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.

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.

Analysis of Chlamydomonas mutants with abnormal expression of CO2 and HCO3- uptake systems.

Eukaryotic microalgae such as Chlamydomonas reinhardtii possess an inducible CO2 concentrating mechanism that operates as a very close interaction between pyrenoid-based Rubisco, various carbonic anhydrases (CAs), and inorganic carbon (Ci) transport systems. While external and internal CAs have been characterised to the molecular level, the biochemistry and molecular biology of Ci uptake mechanisms have not been elucidated. Both Ci species, CO2 and HCO3-, are taken up by the cells and chloroplasts during steady-state photosynthesis. After acclimation to limiting Ci, CO2 and HCO3- transport, measured in whole cells or chloroplasts, change their kinetic characteristics from a constitutive low-affinity state to an inducible high-affinity state. In order to learn more about the genes involved in the signal transduction pathway and in the Ci transport systems, we performed insertional mutagenesis using the arg7 gene as a selectable marker. Application of aqueous membrane inlet mass spectrometry allowed discrimination between CO2 and HCO3- uptake. Data is presented on two mutants, M46 and M21, which show severe damage to the constitutive Ci uptake systems and which are unable to induce a high-affinity state. The mutations might be either in the signal transduction pathway or in the transporters themselves. In addition, we present data that shows a very close connection between high-affinity HCO3- uptake and high-affinity NO3- uptake in cells of C. reinhardtii.