Berendtsen, Nicolai Tubæk

Butt, Mohammad Muddser

Ibsen, Rasmus Nygaard

Jensen, Joachim Wittrup Højris

Sayed, Abid

In this paper, we set out to design a protocol for gene knock-out in GM LS174T human colon cancer cells, with the focus on educating master’s students in gene editing techniques. First, we needed to decide which method to use, between TALEN, ZFN and CRISPR/Cas9. By weighing the pros and cons of each method, we came to the conclusion, that CRISPR/Cas9 was the most suitable method to move on with, due to the low cost and time consumption associated with this method. The experiment utilized a self-cloning CRISPR/Cas9 system, which is essential due to the time-consuming cloning process. The purpose of the experiment is to knock out an inserted YFP-gene in the LS174T cells, by transfecting a sgRNA-plasmid and Cas9-plasmid into the cells. The effect of the YFP-gene knock-out is quantified with fluorescence microscopy and can be visualized as a reduction or removal of the fluorescence. The aim of the protocol is to provide students with knowledge of an advanced gene technological method currently used in molecular biology. We were, in the experiment, effectively able to reduce the fluorescence of the cells by between 42% and 62% depending on the sgRNA sequence chosen. Following this procedure in the lab, students will gain skills in working with human cells, as well as using fluorescence microscopy, and analyzing data with ImageJ. Furthermore, design of sgRNA for CRISPR/Cas9 can be included in the course

Implementering af CRISPR/Cas9 - Genteknologi på den Naturvidenskabelige Uddannelse på RUC

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