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.

中文翻译：

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通过 CRISPR/Cas9 系统将轮状病毒 VP6 基因定点整合至兔 β-酪蛋白位点。


轮状病毒 (RV) 是新生儿病毒性胃肠炎的主要原因，VP6 蛋白已被讨论为潜在的候选疫苗。 CRISPR/Cas9是最新一代的基因编辑工具，可以介导外源基因的定点敲入，为重组蛋白的表达提供强有力的支持。为了设计一种既适合乳腺生产又适合乳汁给药的轮状病毒疫苗，我们选择兔β-酪蛋白（CSN2）基因座作为整合 VP6 基因的靶位点。分析了兔 CSN2 基因座不同靶位点的诱导突变效率，g4 位点似乎是产生突变的最佳位点（g4 72.76 ± 0.32% vs g1 30.14 ± 1.93%、g2 38.53 ± 0.75%、g3 52.26 ± 1.16） %，P < 0.05）。我们进一步比较了兔受精卵细胞质注射两组混合物（含100 ng/μL Cas9 mRNA或Cas9蛋白、20 ng/μL sgRNA4和100 ng/μL供体载体）在兔受精卵中的敲入效率，尽管Cas9 mRNA组当Cas9蛋白用于CRISPR/Cas9系统时，其诱导的HDR效率比Cas9蛋白组（10.3%±3.1%）高达20.0%±2.6%，37.5%的敲入事件部分整合在目标位点中，所有阳性囊胚均显示完全整合，结果表明使用Cas9蛋白比Cas9 mRNA更好地将正确的外源基因整合到目标位点。此外，利用Cas9蛋白组获得了VP6基因正确整合的转基因兔，并用于生产实验性乳基轮状病毒疫苗。 我们的研究提供了一种生产轮状病毒亚单位疫苗的新策略，并为建立针对其他病原体的更广泛的牛奶疫苗保护奠定了基础。