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.
Highlights
MicroRNAs, one of the most important types of RNA, are endogenous small non-coding RNAs
Based on a co-transfection and virus infection strategy, the gene editing efficacies of these gRNA combinations targeting to RB-1B miRNA genes were tested and the result showed that all the four clustered regularly interspaced short palindromic repeat (CRISPR)/gRNA
As for the avian herpesviruses, such as Marek’s disease virus serotype 1 (MDV-1), MDV-2, and HVT, the bacterial artificial chromosome (BAC) mutagenesis has contributed a lot to editing the viral genomes [59,60,61,62]
Summary
MicroRNAs (miRNAs), one of the most important types of RNA, are endogenous small (approximately 22–24 nt) non-coding RNAs. In the past two decades, thousands of miRNAs have been characterized, identified and/or annotated in the genomes of 271 organisms [1], including humans, metazoans, plants and some lower eukaryotes. In the past two decades, thousands of miRNAs have been characterized, identified and/or annotated in the genomes of 271 organisms [1], including humans, metazoans, plants and some lower eukaryotes These small molecules have been demonstrated to play critical post-transcriptional regulatory roles through the RNA-induced gene silencing in various biological processes, such as cellular development, differentiation, disease progress and all aspects of cancer biology [2,3]. A large number of viral miRNAs have been recently reported in the genomes of avian herpesvirus species [8,9,10,11,12], including all the three serotypes of Marek’s disease virus, MDV-1, MDV-2, and MDV-3
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