSN：

年卷期： 2020 年 12 卷 4 期

页码：

收录情况： SCI

摘要： 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-1BDMeq-miRs, RB-1BDM9-M2, RB-1BD M4, RB-1BD M9 and RB-1B DM11, 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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