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

Figure 1. Optimization of gene knock-in via CRISPR/Cas9-induced NHEJ repair. (A) HRM analysis to select the most efficient sgRNA; (B) schematic presentation of insertion of GFP expression cassette between DEV UL26 and UL27; (C) DEV-GFP plaque under UV excitation (1) or phase contrast (2); (D) the efficiency of GFP cassette knock-in with different DEV infection dose; (E) the efficiency of GFP cassette knock-in with different infection time post transfection. Error bar = standard error of mean. * p < 0.05.

Figure 2. Schematic presentation of the application of NHEJ-CRISPR/Cas9 and Cre-Lox systems in construction of recombinant DEV-HA. (A) The live attenuated DEV virus genome with sg2 targeting the intergenic region between viral gene UL26 and UL27; (B) HA expression donor plasmid with GFP expression cassette flanked with Lox site. Bait sequence of SgU was introduced to both side of the insertion segment; (C) the recombinant DEV virus expressing HA and the reporter GFP; (D) the vaccine candidate with the GFP reporter expression cassette removed by recombinant Cre enzyme.

Figure 3. NHEJ-CRISPR/Cas9-based knock-in is non-directional and the unintended indels are minimal. (A) The schematic of sg2 target site in the intergenic region between DEV UL26 and UL27; (B) the target sequence of sgU; (C) the sequencing results of 5′ junction of 4 clones of DEV-GFP-HA; (D) the sequencing results of 3′ junction of 4 clones of DEV-GFP-HA. The DEV genome is colored in black; the sgU target sequence is colored purple; the insertion sequence is colored green; and the indel is colored in red.

Figure 4. Optimization of GFP reporter removal via Cre-Lox system. CEF cells were transfected with Cre or control plasmids. At 24 h post transfection, the CEF cells were infected with DEV-GFP-HA at MOI 0.0025 or 0.01 respectively. Images were taken at 48 h post infection. (A) Empty vector with 0.0025 MOI infection; (B) Cre plasmid with 0.0025 MOI infection; (C) empty vector with MOI 0.01 infection; (D) Cre plasmid with 0.01 MOI infection; (E) the efficiency of GFP reporter removal with different infection DEV dose. The virus was harvested at 48 h post infection and was used to infect CEF cells; the percentage of GFP positive and negative plaques was calculated.

Figure 5. Characterization of DEV-HA. (A) Detection of HA cassette insertion by PCR; (B) detection of HA expression by immunochemistry; (C) detection of HA expression by Western Blot. Comparable protein loading in each lane was demonstrated by alpha tubulin detection.

Figure 6. Plaque morphology and replication kinetics of wild type and recombinant DEV viruses. (A) Plaque morphology of DEV wild type, DEV-GFP and DEV-HA; (B) the plaque size of DEV wild type, DEV-GFP and DEV-HA; (C) multi-step growth curve of DEV wild type, DEV-GFP and DEV-HA. CEF cells were infected with DEV at MOI 0.01. Viruses were harvested at 6 h, 12 h, 24 h, 48 h and 72 h post-infection. ** p < 0.001.