In order to understand and eventually cure genetic disease, it is essential to understand which transcriptional and cellular mechanisms lead to the symptoms experienced by patients. Over the past few decades, the introduction of variants into the genome of laboratory mice as model organisms has been a central part of disease research. The process of generating mouse mutants has been accelerated by the discovery of the genome editing technology CRISPR/Cas9. However, the complete analysis of a mouse mutant with conventional phenotyping methods remains laborious. Another layer of complexity has been the discovery that non-coding mutations can be disease causing. Studies in cancer and rare skeletal phenotypes have demonstrated that variants involving the boundaries of the genome organizational units of topologically associating domains (TADs) can cause disruption of the functional units of cis-regulatory elements (CREs), leading to misexpression of genes within the locus and consequent development of disorders or diseases. In this work, we have addressed two central cornerstones of disease research. First, the development and application of a new strategy for phenotyping mouse mutants, in which we tested single-cell RNA sequencing as a technology for unbiased and comparative study of whole mouse mutants. Second, we extended the role of TAD variants to the highly prevalent field of neurological disorders. In the first project, we generated the mouse mutant cell atlas (MMCA) composed of 22 mouse mutants of varying severity. We developed novel analysis tools to detect and visualize mutant phenotypes at different granularities. By applying scRNA-seq to all mutants in a single experiment, we gained new knowledge about previously studied mutants, uncovered new phenotypes, unraveled the cellular dynamics of disease progression, and established the toolkit for comparative and unbiased whole-embryo analysis. In the second project, we performed an in-depth analysis of the Lmnb1 related neurodegenerative disorder acute demyelinating leukodystrophy (ADLD). We generated mouse models of a patient derived duplication of the gene itself and a deletion upstream of the gene. We discovered 3D conformational changes within the locus caused by the deletion, early onset of molecular changes, and morphological changes in the adult mice, together indicating differences in disease progression between the two variants. In summary, both projects contribute to the field of disease research by establishing scRNA-seq as a tool to generate and analyze mutants and by providing an in-depth analysis of a neurodegenerative disease, challenging the current categorisation of this group of diseases as "late-onset".
