A Course-Based Undergraduate Research Experience in CRISPR-Cas9 Experimental Design to Support Reverse Genetic Studies in Arabidopsis thaliana

Abstract

Gene-editing tools such as CRISPR-Cas9 have created unprecedented opportunities for genetic studies in plants and animals. We designed a course-based undergraduate research experience (CURE) to train introductory biology students in the concepts and implementation of gene-editing technology as well as develop their soft skills in data management and scientific communication. We present two versions of the course that can be implemented with twice-weekly meetings over a 5-week period. In the remote-learning version, students performed homology searches, designed guide RNAs (gRNAs) and primers, and learned the principles of molecular cloning. This version is appropriate when access to laboratory equipment or in-person instruction is limited, such as during closures that have occurred in response to the COVID-19 pandemic. In person, students designed gRNAs, cloned CRISPR-Cas9 constructs, and performed genetic transformation of Arabidopsis thaliana. Students learned how to design effective gRNA pairs targeting their assigned gene with an 86% success rate. Final exams tested students' ability to apply knowledge of an unfamiliar genome database to characterize gene structure and to properly design gRNAs. Average final exam scores of ∼73% and ∼84% for in-person and remote-learning CUREs, respectively, indicated that students met learning outcomes. The highly parallel nature of the CURE makes it possible to target dozens to hundreds of genes, depending on the number of sections. Applying this approach in a sensitized mutant background enables focused reverse genetic screens for genetic suppressors or enhancers. The course can be adapted readily to other organisms or projects that employ gene editing.

Keywords: CRISPR-Cas9; CURE; course-based undergraduate research experience; plant biology; remote learning.

FIG 1

Strategy to identify genetic modifiers through CRISPR-Cas9. Proteins that potentially interact with a protein of interest (POI) are identified through yeast two-hybrid library screens or affinity-purification mass spectrometry. Each candidate interactor gene is assigned to two students. Students identify homologs that may be functionally redundant with a candidate interactor and select two guide RNA (gRNA) sequences to target the candidate and its close homologs, if any. Students use PCR and Golden Gate cloning to insert both gRNAs into a CRISPR-Cas9 construct. Correct constructs are identified after E. coli transformation with colony PCR, plasmid preparation, and sequencing. Constructs are then transformed into the Arabidopsis thaliana poi mutant background. Transformed seed (T1) are selected and selfed to produce T2 seed. Pooled T2 seed from different T1 lines carrying a single construct are phenotyped for either suppression or synthetic enhancement of the poi mutant phenotype. CRISPR-induced mutations are then validated by sequencing of the target gene(s). Black boxes around “candidate interactors” and “CRISPR-Cas9 constructs” indicate CURE contributions specific to this research.

FIG 2

Timeline of the CURE. Steps shown in the left column are recommended prerequisite lessons before beginning the research project. Middle column steps outline the remote-learning CURE while right column steps outline the in-person CURE and require laboratory facilities.