First Year Course-Based Undergraduate Research Experience (CURE) Using the CRISPR/Cas9 Genome Engineering Technology in Zebrafish

Abstract

Genetic analysis in model systems can provide a rich context for conceptual understanding of gene structure, regulation, and function. With an intent to create a rich learning experience in molecular genetics, we developed a semester-long course-based undergraduate research experience (CURE) using the CRISPR-Cas9 gene editing system to disrupt specific genes in the zebrafish. The course was offered to freshman students; nine students worked in four groups (two to three members per group) to design, synthesize, and test the nuclease activity of the CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/sgRNAs for targeted disruption of specific genes in the zebrafish. Each group worked with a gene with an already known mutant phenotype that can be visually scored and a gene that had not been studied in zebrafish previously. Embedded in the course were a series of workshop-styled units or tutorials, including tours to core facilities. The focus was on introducing and developing skills that could be accommodated within the span of a semester. Each group successfully cloned at least one plasmid-encoding CRISPR/sgRNA template, visually analyzed injected embryos, and performed genotyping assays to detect CRISPR-Cas9 activity. In-class discussions, a final end-of-semester written test, and group oral presentations were assessed for an understanding of the CRISPR-Cas9 system, application of the CRISPR-Cas9 system as a gene manipulation tool, and experimental methods used to create plasmid vectors and synthesize sgRNA. In addition, poster presentations were evaluated by faculty, graduate students, and senior undergraduate students at a University research exposition. Self-reflections in the form of group conversations were video recorded. All students (9/9) distinctly showed learning gains after completing the activity, but the extent of the gains was variable, as seen from results of a written test and poster presentation assessment. Qualitative analysis of evaluations and self-reporting data indicated several gains, suggesting that all students found many aspects of the CURE valuable and gained project-specific (conceptual) and transferrable skills (science process and science identity).

FIGURE 1

Course overview with modules, objectives, and outcomes. The orange boxes show the preparatory and core laboratory modules leading up to the poster presentations. The violet boxes show the modules that support the core laboratory modules.

FIGURE 2

Workflow of the molecular biology lab module. Experiments included construction and linearization of a CRISPR/sgRNA template, in vitro transcription of sgRNA, injection of sgRNA with Cas9 protein, observation and analysis of development in injected embryos, and genotyping injected embryos by PCR to detect CRISPR-Cas9 nuclease activity. Numbers indicated in blue circles represent the week in which the activity/procedure was done. CRISPR = clustered regularly interspaced short palindromic repeats; PCR = polymerase chain reaction.

FIGURE 3

Assessment of student learning from poster presentations at the undergraduate research exposition. Student presentations were assessed based on their demonstrated understanding in four areas. The x-axis represents the total number of poster evaluations.

FIGURE 4

Post-course survey responses. Student responses (n=7 students) on the value of activities performed during the course. CRISPR = clustered regularly interspaced short palindromic repeats; PCR = polymerase chain reaction.