Avian influenza is highly contagious poultry disease and the mortality can reach 100 %, leading to huge economic burden and threat to food security. Vaccination is the most effective strategy for prevention and control of influenza. Recombinant vector vaccines are effective promising vaccines capable of immunizing against multiple pathogens. Traditional methods for constructing recombinant vaccines involve homologous recombination or bacterial artificial chromosomes but these methods can be time-consuming and labour-intensive. The clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 system is a recently developed gene editing technology which has proven beneficial to gene modification and offers an alternative for constructing recombinant vaccines. There are mainly two methods used for gene insertion; error-prone non-homologous end joining (NHEJ) and the high-fidelity homology-directed repair (HDR) pathway. Owing to its high fidelity, most studies focus on using HDR for vaccine development but NHEJ offers some attractive advantages through its high efficiency. In our study, both HDR and NHEJ dependent CRISPR/Cas9 systems were explored for the rapid generation of recombinant influenza vaccines using duck enteritis virus and herpesvirus of turkeys as vectors.

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