CRISPR-based editing strategies to rectify EYA1 complex genomic rearrangement linked to haploinsufficiency

Pathogenic structure variations (SVs) and genomic rearrangements are associated with various types of cancer and rare genetic diseases. Recent studies have used Cas9 nuclease with paired guide RNAs (gRNAs) to generate targeted chromosomal rearrangements. Studies on Cas9-mediated translocations and inversions have mainly focused on producing fusion proteins that cause cancer, whereas research on precision genome editing for rectifying SVs is limited. In this study, through whole- genome sequencing, we identified a novel complex genomic rearrangement (CGR), specifically an EYA1 inversion with a deletion, implicated in branchio-oto- renal/branchio-oto (BOR/BO) syndrome. The CGR results in a loss-of-function allele, leading to haploinsufficiency. To address this, two CRISPR-based editing approaches were tested. First, we engineered Cas9 nuclease and paired gRNAs tailored to the patients genome. The dual CRISPR/Cas9 system induced efficient editing at sites with paracentric inversion in patient-derived fibroblasts (up to 1.6%), and effectively restored the expression levels of the EYA1 gene and its downstream targets, restoring overall transcriptional functionality. Additionally, we engineered gene-activating CRISPR-Cas modules (CRISPRa), which increased EYA1 mRNA and protein expression to wild-type levels in human EYA1 monoallelic knockout cells that mimic the haploinsufficiency. Moreover, CRISPRa significantly improved transcriptional activity essential for target gene expression. This suggests that CRISPRa-based gene therapies may offer substantial translational potential for approximately 70% of disease-causing EYA1 variants responsible for haploinsufficiency. In parallel to deciphering the complexities of the genomic landscape related to human genetic disorders, our findings demonstrate the potential of CIRSPR-guided genome editing for correcting SVs, including those with EYA1 CGR linked to haploinsufficiency.