Crowd‐sourcing CRISPR: A Course‐Based Research Project to Investigate the Impact of Chromatin Environment on Double‐Strand Break Repair While Enhancing Student Learning

Chromatin structure is an active player in the cellular response to DNA double‐strand breaks (DSBs). The chromatin signals surrounding a DSB are thought to govern which repair pathway, homologous recombination or non‐homologous end joining, is engaged and if cell cycle arrest or cellular death is induced. However, the logic of how combinatorial chromatin signals are integrated to decide cellular repair outcome is unclear and is a major limitation in our understanding of regional predisposition to genomic change. To test the model that certain chromatin signatures (combinations of various histone post‐translational modifications) impact DSB susceptibility and repair, we have optimized a CRISPR‐Cas9 system for inducing DSBs, which we can monitor for DSB frequency and repair using quantitative PCR and plating assays using Saccharomyces cerevisiae . To curate a collection of genome‐wide DSB sites and to create custom guide RNAs (gRNAs), students in a spring semester 16‐week Molecular Genetics laboratory course select different putative DSB sites using publicly available genome‐wide chromatin modification maps and transcription data. Students then design and clone unique gRNA constructs for Cas9 targeting to their selected site. After sequence verification, students introduce the gRNA constructs into a yeast strain containing fluorescent DNA repair proteins. Students then examine break induction and repair pathway using qPCR across the break site, as well as using cell‐based assays. Follow up work to measure differences in repair pathway choice, checkpoint activation, and cell viability is completed by summer research students. The educational goals of the project are to increase students' content knowledge, communication skills and technical and analytical skills, and are assessed using backward‐designed course materials. This project provides the five defining characteristics of a successful course‐based undergraduate research experience (CURE), as defined by CUREnet: 1. Element of discovery and production of novel data; 2. Iteration built into the lab; 3. High levels of collaboration; 4. Students learn authentic scientific practices and 5. The work is broadly relevant/publishable (1). A pedagogical research question we wish to address is: what elements of a CURE lead to specific outcomes? We would like to assess the incorporation of these CURE design elements in the course using Laboratory Course Assessment Survey (2) and test the relationship between CURE elements and mid‐ and long‐term outcomes such as self‐efficacy and persistence. A recently published assessment tool, the Persistence in the Sciences survey, measures project ownership, self‐efficacy, science identity and community values (3) and will be deployed in parallel with LCAS. Incorporating this research into a CURE will not only leverage the power of parallel projects to collect data for greater scientific understanding, but it will bring this high‐impact practice to a larger diversity of our students, enhance student learning, and aims to inspire the next generation of scientists. This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal .