基于CRISPR/Cas9基因编辑技术的本科教学实验体系的创建与实践

CRISPR/Cas9是新兴的基因编辑技术，在生命科学研究中发挥着重要的作用。将它引入本科生的实验教学，使本科生了解这项前沿科研技术很有意义。我们创建了一个基于CRISPR/ Cas9技术的本科教学实验体系。该实验体系侧重CRISPR/Cas9技术在哺乳动物细胞中的应用，选用一株基因组上被插入mCherry基因的小鼠胚胎成纤维细胞为实验材料，命名为STO-82。首先设计靶向mCherry的sgRNA，构建CRISPR-Cas9/sgRNA共表达质粒。经测序验证无误后，转染到STO-82细胞。采用流式细胞仪分析检测mCherry阴性和阳性两群细胞，分选出阴性单细胞并扩大培养。最后用测序检验单克隆细胞中靶标DNA序列的编辑情况。结果显示，靶位点有插入或缺失突变，说明体系创建成功。该实验体系将sgRNA设计、CRISPR-Cas9/sgRNA共表达质粒的构建、细胞转染、单细胞分选、单克隆细胞培养、测序序列分析等内容融合为一个综合实验，用于高年级本科生的实验教学。根据实际情况，将教学实践内容分解分块教学，也可以做完整性项目教学。本教学实践采用10人左右的小班分块教学，2人一组，经过3个班（共13组）的实践，绝大部分学生都能完成实验，得到预期结果。通过这个实验，学生加深了对CRISPR/Cas9技术的原理和实验流程的理解，锻炼了实验操作能力和严谨的科研思维，也使学生对该技术的医疗应用风险有了一些认识。

Construction and Practice of Experimental System for Undergraduate Teaching Based on CRISPR/Cas9 Gene Editing Technology

CRISPR/Cas9 is an emerging gene editing technique, which plays an important role in life science research. It is of great significance to introduce this cutting-edge scientific technique into the experimental teaching for undergraduates. Therefore, we established an undergraduate experiment system based on CRISPR/Cas9 technology. This experiment system focuses on the application of CRISPR/Cas9-mediating gene editing in mammalian cells. An engineered mouse embryonic fibroblast which genome were inserted with fluorescein mCherry gene was selected as the experimental model, and called STO-82. Firstly, sgRNAs targeting mCherry gene were designed to construct CRISPR-Cas9/sgRNA co-expression plasmids. After being confirmed by sequencing, they were transfected into STO-82 cells. Two groups of cells with mCherry negative and positive signals were detected by fluorescence-activated cell sorting. Single cells with negative fluorescence were separated and then cultivated to become monoclonal cells. The mutation status of mCherry gene in monoclonal cell lines was detected by sequence analysis. The result showed that there were mutations of insertion or deletion at target sites, indicating that the experimental system was successfully established. Therefore, this comprehensive experiment is comprised of sgRNA design, construction of CRISPR-Cas9/sgRNA co-expression plasmids, cell transfection, cell sorting, monoclonal cell cultivation and sequence analysis. This experiment system is used for experimental teaching for senior undergraduates. Teaching practice can either be decomposed into content modules or be taken as a whole program in light of actual situation. In the teaching practice at 3 classes (13 groups in total, two students every group), which adopted the model of small-class teaching (about 10 students per class), the majority completed the content modules and the expected outcomes were achieved. Through the design and teaching practice of this experiment system, the students acquire a deeper understanding for the principle and experimental procedure of CRISPR/Cas9 technology, an enhanced experimental ability and rigorous scientific thinking and also some knowledge in the risk of its medical application.