CRISPR-MEDIATED TARGETED MODULATION OF REGULATORY ELEMENTS IN DISEASE MODELS

Abstract

Enhancers and promoters are regulatory factors which are dominant factors that regulate the expression of genes, and dysregulation of these factors has continued to be related with the emergence of complex diseases, cancer and other neurological disorders. Despite the fact that most of the disease-related variants are found within non-coding regions, their specific functional roles are not well comprehended. The development of CRISPR-Cas9-based technologies has made it possible to specifically investigate these regulatory sequences and has made possible new studies dissecting gene regulatory processes in disease settings. The aim of this work was to measure the functional importance of chosen regulatory factors through CRISPR based targeted regulation in models of disease. The publicly available genomic databases were used to identify specific enhancers and promoter regions and targeted them with the help of CRISPR interference (CRISPRi) and CRISPR activation (CRISPRa) systems. The use of guide RNAs to regulate regulatory activity using experimental validation in cultured cell models was done with experimental validation in cultured cell models placing guide RNAs in the cell models to regulate the activity without potential irreversible alterations in the genome. Quantitative PCR and RNA sequencing were used to measure changes in gene expression, and the proliferation and apoptosis tests to measure the phenotypic effects. The data showed that the specific regulation of the regulatory elements resulted in meaningful locus-dependent changes in the expression of the genes with a corresponding locus-specific modification in the disease-related cellular phenotypes. These results point to the functional significance of non-coding regulatory regions in the regulation of genes. To sum up, CRISPR-mediated modulation is an effective approach to understand the role of regulatory elements and provides possible prospects of therapeutic intervention.