This laboratory module involves a variation of CRISPR, CRISPR interference. CRISPRi does not cut or edit DNA but utilizes the specificity aspect of CRISPR to target desired sequences. Therefore, the learning focus is on trickier concepts of gene expression and regulation. Students hypothesize how to target and inhibit Escherichia coli genes or operons of choice, aiming to alter clear, screenable phenotypes, such as lethality or auxotrophy. Using databases and literature, students analyze their gene’s expression in the context of promoters or other regulatory elements. Students then design a single guide RNA to block the gene’s expression using CRISPRi. Ultimately, students design PCR primers, clone sgRNA genes, sequence, and test their system in vivo using relevant growth medias. This student-driven experiment also addresses critical learning goals like designing controls, media supplementation, and lab calculations. Overall, it is a straightforward way to introduce CRISPR to undergraduates while still obtaining large learning gains.
