Non-genetically modified organism in vitro CRISPR/Cas9 gene editing of the lacZα gene: A 4.5 h laboratory course for senior high-school students

Abstract

The CRISPR/Cas9 system opens new horizons (M. Adli, Nat Commun, 2018) regarding genetic modifications of living organisms but also as an in vitro tool in laboratory protocols. Therefore, it boosts possibilities in research and future medical treatments. As the controversial claim of genomically edited babies by He Jiankui (Cyranoski D., Nature, 2019) demonstrates, the new gene editing potentials entail ethical discussions. A public or social discussion presupposes not only a theoretical knowledge or understanding of the system, but also profits from direct laboratory experiences showing how easy these techniques can be applied. Introducing numerous students and classes into these emerging techniques in a modern biology classroom depends on a suitable course concept, which fits legal and organizational requirements at the same time. Therefore, we implemented an appropriate hands-on laboratory course for senior high-school students, lasting just 4.5 h. Particularly with regard to European regulations concerning the handling of genetically modified organisms, the constructs and protocols avoid the transfer of Cas9 DNA. This normally mandatory transfer was replaced by in vitro gene-editing. This leads to Cas9 induced gene knock-outs due to frame shifts and/or the excision of DNA fragments in common Escherichia coli (E. coli) plasmids, such as pUC19. This gene knock-out concept covers various steps: In vitro plasmid editing with Cas9, ligation and transformation of E. coli cells with the modified plasmid DNA and finally the spread plating of transformed E. coli cells in order to analyze colonies after overnight incubation. The successful excision of DNA fragments by in vitro Cas9 treatment was determined by subsequent gel electrophoresis.