An Undergraduate Research Project Utilizing CRISPR-Cas9 Gene Editing Technology to Study Gene Function in Arabidopsis thaliana

摘要

The CRISPR-Cas9 system functions in microbial viral pathogen recognition pathways by identifying and targeting foreign DNA for degradation. Recently, biotechnological advances have allowed scientists to use CRISPR-Cas9-based elements as a molecular tool to selectively modify DNA in a wide variety of other living systems. Given the emerging need to bring engaging CRISPR-Cas9 laboratory experiences to an undergraduate audience, we incorporated a CRISPR-based research project into our Genetics class laboratories, emphasizing its use in plants. Our genetic manipulations were designed for Arabidopsis thaliana, which despite serving as a plant research model, has traditionally been difficult to use in a classroom setting. For this project, students transformed plasmid DNA containing the essential CRISPR-Cas9 gene editing elements into A. thaliana. Expression of these elements in the plant genome was expected to create a deletion at one of six targeted genes. The genes we chose had a known seedling and/or juvenile loss-of-function phenotype, which made genetic analysis by students with a limited background possible. It also allowed the project to reach completion in a typical undergraduate semester timeframe. Assessment efforts demonstrated several learning gains, including students' understanding of CRISPR-Cas9 content, their ability to apply CRISPR-Cas9 gene editing tools using bioinformatics and genetics, their ability to employ elements of experimental design, and improved science communication skills. They also felt a stronger connection to their scientific education and were more likely to continue on a STEM career path. Overall, this project can be used to introduce CRISPR-Cas9 technology to undergraduates using plants in a single-semester laboratory course.