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

A global assembly of cotton ESTs

Approximately 185,000 Gossypium EST sequences comprising >94,800,000 nucleotides were amassed from 30 cDNA libraries constructed from a variety of tissues and organs under a range of conditions, including drought stress and pathogen challenges. These libraries were derived from allopolyploid cotton (Gossypium hirsutum; A(T) and D(T) genomes) as well as its two diploid progenitors, Gossypium arboreum (A genome) and Gossypium raimondii (D genome). ESTs were assembled using the Program for Assembling and Viewing ESTs (PAVE), resulting in 22,030 contigs and 29,077 singletons (51,107 unigenes). Further comparisons among the singletons and contigs led to recognition of 33,665 exemplar sequences that represent a nonredundant set of putative Gossypium genes containing partial or full-length coding regions and usually one or two UTRs. The assembly, along with their UniProt BLASTX hits, GO annotation, and Pfam analysis results, are freely accessible as a public resource for cotton genomics. Because ESTs from diploid and allotetraploid Gossypium were combined in a single assembly, we were in many cases able to bioinformatically distinguish duplicated genes in allotetraploid cotton and assign them to either the A or D genome. The assembly and associated information provide a framework for future investigation of cotton functional and evolutionary genomics