靶向小鼠CREPT基因134位点的CRISPR/Cas9载体构建及效率检测  

Construction and efficiency detection of CRISPR/Cas9 gene editing vectors targeting serine at position 134 of mouse CREPT gene

作　　者：刘素丽 邹晓辉 范柠粼 孙继超 武会娟 LIU Su-li;ZOU Xiao-hui;FAN Ning-lin;SUN Ji-chao;WU Hui-juan(Department of Technology,Beijing Laboratory Animal Research Center Co.,Ltd.,Beijing 102609,China;Laboratory of Clinical Microbiology and Infection,China-Japan Friendship Hospital,Beijing 100029,China)

机构地区：[1]北京实验动物研究中心有限公司技术部,北京102609 [2]中日友好医院临床微生物与感染实验室,北京100029

出　　处：《生物技术》2022年第4期403-408,420,共7页Biotechnology

摘　　要：[目的]构建靶向小鼠CREPT基因134位丝氨酸的CRISPR/Cas9载体并检测其编辑效率。[方法]利用在线网站设计sgRNAs并克隆入体外转录载体pUC57-sgRNA,桑格测序验证重组载体是否构建成功;以上述重组载体为模板体外转录获得的sgRNAs与商品化Cas9蛋白和靶片段于37℃孵育30 min,随后产物进行琼脂糖凝胶电泳以检测sgRNAs编辑效率。[结果]设计了2条靶向小鼠CREPT基因134位丝氨酸的sgRNAs,并分别准确插入pUC57-sgRNA载体,插入序列无突变;2条sgRNAs均能够与Cas9蛋白正确结合从而介导Cas9蛋白对靶片段的切割,编辑效率分别为0.30、0.36。[结论]成功构建了靶向小鼠CREPT基因134位丝氨酸的CRISPR/Cas9基因编辑载体,且由此转录出的sgRNAs均具有编辑活性,可直接用于小鼠受精卵显微注射制备点突变小鼠模型。[Objective]To construct CRISPR/Cas9 gene editing vectors targeting serine at position 134 of mouse CREPT gene and detect their gene editing efficiency.[Method]sgRNAs were designed on online websites and were cloned into the in vitro transcription vector pUC57-sgRNA,Sanger sequencing was used to verify whether the recombinant vectors were successfully constructed;sgRNAs transcribed in vitro from the recombinant vectors were incubated with commercial Cas9 protein and target fragment at 37℃for 30 minutes,then the products were subjected to agarose gel electrophoresis to examine sgRNA efficiency.[Result]Two sgRNAs targeting serine at position 134 of mouse CREPT gene were designed and were accurately cloned into pUC57-sgRNA vector without mutations;Moreover,the above two sgRNAs were demonstrated to bind to Cas9 protein and mediate cleavage of target fragment,with efficiency of 0.30 and 0.36 respectively.[Conclusion]CRISPR/Cas9 gene editing vectors targeting serine at position 134 of mouse CREPT gene were successfully constructed,and sgRNAs transcribed from them all have gene editing activity,which could be directly used for microinjection of mouse zygotes to prepare point mutation mouse model.

关 键 词：CRISPR/Cas9 CREPT基因 基因编辑 点突变 

分 类 号：R730.2[医药卫生—肿瘤]