Thus far we have implemented activities on all four work packages designed for the project.
In WP1, we expanded previous research by incorporating single-cell data to better define the molecular signatures of holoclone-forming stem cells (KSC), transient amplifying progenitors (TAC), and differentiated cells. New KSC markers identified include H1B, NUSAP1, RRM2 and STMN1. Notably, H1B is a novel target gene of FOXM1, crucial for self-renewal and differentiation in epidermal stem cells. These markers help distinguish KSCs from TACs. In addition, we demonstrated that KSCs uniquely repair DNA damage mainly through homologous recombination. FOXM1, downstream of YAP, coordinates the response to genotoxic stress and sustains epithelial self-renewal.
In WP2, we used a new reference to predict genes involved in intra-lineage reprogramming, employing two bioinformatics tools. This approach identified 10 genes. We evaluated the expression of these genes at RNA and protein levels in keratinocyte cultures. To test the effects of these genes, we are setting up a single-cell CRISPR activation (sc-CRISPRa) screening. The experiments are ongoing.
In WP3, we are developing a "selection-based directed evolution" to develop Cas9 variants with specific PAM sequence recognition. We switched from luminescence-based screening to antibiotic selection and focused on mutations to improve Cas9 specificity and overcome the same technical concerns. We also created a user-friendly platform called “AlPaCas” to screen Cas proteins for allele-specific targeting. AlPaCas identifies SNV-derived PAMs and suggests Cas enzymes or mutational changes to enhance specificity towards specific PAM.
In WP4, we are developing He-RASE, a HEK293 cell line model mimicking a "biallelic genomic context" of a dominant disease for testing CRISPR/Cas systems identified by WP3. This model helps test new CRISPR tools due to the scarcity of primary EBS cells and aims to develop similar models for other dominant diseases, such as Dominant Dystrophic EB (DEB).
