基于密码子优化mSaCas9蛋白的重组表达与应用  

Recombinant Expression and Application of mSaCas9 Protein Based on Codon-optimization

作　　者：陈若雪 蒋月雯 王梦洋 许朝然 梁晶婕 陈天圣 CHEN Ruoxue;JIANG Yuewen;WANG Mengyang;XU Zhaoran;LIANG Jingjie;CHEN Tiansheng(State Key Laboratory of Mariculture Breeding(Jimei University)/Engineering Research Center of the Modern Technology for Eel Industry,Ministry of Education/Key Laboratory of Healthy Mariculture for the East China Sea,Ministry of Agriculture and Rural Affairs/Fisheries College of Jimei University,Xiamen 361021,China;College of Fisheries of Huazhong Agricultural University,Wuhan 430070,China)

机构地区：[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,福建厦门361021 [2]华中农业大学水产学院,湖北武汉430070

出　　处：《广东海洋大学学报》2024年第4期19-26,共8页Journal of Guangdong Ocean University

基　　金：国家自然科学基金(32273127,31771648);集美大学科研基金(ZQ2020003)。

摘　　要：【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。【Objective】Using SaCas9 from Staphylococcus aureus to study gene editing proteins that can replace commercial Cas9,to provide a reference for the production of proteases suitable for gene editing in fish.【Method】 Through optimizing SaCas9(named mSaCas9) with the codon preference of medaka(Oryzias latipes),cloning and constructing recombinant plasmid pET28a-mSaCas9,and obtaining prokaryotic expression of mSaCas9 recombinant protein by using Escherichia coli BL21(DE3),the activity of the purified protein was verified by in vitro enzyme digestion;the applicability of the mSaCas9-RNP delivery method was verified by microinjection using the purified protein.【Result】The recombinant plasmid pET28a-mSaCas9 was successfully constructed,and its recombinant protein had a molecular mass of 130 ku;2.5 mg of mSaCas9 protein was purified in 1 L of medium with a purity of 95%;in vitro enzyme digestion showed that the PCR products of tyr and oca2 genes could be edited at a final concentration of 30 ng/μL,which demonstrated that the purified protein possessed editing activity in vitro.The mSaCas9 protein and oca2-gRNA of melanin synthesis-related gene were injected into medaka embryos,and the eyes of the embryos showed pigmentation loss,which indicated that mSaCas9-RNP could be applied to medaka genome editing.【Conclusion】 The active Cas9protein with smaller molecular mass was obtained by in vitro expression and purification,and the effectiveness of gene editing at the individual level by delivering SaCas9 protein and gRNA,i.e.,SaCas9-RNP(SaCas9 ribonucleoprotein),was demonstrated in fish.

关 键 词：mSaCa9 基因编辑蛋白 CRISPR/Cas9 原核表达 SaCas9-RNP 显微注射 

分 类 号：Q812[生物学—生物工程]