Generation of cellular clones for APOBEC1 enzyme and auxiliary cofactors

In this study, we aimed the generation of a cellular model suitable for the analysis of RNA editing induced by the RNA editing enzyme APOBEC1. The term RNA editing has been used to describe numerous cellular processes that result in the modification of RNA sequences differing from that designated by their DNA templates. These processes, affecting RNAs can alter the function or coding potential of the modified transcripts, and significant advances have been made in characterizing the largely unrelated cellular mechanisms by which these modifications are achieved. Overall, my study focused on two aspects of this research. The first one regards the setting up of the conditions for the cytodifferentiation of Caco-2 cells on Transwell filters in specific culture medium to allow them to resemble phenotypically the small intestine enterocytes where the mRNA editing of apolipoprotein B takes place in vivo. Therefore, we have generated clones of Caco-2 cells which are deficient for APOBEC1 and its cofactors namely ACF (Apobec1 Complementation Factor) and RBM47 (RNA Binding Molecule 47). The other aspect regards the work to inactivate the genes that code for the elements of editosome, namely APOBEC1, ACF and RBM47. During this process, we used a very recent and popular genome editing tool derived from the bacterial CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats)/Cas9 (CRISPR-associated 9) system. At the final step, APOBEC1, ACF and RBM47 deficient Caco-2 cells have been cultured in specific media ultimately for the sequence analysis of knocked-out genes.