Clustered regularly interspaced short palindromic repeats (CRISPR) technology has revolutionized gene editing, offering vast applications in genetic research across various organisms and industries. This study focuses on integrating CRISPR-Cas9 and homology directed repair (HDR) techniques to edit two known genes in Saccharomyces cerevisiae, namely Adenine2 (ADE2) and Sterile12 (STE12). These genes influence metabolic and developmental processes, demonstrating distinct phenotypes upon successful editing. The aim is to familiarize upper division undergraduate students with cutting-edge biotechnological tools like CRISPR, emphasizing practical applications and implications in genetic studies.
Utilizing the budding yeast S. cerevisiae as a model organism in laboratory exercises presents an economical, straightforward, and effective approach to teach CRISPR methodologies in an educational setting. The experiment allows students to engage in all stages of the gene editing process, from selecting guide RNAs to analyzing mutant phenotypes. The protocol’s flexibility enables editing of various expressed genes within the yeast genome, facilitating customizable learning experiences for students. Moreover, the study highlights the significance of metabolic and developmental pathway mutants in eukaryotic cellular functions and their implications on genetic studies and biotechnology applications.
CRISPR gene editing in yeast serves as a practical demonstration of genetic engineering principles, offering insights into forward genetics and gene editing concepts. The use of S. cerevisiae as a model organism underscores its adaptability and relevance in genetic research, bridging fundamental genetic mechanisms across eukaryotic organisms. The incorporation of guide RNAs into the pCAS plasmid, along with the preparation of gap repair templates, demonstrates the hands-on application of CRISPR techniques in manipulating genetic sequences within living cells.
The experimental procedure, designed for upper-division undergraduate laboratory courses, involves detailed protocols for guide RNA incorporation, gap repair template preparation, and large-scale competent yeast cell production. The study’s outcomes are evaluated based on student performance, understanding of CRISPR terminologies, and successful gene editing results. The analysis also includes student feedback, emphasizing the significance of practical applications in enhancing comprehension of complex genetic techniques and fostering independent research skills.
Key Takeaways:
– CRISPR-Cas9 and HDR techniques can be effectively integrated into undergraduate laboratory courses for hands-on gene editing experiences.
– The use of S. cerevisiae as a model organism provides a cost-effective and adaptable platform for teaching genetic engineering concepts.
– Practical applications of CRISPR methodologies enhance student comprehension of genetic mechanisms and cellular processes.
– Flexibility in the experimental protocol allows for customization and exploration of various gene editing scenarios within the yeast genome.
Tags: fungi, yeast, bioinformatics
Read more on pmc.ncbi.nlm.nih.gov