Using CRISPRa-Mediated Activation of Endogenous Target Genes in Peripheral Blood Mononuclear Cell to Characterize Splicing Site Variants in Patients with Genetic Disorders

RNA-level detection is a critical approach for determining whether genetic variants affect splicing and for identifying potential pathogenic variants. However, tissues where disease-causing genes are expressed are often difficult to obtain in routine testing, while readily accessible tissues like blood do not express all disease-causing genes. CRISPR activation (CRISPRa) is a powerful technique for activating endogenous gene expression, and its application in peripheral blood mononuclear cells (PBMCs) offers an effective strategy for RNA analysis. In this study, we used CRISPRa to activate the expression of three disease-associated genes— FBN1 , F8 , and DMD —in PBMCs, achieving upregulation of target gene expression within 48 hours. In patient-derived cells, activation of FBN1 expression revealed that a deletion of exons 48–53 leads to a 708-bp in-frame deletion variant at the mRNA level. Notably, this CRISPRa-based method enabled the validation of splicing site variants within 3 days, significantly reducing turnaround time for clinical testing. Furthermore, we successfully activated 83 common disease-causing genes and established a single-guide RNA (sgRNA) library platform for activatable genes. This resource facilitates broader validation and screening of splicing site variants. Overall, this is the first study to demonstrate the use of CRISPRa for RNA-level variant detection in patient PBMCs. Our approach provides a novel approach for validating splicing site variants, including variants of unknown significance, as pathogenic in real clinical settings.