A simple and rapid approach to develop recombinant avian herpesvirus vectored vaccines using CRISPR/Cas9 system

Abstract

HighlightsDevelopment of a rapid pipeline for generating recombinant HVT-based vaccines.NHEJ repair pathway makes targeted insertion of the foreign gene more efficient.Incorporation of RFP cassette enables the easy identification of recombinant virus.The recombinant virus has similar growth rate as parental virus with stable inserts.

Generation of A Triple Insert Live Avian Herpesvirus Vectored Vaccine Using CRISPR/Cas9-Based Gene Editing

“…The detailed procedures for generation of recombinant HVT are described previously [23]. For the generation of HVT-VP2-gDgI, donor plasmid pGEM-sgC-Lox2272-GFP-gDgI and Cas9/gRNA expression plasmids targeting both HVT65/66 region of HVT-VP2 and the donor plasmid [23] were co-transfected into CEF followed by HVT-VP2 virus infection. GFP fluorescent single cells were isolated by fluorescence activated cell sorting, as described previously [25].…”

“…To generate triple insert HVT recombinant, we inserted ILTV gDgI and AIV H9HA expression cassettes sequentially into the HVT-VP2 virus, reported previously [23], to generate HVT-VP2-gDgI-HA. In order to distinguish between the different donor templates and to ensure that the reporter cassette was correctly excised at each stage, different bait sequences (sgB target sites for HA and sgC target sites for gDgI) were introduced at the ends of the donor templates for releasing the donor sequences.…”

“…Different variant Lox sequences (LoxN for HA and Lox2272 for gDgI) facilitated marker gene excision by Cre recombinase (Figure 1a). Insertion of the gDgI and HA expression cassettes into the HVT-VP2 genome at HVT65/66 and US2 loci respectively were carried out sequentially using the strategy described previously [23]. After each insertion and reporter excision, we examined the correct location and orientation of the insert by junction PCR (5 and 3 ) and the purity of the purified plaque was determined by PCR using primers outside of the insertion sites (Figure 1b).…”

“…Previously we have shown that the VP2 expression cassette was stably integrated into the HVT genome during repeated cell passages [23]. To determine the stability of the newly inserted two expression cassettes during continuous passage of triple insert recombinant virus, HVT-VP2-gDgI-HA was sequentially passaged on primary CEFs and infected cells at the 15th passage were examined by fluorescence imaging.…”

Live poultry vaccines against highly pathogenic avian influenza viruses

“…Tang et al suggested that CRISPR/Cas9-based genome editing could be used as a powerful tool for the generation of the HVT recombinants expressing viral antigens [22].…”