Abstract: Objective To construct CRISPR / Cas9 gene knockout plasmids targeting mouse CREPT gene. Method sgRNAs targeting mouse CREPT gene were designed according to the CRISPR / Cas9 target design principles and were cloned into the in vitro transcription vector pUC57-sgRNA to construct CREPT gene knockout plasmids. Result Three sgRNAs were designed and cloned into puc57-sgRNA, in vitro
digestion experiment showed that the above three sgRNAs could mediate Cas9 protein to cleave the target fragments. Conclusion Mouse CREPT gene knockout plasmids and in vitro transcribed sgRNAs were prepared, laying an important foundation for construction of gene knockout mouse model.

Key words: CREPT, CRISPR / Cas9, gene knockout

摘要： 目的 利用规律间隔成簇短回文重复序列 / 相关蛋白 9( CRISPR / Cas9) 系统构建靶向小鼠 CREPT 基因的敲除质粒。 方法 根据 CRISPR / Cas9 靶点设计原则靶向小鼠 CREPT 基因的 sgRNAs,与体外转录载体 pUC57-sgRNA连接,构建 CREPT 基因敲除质粒;体外切割实验验证设计的 sgRNAs 是否具有活性。 结果 设计了 3 条 sgRNAs 并分别准确插入 pUC57-sgRNA 载体;体外切割实验结果显示,以上 3 条 sgRNAs 均能够介导 Cas9 蛋白对靶片断进行切割。 结论 制备了小鼠 CREPT 基因敲除质粒和体外转录的 sgRNAs,为构建 CREPT 基因敲除小鼠模型奠定了重要基础。

关键词: CREPT, CRISPR / Cas9, 基因敲除