Application of CRISPR/Cas9 Gene Editing System on MDV-1 Genome for the Study of Gene Function

Abstract

Marek’s disease virus (MDV) is a member of alphaherpesviruses associated with Marek’s disease, a highly contagious neoplastic disease in chickens. Complete sequencing of the viral genome and recombineering techniques using infectious bacterial artificial chromosome (BAC) clones of Marek’s disease virus genome have identified major genes that are associated with pathogenicity. Recent advances in CRISPR/Cas9-based gene editing have given opportunities for precise editing of the viral genome for identifying pathogenic determinants. Here we describe the application of CRISPR/Cas9 gene editing approaches to delete the Meq and pp38 genes from the CVI988 vaccine strain of MDV. This powerful technology will speed up the MDV gene function studies significantly, leading to a better understanding of the molecular mechanisms of MDV pathogenesis.

Targeted Editing of the pp38 Gene in Marek’s Disease Virus-Transformed Cell Lines Using CRISPR/Cas9 System

“…A dual gRNA construct pp38-gNC, which expresses two gRNAs targeting both ends of pp38 gene and Cas9 nuclease in pX330A-1 × 2 vector was used for pp38 deletion in MSB-1 [39]. Two-part guide RNA system containing crRNA:tracrRNA guide complex was used for HP8 editing.…”

“…MDV-encoded phosphoprotein pp38, strongly associated with lytic replication of the virus in B cells, is thought to play an important role in maintaining the transformed status of lymphocytes in vivo by preventing apoptosis, although its role in reactivation has been shown to be debatable [38]. Previously, we have reported deletion of pp38 from the vaccine strain CVI988 using CRISPR/Cas9 editing [39] in infected CEF (primary chick embryo fibroblasts). In this report, we have used a new approach with the same gRNA sequences to delete pp38 and insert green fluorescent protein (GFP) into pp38 in MDV-transformed LCLs MSB-1 and HP8 to examine its functional roles.…”

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

“…The expressions of viral proteins Meq, gB, and pp38 in CEF cells infected with the RB-1B miR-KO mutants were determined by IFA using inverted immunofluorescence microscopy as described previously [48,49]. For IFA analysis, the rabbit anti-Meq polyclonal antibody and MDV-1 specific mAbs HB3 and BD1 were used as primary antibodies, and the Alexa Fluor TM 568 Goat anti-Rabbit IgG (H+L) and 488 Rabbit anti-Mouse IgG (H+L) (Invitrogen) were used as secondary antibodies for staining the expression of Meq, gB, and pp38 proteins, respectively.…”

“…The relative expression levels of miR-M4-5p, miR-M9-5p, miR-M11-5p, miR-M12-3p, and miR-M31-3p in RB-1B or miR-KO virus infected CEFs were determined by qRT-PCR as previously described [49]. The TaqMan MicroRNA Assay System (Thermo Fisher Scientific, Basingstoke, United Kingdom) was used for reverse transcription reaction, each of which was performed twice independently using 10 ng total RNA as a template.…”

“…It has not only been widely applied for generating gene knockout (KO) cell lines and animal models, but has also been used for manipulating large viral DNA genomes, such as Epstein-Barr virus (EBV) [36], herpes simplex virus type I (HSV-1) [32,37,38], guinea pig cytomegalovirus (GPCMV) [39], pseudorabies virus (PrV) [40][41][42], vaccinia virus (VACV) [43,44], and duck enteritis virus (DEV) [45,46]. We have successfully demonstrated the manipulation of the HVT and MDV-1 genomes using the CRISPR/Cas9 system both for generating recombinant vaccines and for studying the viral protein-coding gene functions respectively [47][48][49][50][51].…”