利用CRISPR/Cas9技术构建能克隆难度序列的Stbl2^(TM)菌株  

Construction of Stbl2^(TM) strain capable of cloning difficult sequence by CRISPR/Cas9 technology

作　　者：覃鸿妮[1] 谢亦潇 汤淑伟 薛高旭 谢悦 张勇 QIN Hongni;XIE Yixiao;TANG Shuwei;XUE Gaoxu;XIE Yue;ZHANG Yong(School of Biotechnology,Suzhou Industrial Park Institute of Services Outsourcing,Suzhou 215123,China;Genewiz Biological Technology Company Limited,Suzhou 215123,China;Ruitebai Biological Technology Company Limited,Suzhou 215123,China)

机构地区：[1]苏州工业园区服务外包职业学院生物科技学院,江苏苏州215123 [2]金唯智生物科技有限公司,江苏苏州215123 [3]瑞特佰生物科技有限公司,江苏苏州215123

出　　处：《扬州大学学报（农业与生命科学版）》2022年第5期87-93,100,共8页Journal of Yangzhou University：Agricultural and Life Science Edition

基　　金：国家自然科学基金面上项目(22077092);江苏省产学研合作项目(BY2020521);江苏省高校“青蓝工程”项目(2020)。

摘　　要：Stbl2^(TM)菌株适合克隆不稳定的外源DNA片段,但该菌株易受噬菌体侵染,且在构建某些高拷贝质粒时,难以克隆成功。利用CRISPR/Cas9技术将Stbl2^(TM)菌株的抗噬菌体相关基因fhuA和控制拷贝数的基因pcnB进行编辑,降低克隆难度,提高克隆效率。针对2个基因分别设计sgRNA,构建含有sgRNA的质粒pTarget-fhuA和pTarget-pcnB,分别将2个质粒与pCas9-Red质粒转入Stbl2^(TM)菌株中,经菌落PCR和测序验证基因正确编辑后再将2个外源质粒消除,并将2 kb的线粒体基因组序列作为外源基因,转入菌株进行最终的功能验证。结果表明:fhuA基因被成功敲除103 bp,pcnB基因被成功敲除802 bp,将高拷贝pUC57质粒转入基因编辑后的Stbl2^(TM)菌株,可明显降低质粒的拷贝数,且能正确克隆线粒体基因难度序列。该研究构建的菌株可作为后续克隆难度序列的感受态使用,还可为难度序列的基因克隆提供借鉴方法。Stbl2^(TM) strain is suitable for cloning unstable foreign DNA fragments,but it is easy to be infected by phage and difficult to clone successfully when constructing some high copy plasmids.The anti-phage related gene fhuA and the copy number controlling gene pcnB of Stbl2^(TM) strain were edited by CRISPR/Cas9 technology to reduce the difficulty of cloning and improve the cloning efficiency.sgRNA was designed for the two genes,and plasmids pTarget-fhuA and pTarget-pcnB containing sgRNA were constructed.The two plasmids and pCas9-Red plasmids were transferred into Stbl2^(TM) strain respectively.The correct editing of the genes was verified by colony PCR and sequencing,and then the two foreign plasmids were eliminated.Mitochondrial genome sequence(2 kb)was used as foreign gene,and the function of the transferred strain was verified.The results showed that fhuA gene was successfully knocked out by 103 bp and pcnB was successfully knocked out by 802 bp.Transferring the high copy pUC57 plasmid into the gene edited Stbl2^(TM) strain could significantly reduce the copy number of the plasmid and clone the mitochondrial gene sequence correctly.The strain constructed in this study can be used as a receptive state for subsequent cloning of difficult sequences,and can provide a method for gene cloning of difficult sequences.

关 键 词：CRISPR/Cas9技术 Stbl2^(TM)菌株 fhuA基因 pcnB基因 

分 类 号：Q78[生物学—分子生物学]