Site-specific Intergration Of Rotavirus VP6 Gene In Rabbit ?-casein Locus By CRISPR/Cas9 System

Rotavirus(RV)is the leading cause of viral gastroenteritis in neonates and every child experienced at least one episode of rotavirus-induced diarrhea under the age of five.Being the major inner-capsid protein of rotavirus,VP6 accounts for 51% of the viral protein and is the group specific antigen,Although the protective mechanisms of VP6 have not been completely clarified,recombinant VP6 protein is shown to induce heterotypic cross-protective rotavirus immunity responses and to confer protection against rotavirus shedding in animal models,and has been discussed as an alternative vaccine candidate.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 ?-casein(CSN2)locus was chosen as the target site to integrate the VP6 gene.The ultimate aim of this study was to efficiently generate genetically modified rabbits though injection of Cas9 m RNA,sg RNA and donor plasmid into one-cell embryo.1.Targeted mutagenesis efficiency analysis of CRISPR/Cas9 systemFour sg RNAs target CSN2 locus in the rabbit genome were designed though the website http//:crispr.mit.com,and the four corresponding RGS vectors were also designed.The efficiencies of these sg RNAs were first tested by co-transfection with plasmid expressing Cas9,g RNA vector and RGS vector into the 293 T cells.Flow cytometry showed that all four g RNA had cutting activity.After that,REF cell lines derived from rabbit fetal ear were transfected by Cas9 plasmid and g RNA vector individually.PCR products,including the target site,was amplified and analyzed by T7 EI cleavage assay for mutagenesis identification.Results showed that four targets have cleavage band,and the efficiency of these sg RNAs were significant difference(g1,37.1%;g2,42.0%;g3,60.7%;g4,62.4%;respectively)between the four sites.Then all four PCR products were sub-cloned and sequenced to characterize the modifications.In view of the different chromatin environment around the target sites between zygotes and cell lines,we further verified the genome editing efficacy by cytoplasmic injection of Cas9 m RNA and sg RNA into rabbit fertilized eggs on the basis of RFF results.The results showed that there was no significant difference(g1: 72.77±1.12%,g2: 70.33±0.56%,g3: 73.33±1.84%,g4: 73.44±0.86% vs control group: 73.25±1.03%,p>0.05)in the development rate of blastocysts injected with four g RNAs and the gene editing efficiency injected with Cas9 m RNA and sg RNA was significant difference(g4: 72.76±0.32% vs g1: 30.14±1.93%,g2: 38.53±0.75%,g3: 52.26±1.16%,p<0.05)between the four sites,suggesting that g RNA4 was the most favorable sg RNA for the CRISPR/Cas9 system in this target region.Therefore,we used the g RNA4 in subsequent experiments.2.Site-specific integration of exogenous gene in CSN2 locus with different length homology arm of donor vectorThe DSB mediated by CRISPR/Cas9 can stimulate a homologous recombination in the presence of a DNA donor with the appropriate homology arms.After verifying the efficiencies of sg RNAs,we constructed two homologous donor vectors with different length arm though amplify left and right homology arms from rabbit genome,and EGFP CDS sequence from p EGFP-N1 vector.Then we connect those fragments onto double digestion p MD-18 T vector and obtained Donor-EGFP1(200bp)and Donor-EGFP2(1000bp).The activity of the two donor vectors were tested by co-transfection with plasmid expressing Cas9 and g RNA4 vector into the REF cells lines.PCR products,including the 5'J and 3'J was amplified and analyzed the knock-in events.Then the positive PCR products were sub-cloned and sequenced.Results showed that the two donor vectors have activity,and integrate the EGFP gene into the target site.Based on this,we tried to illustrate homologous recombination efficiency by cytoplasmic injection of Cas9 m RNA,sg RNA4 and the two donor vector into rabbit zygotes,results showed that there was no significant difference of knock-in efficiency(17.25±1.60% vs 19.23±1.73%,p>0.05)between the two groups.But all of the knock-in events by Donor-EGFP1 were partial integration in the target site,when Donor-EGFP2 used in the CRISPR/Cas9 system,75% of the positive blastocysts showed completely integrated,results showed that the 1000 bp homology arm lengh is better than 200 bp group to integrate the correct exogenous gene into the target site.3.Generation of VP6 knock-in rabbitsAfter selection of highly active sg RNA in CSN2 locus,we further verified the CRISPR/Cas9-mediated site-specific integration of VP6 gene by cytoplasmic injection of Cas9 m RNA,sg RNA4 and donor vector into rabbit zygotes.The injected zygotes were in vitro cultured for 4 days and the blastocysts were selected for knock-in events detection.Minimal toxic effect of the CRISPR/Cas9 system containing VP6 donor vector was found on the embryo development.We further compared the knock-in efficiency through cytoplasmic injection of two group mixture(containing 100 ng/?L Cas9 m RNA or Cas9 protein,20 ng/?L sg RNA4 and 100 ng/?L donor vector)in rabbit zygotes,though the Cas9 m RNA 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 m RNA 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.But the positive rabbit was died lived four months,and we test all the tissues genotype,results showed that the rabbit we obtained was not a chimera but a heterozygote.To rule out random integration of the transgene,Southern blot analysis was also used to confirm successful targeting.These results,together with those of the PCR and Southern blotting firmly demonstrate the capacity of Cas9 endonuclease activity to trigger homology-directed insertion of a functional expression unit at a native locus in the rabbit genome.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.