病原结构变异(SVs)与各种类型的癌症和罕见的遗传疾病有关。最近的研究已经使用Cas9核酸酶与配对的引导RNA(gRNA)来产生靶向的染色体重排,专注于生产导致癌症的融合蛋白,而用于整流SV的精确基因组编辑研究有限。在这项研究中,我们发现了一种新的复杂基因组重排(CGR),特别是带有删除的EYA1倒置,与分支-器官-肾/分支-器官综合征有关。为了解决这个问题,测试了两种基于CRISPR的方法。首先,我们使用Cas9核酸酶和针对患者基因组定制的配对gRNA。双重CRISPR-Cas9系统可有效纠正患者来源的成纤维细胞的中心旁倒置,有效恢复EYA1mRNA和蛋白的表达,以及它的转录活性需要调节靶基因的表达。此外,我们使用了CRISPR激活(CRISPRa),这导致患者来源的成纤维细胞中EYA1mRNA表达上调。此外,CRISPRa显著提高了靶基因表达所必需的EYA1蛋白表达和转录活性。这表明基于CRISPRa的基因治疗可以为大约70%的引起单倍体不足的EYA1变异体提供实质性的翻译潜力。我们的研究结果证明了CRISPR指导的基因组编辑纠正SV的潜力,包括那些与单倍体不足相关的EYA1CGR。
Pathogenic structure variations (SVs) 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, focusing on producing fusion proteins that cause cancer, whereas research on precision genome editing for rectifying SVs is limited. In this study, we identified a novel complex genomic rearrangement (CGR), specifically an EYA1 inversion with a deletion, implicated in branchio-oto-renal/branchio-oto syndrome. To address this, two CRISPR-based approaches were tested. First, we used Cas9 nuclease and paired gRNAs tailored to the patient\'s genome. The dual CRISPR-Cas9 system induced efficient correction of paracentric inversion in patient-derived fibroblast, and effectively restored the expression of EYA1 mRNA and protein, along with its transcriptional activity required to regulate the target gene expression. Additionally, we used CRISPR activation (
CRISPRa), which leads to the upregulation of EYA1 mRNA expression in patient-derived fibroblasts. Moreover,
CRISPRa significantly improved EYA1 protein expression and transcriptional activity essential for target gene expression. This suggests that
CRISPRa-based gene therapies could offer substantial translational potential for approximately 70% of disease-causing EYA1 variants responsible for haploinsufficiency. Our findings demonstrate the potential of CRISPR-guided genome editing for correcting SVs, including those with EYA1 CGR linked to haploinsufficiency.