Site-specific integration of rotavirus VP6 gene in rabbit β-casein locus by CRISPR/Cas9 system

Rotavirus (RV) is the leading cause of viral gastroenteritis in neonates and VP6 protein has been discussed as a potential candidate vaccine. CRISPR/Cas9 was the latest generation of gene editing tools that can mediate the site-specific knock-in of exogenous genes, providing strong support for the expression of recombinant proteins. Here, seeking to design a rotavirus vaccine that would be suitable for both mammary-gland-based production and milk-based administration, rabbit β-casein (CSN2) locus was chosen as the target site to integrate the VP6 gene. The efficiency of inducing mutations in different target sites of rabbit CSN2 locus was analyzed and g4 site seems to be the best one to generate mutations (g4 72.76 ± 0.32% vs g1 30.14 ± 1.93%, g2 38.53 ± 0.75%, g3 52.26 ± 1.16%, P < 0.05). We further compared the knock-in efficiency through cytoplasmic injection of two group mixtures (containing 100 ng/μL Cas9 mRNA or Cas9 protein, 20 ng/μL sgRNA4, and 100 ng/μL donor vector) in rabbit zygotes, though the Cas9 mRNA group induced an HDR efficiency as high as 20.0% ± 2.6% than Cas9 protein group (10.3% ± 3.1%), 37.5% of the knock-in events were partial integration in the target site, when Cas9 protein used in the CRISPR/Cas9 system, all of the positive blastocysts showed completely integrated, results showed that the use of Cas9 protein is better than Cas9 mRNA to integrate the correct exogenous gene into the target site. Moreover, the transgenic rabbit that harbored correct integration of VP6 gene was obtained using Cas9 protein group and was used to produce an experimental milk-based rotavirus vaccine. Our research provides a novel strategy to produce rotavirus subunit vaccine and make a foundation for building broader milk-based vaccine protection against other pathogens.