Rapid construction of recombinant HVT-HA（Y280） using CRISPR/Cas9 combined with the Cre-lox system

A new threat to the poultry industry was posed by the emergence of a novel variant of the H9N2 subtype avian influenza virus belonging to the Y280 lineage， and complete protection was not provided by the existing commercial vaccines. A recombinant virus expressing the hemagglutinin （HA） protein of the H9N2 Y280 isolate was constructed by using CRISPR/Cas9 gene editing technology with turkey herpesvirus （HVT） as the vector. First， a guide RNA （gRNA） targeting the US2 non‑essential region of the HVT genome was designed and synthesized， and a CRISPR/Cas9 expression plasmid as well as a donor plasmid carrying the red fluorescent protein （RFP） and HA genes were constructed. These recombinant plasmids were co‑transfected into chicken embryo fibroblasts， and after puromycin selection， the cells were infected with HVT. After plaques expressing RFP were observed， three consecutive rounds of plaque purification were performed， and the recombinant virus HVT‑RFP‑HA expressing the HA protein was obtained. Subsequently， the RFP marker gene was excised by the Cre‑lox system， and after three additional rounds of non‑fluorescent plaque purification， a marker‑free recombinant virus， designated HVT‑HA， was finally obtained. The insertion site of the exogenous gene was identified by PCR and sequencing， and the recombinant virus was serially passaged 20 times to evaluate its genetic stability. The results showed that the HA gene was accurately inserted into the US2 site of the HVT genome and was stably retained after 20 consecutive passages. In conclusion， a rapid platform for the construction of recombinant HVT‑vectored vaccines based on the NHEJ‑CRISPR/Cas9 and Cre‑lox systems was successfully established， and the good genetic stability was exhibited by the resulting recombinant virus HVT‑HA. A candidate vaccine strain was thus provided for the prevention and control of the H9N2 Y280 variant； however， its immunoprotective efficacy remained to be further validated.