基于酵母杂交的CRISPR/Cas9基因编辑教学实验设计与应用效果

Design and application effect of CRISPR/Cas9 gene-editing teaching experiment based on mating yeast

综合制药工程专业实践教学要求和学科前沿方向,设计了"基于酵母杂交的CRISPR/Cas9基因编辑"实验。该实验先采用基因重组技术,分别构建CRISPR/Cas9基因编辑载体,以及重组酵母yPH1和y PH6-8,再利用酵母杂交平台研究CRISPR/Cas9基因编辑载体的靶向性和脱靶机率。该实验内容包括微生物培养、微生物生长曲线绘制、基因重组技术、基因表达和CRISPR/Cas9基因编辑技术,以及学习Origin作图软件和荧光成像技术。该实验具有综合性、设计性和研究性特点,同时具有材料成本低、过程简化和结果明显的优势,不仅方便学生学习基因编辑原理,掌握基因编辑核心技术,了解脱靶效应的普通性,而且有利于培养学生的创造性思维,提高分析、解决问题的能力。

Combined with the practical teaching requirements of pharmaceutical engineering specialty and the cutting-edge direction of the discipline, a comprehensive and exploratory CRISPR/Cas9 gene-editing experiment based on mating yeast is designed. The gene-editing vectors with on-target/off-target oligonucleotides and recombinant yeast strains named yPH1 and yPH6-8 respectively are constructed through DNA recombinant technique, and then the on-target and off-target efficiency are studied based on yeast hybridization. The project includes the microbial cultivation, measurement of microbial growth curve, DNA recombinant technique, gene expression, genome-editing technique, practicing origin graphics mapping software and the fluorescence microscopy. The low-budget experimental materials and distinct results can not only provide easier ways for students to study genome-editing technique and learn more about the popularity of off-target effects, but also can stimulate students’ creative thinking creativity and improve their problem-solving skills.