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Efficient Mutagenesis of Marek's Disease Virus-Encoded microRNAs Using a CRISPR/Cas9-Based Gene Editing System.
- Source :
-
Viruses [Viruses] 2020 Apr 20; Vol. 12 (4). Date of Electronic Publication: 2020 Apr 20. - Publication Year :
- 2020
-
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.
- Subjects :
- Animals
Base Sequence
Cells, Cultured
Chick Embryo
Cloning, Molecular
Gene Knockdown Techniques
Genome, Viral
Open Reading Frames
RNA, Guide, CRISPR-Cas Systems
RNA, Messenger genetics
Sequence Analysis, DNA
Virus Replication
CRISPR-Cas Systems
Gene Editing
Marek Disease genetics
MicroRNAs genetics
Mutagenesis
RNA, Viral genetics
Subjects
Details
- Language :
- English
- ISSN :
- 1999-4915
- Volume :
- 12
- Issue :
- 4
- Database :
- MEDLINE
- Journal :
- Viruses
- Publication Type :
- Academic Journal
- Accession number :
- 32325942
- Full Text :
- https://doi.org/10.3390/v12040466