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Optimization of CRISPR/CAS9 gene editing in potato

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2020
Özköse, Dorukcan
Increased human population, climate change, and reduced water supply are demanding creative approaches in agricultural production. Now, a new method of gene editing may allow needed improvements possible. This gene editing technology is called CRISPR / Cas9 technique which is less demanding than that of the other programmable editing technologies such as ZFNs and TALENs. The CRISPR / Cas9 model is developed based on the innate immune system observed in bacteria and archaea that directly degrades nucleic acids of bacteriophages. The safe and effective editing of potato genome especially against disease causing pathogens is of a great value in agriculture. The objective of this research is to explore the capacity of CRISPR / Cas9 technique for editing two potato genes; eukaryotic translation initiation factor 4E (eIF4E) and vacuolar invertase enzyme (VInv)). The eIF4E when mutated generates Potyvirus X defense, VInV prevents acrylamide synthesis, thus allowing long-lasting taste and safer food alternative against cancer. For both of the genes guide RNAs were engineered and were tested for effectiveness of generating mutations. In this research, gene editing is planned to be conducted in potato protoplasts. A high percentage of intact and healthy potato protoplasts were obtained by use of one-month-old plantlets grown in soil, long dark treatment (four days), and lower centrifugation speed during the isolation. Overall, it was possible to set up CRISPR / Cas9 tests in vitro and optimize in vivo studies with the use of better quality potato protoplasts that can be used for regeneration.