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.

Keywords: CRISPR-Cas9; Cre-Lox; NHEJ; Pasteurella multocida; duck enteritis virus; fowl cholera; viral vector.

Figure 1

A schematic representation of the cloning strategy for donor plasmid construction containing the OmpH-V5 expression cassette tagged with a removable GFP reporter cassette.

Figure 2

The total positive green cell area measurement with multiple gRNA target sites in first plaque purification of DEV-OmpH-V5. Error bar = standard error of mean. (* p < 0.05, *** p < 0.001).

Figure 3

Schematic illustration of the recombinant DEV-OmpH-V5. (A) Full-length of the attenuated commercial DEV vaccine strain used in this study. The scissor icon represents the gRNA targeting sites in the intergenic region of DEV genome (UL55-LORF11 and UL44-44.5) for OmpH-V5 gene insertion via CRISPR-Cas9 base gene knock-in. (B) Two portions of the genome selected to insert fragment of the GFP and the OmpH-V5 expression cassettes released by Cas9/sgA cleavage from donor plasmid. (C) The recombinant DEV expressing the reporter GFP and OmpH-V5 before the GFP gene is excised by Cre recombinase. (D) After GFP excision, the recombinant vaccine candidate terms are DEV-OmpH-V5 of UL 55-LORF11 and UL44-44.5.

Figure 4

Verification of the recombinant DEV-OmpH-V5 by PCR. (a) The name and position of primers use for verification purification (Square shape) and 5′ and 3′ junction PCR (Oval shape). (b) The purification of DEV-GFP-OmpH-V5 recombinant virus in each insertion site and DEV wild type were detected by PCR. The primers pair between each intergenic region of DEV genome labelled under the panel. (c) 3′ junction PCR verification of DEV-GFP-OmpH-V5 with specific primers in each site to confirm integration and identification insertion sites. Sense orientation insertion of DEV-GFP-OmpH-V5 of UL55-LORF11 was detected by primer pair OmpH-3F & LORF11-R and anti-sense direction of UL44-44.5 was detected by primer pair OmpH-3F and UL44-R. (d) The confirmation of whole OmpH-V5 gene inserted in each recombinant DEV comparing with DEV wild type was identified by PCR using primers OmpH-F and OmpH-V5-R. The molecular size of DNA is indicated in the left lane.

Figure 4

Verification of the recombinant DEV-OmpH-V5 by PCR. (a) The name and position of primers use for verification purification (Square shape) and 5′ and 3′ junction PCR (Oval shape). (b) The purification of DEV-GFP-OmpH-V5 recombinant virus in each insertion site and DEV wild type were detected by PCR. The primers pair between each intergenic region of DEV genome labelled under the panel. (c) 3′ junction PCR verification of DEV-GFP-OmpH-V5 with specific primers in each site to confirm integration and identification insertion sites. Sense orientation insertion of DEV-GFP-OmpH-V5 of UL55-LORF11 was detected by primer pair OmpH-3F & LORF11-R and anti-sense direction of UL44-44.5 was detected by primer pair OmpH-3F and UL44-R. (d) The confirmation of whole OmpH-V5 gene inserted in each recombinant DEV comparing with DEV wild type was identified by PCR using primers OmpH-F and OmpH-V5-R. The molecular size of DNA is indicated in the left lane.

Figure 5

The Excision of the GFP Cassette from DEV-GFP-OmpH-V5 by Cre-Lox system. Before Cre plasmid addition, DEV-GFP-OmpH-V5 plaque under were observed under bright-field (1) and fluorescent microscopy (2). After Cre plasmid treatment (3), this panel shows more than 70% of GFP cassette was removed from the DEV-GFP-OmpH-V5 virus genome. fluorescent microscopy.

Figure 6

Characterization of each recombinant DEV-OmpH-V5 infected CEF cells. (a) Lower panel shows the expression of OmpH protein was analyzed by Western blot using anti-OmpH polyclonal antibody. The positive OmpH expression of CEF cell lysate infected with rDEV-Omp-V5 of UL55-LORF11 (lane 3 and 4) and rDEV-Omp-V5 of UL44-44.5 (lane 5 and 6) were duplicate loaded into each lean. While CEF cells lysate infected with DEV-WT was loaded into lane 2 for negative control. The recombinant OmpH protein was loaded in last lane for positive control. The molecular weight was indicated in first lane. Upper panel shows antibody specific to DEV as a DEV loading control. (b) This panel shows the confirmation the successful of V5 expression by indirect immunofluorescence assay (IFA) with anti-V5 monoclonal antibody (green) sequentially strained with DEV-infected mouse serum (red) for detection of DEV infected-cells. The region of merged images was taken by Incucyte machine. (c) Upper panels display plaque morphology of DEV wild type, DEV-OmpH-V5 of UL55-LORF11 and DEV-OmpH-V5 of 44-44.5. Lower graph shows the plaque diameters of CEF cells infected with recombinant DEVs and DEV wild type were measured at 6 days post infection. (d) The multi-step growth kinetic curve of recombinant DEVs and DEV wild type infected CEF cells at MOI 0.01. Supernatants were collected and viral titers were determined at the indicated time points post-inoculation by plaque assay. (p < 0.01).

Figure 6

Characterization of each recombinant DEV-OmpH-V5 infected CEF cells. (a) Lower panel shows the expression of OmpH protein was analyzed by Western blot using anti-OmpH polyclonal antibody. The positive OmpH expression of CEF cell lysate infected with rDEV-Omp-V5 of UL55-LORF11 (lane 3 and 4) and rDEV-Omp-V5 of UL44-44.5 (lane 5 and 6) were duplicate loaded into each lean. While CEF cells lysate infected with DEV-WT was loaded into lane 2 for negative control. The recombinant OmpH protein was loaded in last lane for positive control. The molecular weight was indicated in first lane. Upper panel shows antibody specific to DEV as a DEV loading control. (b) This panel shows the confirmation the successful of V5 expression by indirect immunofluorescence assay (IFA) with anti-V5 monoclonal antibody (green) sequentially strained with DEV-infected mouse serum (red) for detection of DEV infected-cells. The region of merged images was taken by Incucyte machine. (c) Upper panels display plaque morphology of DEV wild type, DEV-OmpH-V5 of UL55-LORF11 and DEV-OmpH-V5 of 44-44.5. Lower graph shows the plaque diameters of CEF cells infected with recombinant DEVs and DEV wild type were measured at 6 days post infection. (d) The multi-step growth kinetic curve of recombinant DEVs and DEV wild type infected CEF cells at MOI 0.01. Supernatants were collected and viral titers were determined at the indicated time points post-inoculation by plaque assay. (p < 0.01).

Figure 7

Stability of recombinant DEV-OmpH-V5. (a) Detection of OmpH-V5 gene insertion in DEV-OmpH-V5 was confirmed by PCR using primer at both ends of each insertion site during serial 5 passages compared with DEV-WT infected CEF as controls, indicating that the recombinant virus is stable. The lane numbers represent the passage numbers of the recombinant virus. (b) Detection of V5 expression of recombinant DEV was assessed by IFA. Merged images of positive double stained DEV-OmpH-V5 infected CEF with anti-V5 (Green) and anti-DEV (Red) was taken by Incucyte at 5th, 10th, and 15th passages.