You Had Me at CRISPR: Exploring Gene Knockout Techniques in an Undergraduate Laboratory Course

Abstract

A research project‐based Cell Biology laboratory serves as a capstone course for undergraduate students in the Biochemistry/Molecular Biology program at Benedictine university. This year, the students worked on a project to knockout a gene to study its function. Previous studies have implicated a role for CFTR in bile acid‐induced diarrhea. The gene‐editing technique, CRISPR (clustered regularly interspaced short palindromic repeats), was used to knockout the cystic fibrosis transmembrane conductance regulation (CFTR) gene to confirm its role in bile aciddiarrhea. In mammalian intestines, excess luminal bile acids affect the growth of colonic epithelial cells and increase fluid secretion resulting in fluid loss.The students were challenged to generate a cell line model ± CFTR. A common intestinal cell model, the T84 cell line derived from human colon carcinoma, was used to study CFTR function. However, these cells do not lend easily to genetic manipulation. Thus, the goal of the students in this course was to use the novel, efficient CRISPR/Cas9 technology to knockout CFTR function in T84 cells and compare the transfection efficiencies of two methods i) CRISPR/Cas9 system, utilizing an sgRNA expressing plasmid and ii) sgRNA in vitro transcription system, to deliver a complete transcribed molecule without a plasmid intermediary.Four groups of students having prior basic molecular biology technique experience learned cell culture techniques, read primary literature, including CRISPR and designed the primers for the constructs necessary for these studies. The CRISPR/Cas9 or the sgRNA construct with a Cas9 plasmid were introduced into T84 cells and the transcription efficiencies of both techniques were evaluated using real time PCR, Western blotting and T7 endonuclease assay. Following successful knockdown of CFTR, WT and CFTR−/− cells, grown in 12‐well plates, were incubated with primary and secondary bile acids and Cl− secretion was measured using MQAE fluorescence.Cell lines provide an excellent reductionist model to study CFTR expression and to introduce students to cell and molecular biology techniques. Students were excited to learn and successfully used innovative techniques to answer research questions in a classroom setting. Presented as a 3‐hour laboratory twice a week, most of the students succeeded in their projected goal to knockout CFTR in one semester, albeit some with low efficiencies. The course was conducted as an active learning classroom, where group activity and participation were essential. Beyond developing scientific expertise, the course further fostered scientific thinking, critical analysis and problem‐solving skills. For example, initial transfection efficiencies of only 3 – 5% were increased to 30–40% by the collaborative trouble shooting efforts of students. Scientific writing skills were honed using revise‐rewrite‐resubmit process whereby students produced multiple faculty‐reviewed and peer‐reviewed drafts of abstracts and publication style reports. They also got the experience of designing poster/slideshow and giving multimedia presentations at research symposia highlighting their findings at local and regional meetings, with documented positive responses given by visiting faculty from other local institutions. Further, these studies helped identify a simple, state of the art method to mediate genome editing in T84 cells, a model that typically has low transfection rates.Support or Funding InformationNSF ‐ MRI: DBI‐1427937 to JS and Institutional Funds, Ben. U. to JS and MPThis abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.