Hereditary defects in the essential and multifunctional transcription and DNA repair factor TFIIH are associated with several distinct, clinically heterogeneous diseases characterized by cancer predisposition, (progressive) neurological defects, developmental failure and segmental progeria, whose pathogenesis is not fully understood. The function and activity of TFIIH has been thoroughly investigated, but it is still not entirely clear how mutations in the same complex can lead to diverse symptoms and human diseases and why the impact of hereditary mutations differs depending on the tissue type. Systematic comparison of TFIIH mutations in cells is difficult because TFIIH is essential and patient cells are not isogenic and often compound-heterozygote. To better understand mutant TFIIH activity in cells, in this project we propose to generate and functionally compare multiple patient-derived mutations in the same TFIIH gene, in isogenic human cells and in C. elegans as in vivo model. To this end, we will label, by knock-in, endogenous TFIIH with the multi-purpose GFP-tag while simultaneously introducing patient-derived mutations in its XPD helicase subunit, using CRISPR-Cas9. Next, using state-of-the-art DNA repair assays, live cell confocal imaging, proteomics and genomics approaches, we will thoroughly investigate the activity of each mutant TFIIH in nucleotide excision repair and transcription, in relation to its phenotypic impact, in both human cells in culture and in vivo, in C. elegans, to study mutant TFIIH mechanism and impact in differentiated cell types such as neurons. As functional TFIIH is essential for life and because its dysfunction causes developmental failure, cancer and aging, it is of major importance to understand its precise activity to be able to promote human health.