Construction and Practice of Experimental System for Undergraduate Teaching Based on CRISPR/Cas9 Gene Editing Technology / 中国生物化学与分子生物学报

ABSTRACT

CRISP R/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.