Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) RNA are DNA-cleaving molecules in bacterial and archaeal degradation pathways meant to defend against viral infection. Recently, biotechnological advances have allowed scientists to use CRISPR as a molecular tool to modify DNA in other living systems, including plants, fruit flies, mice, and humans. This technology creates targeted gene deletions and point mutations, two important mechanisms by which gene function is determined. Given the recent attention to this technology, we sought to incorporate a CRISPR-based research project in our undergraduate Genetics laboratories. The learning objectives of this project emphasize an understanding of bacterial and plant transformation, post-transcriptional gene regulation, gene editing, and phenotype and genotype analysis. Current assessment efforts are designed to evaluate improved student understanding of these topics as well as changes to their feelings of scientific self-efficacy, identity, and value. For this project, each student worked collaboratively to transform a CRISPR plasmid construct into the model plant Arabidopsis thaliana. Expression of a gRNA and the nuclease Cas9 in the plant genome leads to DNA modification at target genes (e.g. Glabrous1, Too Many Mouths, HY5, Phosphoglucomutase (PGM), Scarecrow, and Werewolf). These genes have shown a seedling and/or juvenile plant phenotype when mutated by traditional means, which is necessary to obtain F2 generation CRISPR mutants in a 13-week semester. Our future goal is to expand student involvement in the gene target development process. Additionally, our students will work with SALK deletion lines associated with the unPAK project (NSF grant # 1355106); our highest priority targets will be for SALK seed lines that could not be phenotyped due to multiple T-DNA inserts.
The asset you are trying to access is locked. Please enter your access key to unlock.