CRISPR/Cas-mediated activation of genes associated with inherited retinal dystrophies in human cells for diagnostic purposes

Many patients suffering from inherited diseases do not receive a genetic diagnosis and are therefore excluded as candidates for treatments, such as gene therapies. Analyzing disease gene transcripts from patient cells holds potential for detection and interpretation of causative variants, but may be complicated by unavailability of affected tissue and/or lack of expression of the respective genes in blood or other easily accessible tissues. Using CRISPR/Cas-mediated transcriptional activation (CRISPRa), we developed a robust and efficient approach to activate genes in skin-derived fibroblasts and in freshly isolated peripheral blood mononuclear cells (PBMCs) from healthy individuals. This approach was successfully applied to blood samples from patients with inherited retinal dystrophies (IRDs). We were able to efficiently activate several IRD genes and detect their transcript isoforms using different diagnostically relevant methods such as RT-(q)PCR and long- and short-read RNA sequencing. CRISPRa-mediated transcriptional activation in PBMCs and fibroblasts will contribute to closing the critical gap in the genetic diagnosis of patients with IRD or other inherited diseases.