The Application of NHEJ-CRISPR/Cas9 and Cre-Lox System in the Generation of Bivalent Duck Enteritis Virus Vaccine against Avian Influenza Virus

Abstract

Duck-targeted vaccines to protect against avian influenza are critically needed to aid in influenza disease control efforts in regions where ducks are endemic for highly pathogenic avian influenza (HPAI). Duck enteritis virus (DEV) is a promising candidate viral vector for development of vaccines targeting ducks, owing to its large genome and narrow host range. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a versatile gene-editing tool that has proven beneficial for gene modification and construction of recombinant DNA viral vectored vaccines. Currently, there are two commonly used methods for gene insertion: non-homologous end-joining (NHEJ) and homology-directed repair (HDR). Owing to its advantages in efficiency and independence from molecular requirements of the homologous arms, we utilized NHEJ-dependent CRISPR/Cas9 to insert the influenza hemagglutinin (HA) antigen expression cassette into the DEV genome. The insert was initially tagged with reporter green fluorescence protein (GFP), and a Cre-Lox system was later used to remove the GFP gene insert. Furthermore, a universal donor plasmid system was established by introducing double bait sequences that were independent of the viral genome. In summary, we provide proof of principle for generating recombinant DEV viral vectored vaccines against the influenza virus using an integrated NHEJ-CRISPR/Cas9 and Cre-Lox system.

Application of HDR-CRISPR/Cas9 and Erythrocyte Binding for Rapid Generation of Recombinant Turkey Herpesvirus-Vectored Avian Influenza Virus Vaccines

“…Another attractive trait of NHEJ is that the donor plasmid is free from the restriction of homology arms, and is therefore beneficial for the donor plasmids shared between different virus vectors or different insertion sites inside one virus vector. However, the downside of NHEJ is that small insertion or deletion of nucleotides may occur and the insert is bi-directional [24]. HDR has advantage over these aspects, because of high fidelity and insertion of a cassette in a uni-directional manner.…”

“…CEF cells were infected with HVT-H7HA for 72 h and then immunohistochemical stained as described previously [24]. The expression of H7N9 HA antigen in HVT-AIV vaccine-infected cells was visualized by incubating cells with AIV H7N9 HA mouse monoclonal antibody (generated at the Pirbright Institute and used at 1/200 dilution), followed by horseradish peroxidase-conjugated rabbit anti-mouse secondary antibody (used with 1/200 dilution) (DAKO, Agilent Technologies, Santa Clara, CA, USA).…”

Immune Escape Adaptive Mutations in the H7N9 Avian Influenza Hemagglutinin Protein Increase Virus Replication Fitness and Decrease Pandemic Potential

“…The pH of fusion for H7N9 AIV was determined by syncytium formation assays as previously described with slight modification ( 49 ). To determine the optimal number of viruses to use, the H7N9 AIVs were 2-fold serially diluted in DMEM to infect the Vero cells in 96-well plates for 1 h. The inoculum was then removed and washed with PBS before addition of growth medium for 15 h. The cells were then fixed with a methanol and acetone (1:1) mixture and then immunostained with anti-nucleoprotein (anti-NP) mouse monoclonal antibody followed by horseradish peroxidase-labeled rabbit anti-mouse immunoglobulins (Dako) as previously described ( 50 ). The virus concentration at the highest dilution still infecting 100% of the Vero cells was used to infect Vero cells in 96-well plates for the syncytium formation assays.…”

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

“…Based on the type II CRISPR-Cas system consisting of a single guide RNA (sgRNA), a trans-activating crRNA (tracrRNA) and a RNA-guided endonuclease Cas9 derived from Streptococcus pyogenes, the latest developed CRISPR/Cas9 gene editing technique has been displayed as a simple, efficient and powerful tool for gene editing in biological research [33][34][35]. 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].…”