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Open AccessArticle

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

1
The Pirbright Institute & UK-China Centre of Excellence for Research on Avian Diseases, Pirbright, Ash Road, Guildford, Surrey GU24 0NF, UK
2
Key Laboratory of Animal Immunology, Ministry of Agriculture & Henan Provincial Key Laboratory of Animal Immunology, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
3
UK-China Centre of Excellence for Research on Avian Diseases, Henan Academy of Agricultural Sciences, Zhengzhou 450002, China
4
Ministry of Education Key Lab for Avian Preventive Medicine, Yangzhou University, Yangzhou 225009, China
5
Binzhou Animal Science and Veterinary Medicine Academy & UK-China Centre of Excellence for Research on Avian Diseases, Binzhou 256600, China
6
College of Animal Science and Technology, Guangxi University, Nanning 530004, China
*
Authors to whom correspondence should be addressed.
These authors contributed equally to this work.
Viruses 2020, 12(4), 466; https://doi.org/10.3390/v12040466
Received: 25 March 2020 / Revised: 16 April 2020 / Accepted: 17 April 2020 / Published: 20 April 2020
(This article belongs to the Section Animal Viruses)
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. View Full-Text
Keywords: CRISPR; herpesvirus; Marek’s disease virus; miRNA; gene editing CRISPR; herpesvirus; Marek’s disease virus; miRNA; gene editing
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MDPI and ACS Style

Luo, J.; Teng, M.; Zai, X.; Tang, N.; Zhang, Y.; Mandviwala, A.; Reddy, V.R.A.P.; 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, 466.

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