Universität zu Köln

Daliri, Karim (2024). Developing a Gene Editing Strategy to Target the COL1A1 Promoter: Significance for Fibrotic Disorders and Pioneering Beyond the Boundaries of CRISPRi System. PhD thesis, Universität zu Köln.

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Abstract

CRISPR-Cas9 gene editing is a promising therapy for pathogenic mutations, but current editing approaches are mainly aimed at relatively small groups of patients with specific mutations. Here, we introduce a new application of gene editing to develop an antifibrotic strategy that could potentially be used to address a wide range of organs with fibrosis. In our research on adenine base editing, we successfully targeted the CCAAT box inside the promoter of Col1a1gene promoter in fibroblasts to substitute a twin adenine (AA) with guanine (GG). This innovative modification led to reduction in collagen indicating the precision and efficiency of our editing approach.To assess the broader implications of this intervention; we employed high-throughput multi-omics technologies. Through transcriptomics (RNA-seq) and proteomics (tandem mass spectrometry), we aimed to evaluate our gene editing approach on the molecular signature. Notably, our investigation revealed that the reduction in Col1a1 expression influencing in PI3K-Akt signalling pathway which is linked with various organ fibrosis such as liver, kidney, lung and heart. This suggests the specificity and efficacy of our editing approach for possible antifibrosis goals. By targeting CCAAT promoter region, our method presents a versatile alternative to the CRISPR interference (CRISPRi) system. This addresses its key drawbacks, such as reversible and transient outcomes, which are suboptimal especially in the context of therapeutic applications where permanent cure is desired. Col1a1 promoter editing may thus represent a permanent and optimal therapeutic strategy across diverse fibrotic disorders.