Changing DNA specifically CRISPR-Cas

CRISPR-Cas is an adaptive and heritable prokaryotic immune system which targets invasive genetic elements such as viruses and plasmids. In recent years it has been adapted for the site-specific manipulation of eukaryotic genomes by deleting or exchanging gene copies. The nuclease Cas9 recognizes a given sequence within a host cell by a complementary guide-RNA and inserts a double strand break which is repaired by the cell's own non-homologous recombination repair. Due to this unprecise repair mechanism mistakes are inserted and the gene is disrupted. In our experiment we destruct the gene of a viral thymidine kinase stably inserted into the genome of a human glioblastoma cell line. As the thymidine kinase gene is fused to the gene of Green Fluorescent Protein, the success of the experiment can be monitored by a disappearance of the green fluorescence. At the same time cells producing Cas9 show an orange-red fluorescence, because Cas9 is always produced together with the Orange Fluorescent Protein. Green will become red