An Undergraduate Laboratory Manual for Analyzing a CRISPR Mutant with a Predicted Role in Regeneration

Exposing students to undergraduate research has reportedly improved students’ development of knowledge and skills in the laboratory, self- efficacy, satisfaction with their research, retention, and perseverance when faced with obstacles. Furthermore, utilizing authentic course- based undergraduate research experiences (CUREs) includes all students enrolled in the class, giving those who may not otherwise have access to an independent undergraduate research project an opportunity to engage in the scientific process in context of an original, unanswered question. In the fall of 2016, second semester introductory biology students conducted a semester-long research project on the transcription factor Lin28a to determine the effect of Lin28a on regeneration in a CRISPR mutant. During ten laboratory periods, students completed four experiments: 1) genotyping mutants by PCR and RFLP, 2) neuromast regeneration after copper sulfate treatment, 3) measuring changes in gene expression by RT- PCR after fin clipping, and 4) swimming behavior. In the context of this class, students were challenged to design their own experiments, interpret their own data, and make connections among the experiments to draft a final paper presenting their results and conclusions. Here, we present a student laboratory manual that can be adapted to other relevant CRISPR mutants. Overall, this coursework aligns with Vision and Change, and these experiments gave students a taste of the questions, techniques, and experimental design currently used in the field of regenerative biology.