Editing MicroRNA: mRNA Complementarity Using CRISPR-Cas9

MicroRNAs (miRNAs) are a class of small noncoding RNAs that are approximately 20-22 nucleotides in length and have a prominent role in regulating gene expression. miRNA-based regulation has been shown to significantly impact biological processes such as development, differentiation, proliferation, and stress responses. miRNA regulation is largely dictated by complementarity between the miRNA and its target mRNA. Perfect complementarity between miRNA and mRNA results in cleavage of the targeted mRNA, whereas partial complementarity results in translational inhibition. A specific miRNA, miR-34a, has been shown to regulate genes involved in cell cycle progression and apoptosis, and it is often downregulated in many cancer types. Given the importance of miR-34a as regulator in anti-proliferative biological networks, it is important to unravel its function. The goal of this thesis is to characterize miRNA:mRNA complementarity using synthetic biology-based engineered miRNA sensors and custom CRISPRbased editing of miRNA targets. We experiment with HCT116, a human colorectal carcinoma cell line. Our experiments not only assist in understanding miRNA-mediated gene repression, but also aid in developing miRNA-based therapeutics.