TEMA: Stvaranje SPRTN i TDP deficijentnih animalnih modela pomoću CRISPR/Cas sustava
Detaljnije o projektu:
DNA-protein crosslink (DPC) is a type of DNA lesion in which a protein becomes irreversibly covalently bound to DNA when exposed to endogenous or exogenous crosslink inducers. Endogenous DPC inducers include products of normal cellular metabolism such as reactive oxygen species, aldehydes and helical DNA modification, while exogenous inducers include UV light, ionizing radiation and various chemicals. DNA-protein crosslinks are common DNA lesions that present a physical blockage to all DNA transactions: replication, transcription, recombination and repair. If not repaired, DPCs cause genomic instability and adverse phenotypes in humans including premature aging, neurodegeneration and cancer. Despite the prevalence and severe consequences of DPCs, DNA-protein crosslink repair (DPCR) has been little studied, largely because it was not considered a DNA damage repair pathway in its own right until recently. In 2014 and 2016, we and others identified novel proteases SPRTN and Wss1 that initiate the removal of DPCs through proteolytic digestion of crosslinked proteins. The discovery of proteolysis-coupled DPC repair led to the recognition of the DNA-protein crosslink repair as a separate DNA damage repair pathway. Currently, we do not know how is this pathway orchestrated, while almost nothing is known about the pathway at the organism level.
Using CRISPR/Cas gene manipulation tools, we knock-out or mutate target genes in zebrafish that we suspect are involved in eliminating DNA-protein crosslinks. We are creating Sprtn-deficient zebrafish strain and double mutants with inactivated tyrosyl-DNA phosphodiesterases (Tdp1, Tdp2a, Tdp2b) with the aim of finding the molecular mechanisms behind the phenotype of human diseases. Sprtn hypomorphic mouse, and human syndromes caused by mutations in the SPRTN (Rjuils-Alf), TDP1 (Spinocerebellar ataxia with axonal neuropathy, SCAN 1), and TDP2 genes (Spinocerebellar ataxia autosomal recessive 23, SCAR23) show severe phenotypes including liver cancer and premature aging, neurological dysfunction and cerebellar atrophy and ataxia, respectively.
Prospective MSc candidates will learn wide range of methods including CRISPR/Cas knock-out design and workflow, cloning, CRISPR probe creation, RNA isolation, qPCR, High resolution melting curve analysis, western blot, genotyping embryos and adults
Group leader: dr. sc. Marta Popović; Supervisors: dr. sc. Ivan Antičević, dr. sc. Cecile Otten
Lab website: https://martafry.wixsite.com/popoviclab,
PI profile: https://www.researchgate.net/profile/Marta_Popovic/publications Email: email@example.com