The impact of this research project is manifold. First, I have generated the first endogenous splice reporter of TTN which can be used to dissect alternative isoforms of TTN in unprecedented detail and at single cell resolution. Notably, we seek to perform high-throughput CRISPR interference screens to identify other genes that regulate the splicing of TTN. Other plans are to use this cell line as read-out for saturation mutagenesis screens to uncover mutations of RBM20 that affect splicing. This is subsequently very important to stratify novel disease-causing mutations in patients with DCM. Finally, the TTN splice reporter cell line can be used in combination with small molecule screening platforms to identify drugs that could revert the aberrant splice profile of TTN concomitant with mutations in RBM20. Secondly, in our paper, we developed an approach for correcting Rbm20 mutations directly in the heart of adult mice. We identified a cohort of RBM20-responsive genes which can be used as biomarkers for screening the magnitude of different RBM20 mutations. This project has broad impact on general gene therapy strategies for hereditary cardiac mutations. Finally, a novel method based on combinatorial barcoding and long-read sequencing was established. This method is broadly applicable to analyze alternative splicing in single cells. In contrast to several other methods, there is no cell size limitation which enables the isoform analysis in large cells such as cardiomyocytes, neurons, and others. In summary, this project pushed the technological limits of several aspects in the fields of transcriptomics, cell engineering and gene therapy and therefore has a broad impact in these areas.
