CRISPR/Cas9 Editing of Duck Enteritis Virus Genome for the Construction of a Recombinant Vaccine Vector Expressing ompH Gene of Pasteurella multocida in Two Novel Insertion Sites

Abstract

Duck enteritis virus (DEV) and Pasteurella multocida, the causative agent of duck plague and fowl cholera, are acute contagious diseases and leading causes of morbidity and mortality in duck. The NHEJ-CRISPR/Cas9-mediated gene editing strategy, accompanied with the Cre–Lox system, have been employed in the present study to show that two new sites at UL55-LORF11 and UL44-44.5 loci in the genome of the attenuated Jansen strain of DEV can be used for the stable expression of the outer membrane protein H (ompH) gene of P. multocida that could be used as a bivalent vaccine candidate with the potential of protecting ducks simultaneously against major viral and bacterial pathogens. The two recombinant viruses, DEV-OmpH-V5-UL55-LORF11 and DEV-OmpH-V5-UL44-44.5, with the insertion of ompH-V5 gene at the UL55-LORF11 and UL44-44.5 loci respectively, showed similar growth kinetics and plaque size, compared to the wildtype virus, confirming that the insertion of the foreign gene into these did not have any detrimental effects on DEV. This is the first time the CRISPR/Cas9 system has been applied to insert a highly immunogenic gene from bacteria into the DEV genome rapidly and efficiently. This approach offers an efficient way to introduce other antigens into the DEV genome for multivalent vector.

Efficiency of NHEJ-CRISPR/Cas9 and Cre-LoxP Engineered Recombinant Turkey Herpesvirus Expressing Pasteurella multocida OmpH Protein for Fowl Cholera Prevention in Ducks

“…The individual single guide RNAs (sgRNAs) targeting the intergenic region UL45/46 and SgA targeting the donor plasmid to release the insert fragment were cloned into pX459-v2, which contains Cas9 gene from S. pyogenes for the gRNA cloning vector. The donor plasmid, depicted in Figure 1 B, was adapted from a previous study and includes the GFP reporter gene and OmpH-V5 cassettes flanked by the SgA target sites [ 21 ]. The simultaneous cleavage of both the donor plasmid DNA and the viral genome by Cas9 leads to the insertion of the GFP-OmpH-V5 cassette into the UL45/46 region.…”

“…The purified recombinant HVT-OmpH of the first generation was designated as rHVT-GFP-OmpH ( Figure 1 C). After that, the GFP expression cassette was removed using Cre treatment as described previously [ 21 ]. Finally, the purified new recombinant HVT was named rHVT-OmpH after being identified by PCR using outside specific insertion sites primers UL45-F and UL46-R ( Figure 1 D).…”

“…The oligos of the gRNA targeting the HVT UL45/46 intergenic region and the sg-A sequence were synthesized and cloned into pX459-V2 (Addgene, UK) following the method stated previously [ 20 ]. The donor plasmid containing the removable green fluorescent reporter gene (GFP) and OmpH-V5 gene expression cassettes was designed and constructed previously [ 21 ]. The steps for transfection and HVT infection to create recombinant HVT were conducted according to the previously provided description [ 20 ].…”

Full Issue

“…Vector vaccines using Gallid alphaherpesvirus 2 as a vector against AI, IB, and/or ND were also evaluated (366)(367)(368)(369)(370)(371); a CVI988-ND construct (368,369) was at the end of licensing process in Japan but it was not launched. The duck enteritis virus was evaluated as a vector for AI (372-375), ND (376), IB (377), Pasteurella multocida (147,378), and duck hepatitis A (379) in ducks and/or chickens.…”

Simultaneous Protective Immune Responses of Ducks against Duck Plague and Fowl Cholera by Recombinant Duck Enteritis Virus Vector Expressing Pasteurella multocida OmpH Gene

“…Our data indicate that CRISPR/Cas9 is a promising strategy for screening multiple gene loci in the DEV genome as a stable insertion site of foreign genes in mammalian cells. Moreover, the presence and the expression of the ompH gene were successfully detected by PCR, immunofluorescence and Western blot analysis for both rDEV-ompH-UL55 and rDEV-ompH-UL44 in chicken embryo fibroblasts (CEFs), and the insert could be stably maintained in the recombinant viruses for 15 passages without any adverse effects on viral replication [27].…”