陈若雪,蒋月雯,王梦洋,许朝然,梁晶婕,陈天圣

• 1:海水养殖生物育种全国重点实验室(集美大学)/鳗鲡现代产业技术教育部工程研究中心/农业农村部东海海水健康养殖重点实验室/集美大学水产学院
• 2:华中农业大学水产学院

摘要(Abstract)：

【目的】利用金黄色葡萄球菌（Staphylococcus aureus）来源的SaCas9研究可替代商业Cas9的基因编辑蛋白，为生产适用于鱼类基因编辑的蛋白酶提供参考。【方法】以青鳉（Oryzias latipes）密码子偏好优化SaCas9（命名为mSaCas9），克隆构建重组质粒pET28a-mSaCas9，并利用大肠埃希菌（Escherichia coli）BL21(DE3)原核表达mSaCas9重组蛋白，对纯化蛋白活性进行体外酶切验证；利用纯化蛋白进行显微注射，验证mSaCas9-RNP递送方式的应用性。【结果】成功构建重组质粒pET28a-mSaCas9，其重组蛋白分子质量为130 ku；在1 L培养基中纯化获得2.5 mg mSaCas9蛋白，纯度为95%；体外酶切结果显示，其终质量浓度为30 ng/μL时即可编辑tyr和oca2基因的PCR产物，表明纯化所得蛋白具备体外编辑活性；向青鳉胚胎中注射mSaCas9蛋白和黑色素合成相关基因oca2-gRNA，胚胎眼部出现色素缺失，表明mSaCas9-RNP可应用于青鳉基因组编辑。【结论】通过体外表达纯化获得了有活性且分子质量更小的Cas9蛋白，并在鱼类中证明了以递送SaCas9核糖核蛋白（SaCas9-RNP）形式，实现个体水平基因编辑的有效性。

关键词(KeyWords)： mSaCa9;基因编辑蛋白;CRISPR/Cas9;原核表达;SaCas9-RNP;显微注射

Abstract:

Keywords:

基金项目(Foundation): 国家自然科学基金（32273127,31771648）;; 集美大学科研基金（ZQ2020003）

作者(Author): 陈若雪,蒋月雯,王梦洋,许朝然,梁晶婕,陈天圣

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