The CRISPR (clustered regularly interspaced short palindromic repeats) – CAS (CRISPR associated protein) system is a prokaryotic adaptive immunity mechanism. Emmanuelle Charpentier and Jennifer A. Doudna adapted this system as a tool for gene editing experiments, for which they became the winners of the Nobel Prize 2020 in Chemistry. This new revolutionary technology has become a valuable tool in cell biology.
Students at the School of life sciences and environmental technologies of Avans University of Applied Sciences (Avans UAS, Breda, NL) are trained to become lab-technicians and thereby these students need to learn lab technologies, including CRISPR/CAS. In a newly developed CRISPR course, students of Avans UAS learn the basics of CRISPR/CAS and the use of the CRISPR/CAS toolbox, both in theory and practice. For the practical session on gene editing, a model system developed by Glaser et al. in 2016 is being used. This experiment uses the chronic myeloid leukemia cell line K562 expressing EGFP. A guide RNA was developed in order to either knock-out (Non-Homologous End Joining; NHEJ) the EGFP gene (i.e. green light off), or, using a donor template, to convert EGFP to BFP (Homology Directed Repair; HDR; i.e. green light becomes blue light). A pX330 plasmid was used for expression of both the Cas9 protein and sgRNA in these eukaryotic cells. The plasmid was transfected into K562 cells using the Nucleofector® 2b Device with the program T-016 according to the Lonza Optimized Protocol. This hands-on experience resulted in up to 60% K562-EGFP knock-out cells (via NHEJ) and up to 5% of K562-BFP positive cells (via HDR).
