A DNA-protein crosslink (DPC) is a type of DNA lesion where a protein becomes irreversibly covalently bound to DNA upon exposure to endogenous or exogenous crosslink inducers. Endogenous DPC inducers are products of normal cellular metabolism such as reactive oxygen species, aldehydes and DNA helical alterations, while exogenous inducers include UV light, ionizing radiation and various chemicals. DNA-protein crosslinks are one of the most common DNA lesions and 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 frequency and severe outcomes of DPC formation, DNA-protein crosslink repair (DPCR) has been sparsely studied, mostly because it has not been considered to be a separate DNA damage repair pathway until recently. In 2014 and 2016, several groups identified novel proteases, Wss1 and SPRTN, which initiate the removal of DPCs through the proteolytic digestion of crosslinked proteins. The discovery of proteolysis-coupled DPC repair lead to recognition of DPCR as a separate DNA damage repair pathway. However, we currently do not know how this pathway is orchestrated and which other factors are involved. Indeed, almost nothing is known of DPCR mechanisms in vivo. Therefore, within this project we aim to unravel the orchestration of the DPCR pathway in vivo using zebrafish as a well-characterized vertebrate model. We will use CRISPR/Cas9 gene manipulation tools to knock-out and mutate specific genes in zebrafish which we suspect are involved in DPC removal. The contribution of each protein (and their combinations) to DPCR will be quantified after DPC isolation from transgenic zebrafish embryos and adults. We will also generate a GFP reporter assay in transgenic fish which will enable the quantification of DPCR efficiency in vivo.