Functional Characterization of Malaria Parasite DNA Photolyase Gene Using CRISPR-Cas9 technology


Thesis Type: Post Graduate

Institution Of The Thesis: Istanbul University, Faculty Of Science, Moleküler Biyoloji Ve Genetik Bölümü, Turkey

Thesis Language: English

Student: İlknur YILMAZ

Principal Consultant (For Co-Consultant Theses): Ahmed Sayed Ibrahım Aly

Co-Consultant: Bedia Palabıyık

Abstract:

Ultraviolet light damages DNA by converting any two adjacent thymines into a thymine dimer that is potentially mutagenic, carcinogenic, or lethal to the organism. This damage is repaired in all organisms (except mammals) by the enzyme Photolyase [1,3]. Dr. Aziz SANCAR has won the Noble prize for the characterization of the function of a homologue of the photolyase enzymes in higher eukaryotes. Here, we are going to use the CRISPR-Cas9 technology to investigate function of DNA Photolyase by targeted gene deletion, gene knockin-tagging and gene swap with plants and yeast photolyases. We have designed the knock-out construct of our study to contain the novel monomeric photo-convertible fluorescent protein Eos4b. UV or blue light exposure of less than 1 minute transforms EOS4b protein from bright green to bright red. Thus, we will be able to investigate the effect of UV on knockout parasites using a UV sensitive marker. For the Knockin-tagging study, we have designed the construct to tag the photolyase protein at the C-terminus with the very bright and photostable NeonGreen or Ruby3 fluorescent proteins, which contain the Strep Twin epitope tag. For the gene swap design for to determine function of photolyase gene by gene swapping of different photolyase and Cryptochrome genes found in other organisms into the malaria parasite. In the future, knowing the detailed function of the gene will lead to future studies to develop vaccine against malaria or to develop an inhibitor for use in the treatment of patients