CRISPR-Cas9 Gene Editing in Yeast: A Molecular Biology and Bioinformatics Laboratory Module for Undergraduate and...

CRISPR-Cas9 genome editing technology is widely used in scientific research and biotechnology. As this technology becomes a staple tool in life sciences research, it is increasingly important to incorporate it into biology curricula to train future scientists. To demonstrate the molecular underpinnings and some limitations of CRISPR-based gene editing, we designed a laboratory module to accompany a discussion-based course on genome editing for college and advanced high school biology students. The laboratory module uses CRISPR-Cas9 to target and inactivate the ADE2 gene in Saccharomyces cerevisiae so as to give red colonies, employing an inexpensive yeast model system with a phenotypic readout that is easily detectable without specialized equipment. Students begin by accessing the yeast ADE2 sequence in a genome database, applying their understanding of Cas9 activity to design guide RNA (gRNA) sequences, using a CRISPR analysis tool to compare predicted on- and off-target effects of various gRNAs, and presenting and explaining their choice of an optimal gRNA to disrupt the ADE2 gene. They then conduct yeast transformations using Cas9 and preselected gRNA plasmids with or without donor templates to explore the importance of DNA repair pathways in genome editing. Lastly, they analyze the observed editing rates across different gRNAs targeting ADE2, leading to a discussion of editing efficiency. This module engages students in experimental design, provides hands-on experience with CRISPR-Cas9 gene editing and collaborative data analysis, and stimulates discussion on the uses and limitations of CRISPR-based gene editing technology.