Abstract
Background: Haploinsufficiency, resulting from loss-of-function mutations in one allele of an autosomal gene, underlies many autosomal dominant rare diseases. Current therapeutic strategies, including gene replacement and allele-specific editing, face limitations in delivery, durability, and applicability. CRISPR-Cas9-based transcriptional activation (CRISPRa) offers a mutation-independent approach to upregulate the expression of the remaining wild-type allele, thereby compensating for the deficient protein product. Methods: We designed and validated a dCas9-VP64-based CRISPRa system targeting the promoters of seven haploinsufficient genes implicated in autosomal dominant disorders (e.g., FOXC2, RHO, AFG3L2). Single-guide RNAs (sgRNAs) were screened for activation efficiency in HEK293T and patient-derived cell lines. Off-target effects and cell viability were assessed. Results: Optimal sgRNAs induced 3- to 8-fold increases in target gene mRNA expression, with minimal off-target activity and no significant cytotoxicity. In a cellular model of FOXC2 haploinsufficiency, CRISPRa restored protein levels to 70% of wild-type and rescued functional defects in lymphedema-associated gene expression. Conclusions: CRISPRa-mediated upregulation of wild-type alleles represents a promising, broadly applicable strategy for treating haploinsufficiency-driven autosomal dominant rare diseases, warranting further in vivo evaluation.
Keywords
CRISPR-Cas9, transcriptional activation, haploinsufficiency, autosomal dominant, rare diseases, gene therapy, dCas9-VP64, sgRNA