目的设计靶向小鼠 Ext1和 Ext2基因的CRISPR/Cas9基因编辑系统，并在细胞水平验证打靶效力，旨在建立高效靶向小鼠 Ext1和 Ext2基因的编辑系统，为后续建立基因编辑小鼠模型奠定基础。
方法基于CRISPR/Cas9系统基因打靶的原理，靶向小鼠 Ext1和 Ext2基因分别设计两条sgRNA导向序列，构建了4个sgRNA表达质粒，并与Cas9表达质粒共转染小鼠N2a细胞，经过嘌呤霉素和杀稻瘟霉素药物筛选后收取阳性转染细胞，PCR扩增打靶区域DNA片段，利用Sanger测序鉴定打靶是否成功，最后通过TA克隆测序进行打靶效率分析，并对7次打靶效率结果进行统计学分析。
结果 Ext1基因的Ext1-M-sgRNA1、Ext1-M-sgRNA2以及 Ext2基因的Ext2-M-sgRNA1、Ext2-M-sgRNA2这4个靶点均成功发生突变，通过TA克隆测序结果统计分析得到4个位点的突变效率分别为90%、80%、70%和50%，且Ext1-M-sgRNA1的打靶效率高于Ext1-M-sgRNA2( P值为0.040 8)，Ext2-M-sgRNA1的打靶效率高于Ext2-M-sgRNA2( P值为0.040 1)。
结论本研究成功构建了高效靶向小鼠 Ext1和 Ext2基因的CRISPR/Cas9系统，为后续构建动物模型深入研究遗传性多发性外生骨疣(hereditary multiple eostosis，HME)奠定基础。
ObjectiveDesign a CRISPR/Cas9 gene editing system targeting mouse Ext1 and Ext2 genes and verified the targeting efficacy at the cellular level, aiming to establish an editing system that efficiently targets mouse Ext1 and Ext2 genes, and to lay the foundation for the subsequent establishment of a gene editing mouse model.
MethodsBased on the principle of the CRISPR/Cas9 gene targeting system, two sgRNA guide sequences were designed to target the Ext1 and Ext2 genes in mice. Four sgRNA expression plasmids were constructed and co-transfected with the Cas9 expression plasmid into mouse N2a cells. After selection with puromycin and blasticidin drugs, positive transfected cells were collected. The DNA fragment of the targeting region was amplified by PCR, and the targeting success was identified by Sanger sequencing. Finally, TA cloning sequencing was used for analysis of the targeting efficiency, and statistical analysis was performed on the results of seven targeting efficiency experiments.
ResultsThe four targets, Ext1-M-sgRNA1 and Ext1-M-sgRNA2 for the Ext1 gene, and Ext2-M-sgRNA1 and Ext2-M-sgRNA2 for the Ext2 gene, all successfully underwent mutations. Statistical analysis of the TA cloning sequencing results showed that the mutation efficiencies for the four sites were 90%, 80%, 70%, and 50%, respectively. The targeting efficiency of Ext1-M-sgRNA1 is higher than that of Ext1-M-sgRNA2 ( P=0.040 8), and the targeting efficiency of Ext2-M-sgRNA1 is higher than that of Ext2-M-sgRNA2 ( P=0.040 1).
ConclusionsIn this study, we successfully constructed a CRISPR/Cas9 system that efficiently targeted mouse Ext1 and Ext2 genes, which laid a foundation for further research on hereditary multiple exostosis (HME) by constructing animal models.
王宁,王添贤,岳鹏鹏,等. 靶向小鼠 Ext1和 Ext2基因的CRISPR/Cas9系统设计及打靶效力分析 [J]. 国际遗传学杂志,2025,48(01)：8-16.
DOI:10.3760/cma.j.cn231536-20240912-00083版权归中华医学会所有。
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除非特别声明，本刊刊出的所有文章不代表中华医学会和本刊编委会的观点。
王宁：数据整理与分析，撰写论文；王添贤：开展了质粒构建、细胞培养、基因型分析等实验；岳鹏鹏：实验设计；于鸿浩：实验设计与指导，论文修改
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