利用CRISPR/Cas9系统建立斑马鱼Morn3基因敲除模型

Morn3 knockout zebrafish model established by the CRISPR/Cas9 system

摘要:

目的：采用高效基因编辑系统CRISPR/Cas9对斑马鱼的Morn3基因进行编辑，以期建立Morn3基因敲除斑马鱼模型。方法：针对斑马鱼Morn3基因用生物信息学选取靶位点，构建gRNA表达载体并体外转录，将gRNA和Cas9mRNA混合物显微注射到斑马鱼单细胞期的受精卵中，24~48 h提取基因组DNA，PCR扩增出目的片段，限制性酶切法初步检测基因打靶情况，最后进行测序分析确定编辑效果。结果：取3个靶位点（M1、M2、M3）进行试验，选择gRNA浓度较高的M2和M3进行注射。限制性酶切及测序的结果显示M3已产生基因编辑效应。结论：本研究通过CRISPR/Cas9系统成功获得Morn3基因编辑的突变体，为进一步探讨Morn3基因的作用提供研究基础。

Abstract:

Objective：To establish a Morn3 knockout zebrafish model using the efficient CRISPR/Cas9 gene editing system. Methods：The target sites for the Morn3 gene in zebrafish were chosen by bioinformatics analysis；the gRNA expression vector was constructed，and in vitro transcription was performed；then a mixture of gRNA and Cas9 mRNA was microscopically injected into the one-cell fer－tilized eggs of zebrafish. Genomic DNA was extracted at 24 to 48 hours post microinjection，and target fragments were amplified by PCR. Restriction enzyme digestion was used to detect gene targeting. Finally，sequencing analysis was used to determine the editing effect. Results：Three target sites（i.e.，M1，M2，and M3） were tested，and the two sites with higher concentrations of gRNA（M2 and M3） were injected. Results from restriction enzyme digestion and DNA sequencing revealed that gene editing effect had been produced in M3. Conclusion：The Morn3 knockout zebrafish model has been successfully obtained by the CRISPR/Cas9 gene editing system，which provides a basis for further study of the Morn3 gene.