CRISPR AS A TOOL FOR TARGET DISCOVERY AND TECHNOLOGICAL INNOVATION: HIGH-THROUGHPUT CBE SCREENING TO EVALUATE M6A MODIFICATION PATHWAY IN CANCER PROLIFERATION AND DRUG RESISTANCE, AND INNOVATIVE PRIME EDITING STRATEGIES FOR DISEASE MODELLING AND THERAPY

N6-methyladenosine (m6A) is the most abundant internal RNA modification of mRNA in mammals. The m6A -pathway comprises numerous effectors, which are classified based on their function. m6A is deposited co-transcriptionally by “writers”, including methyltransferase-like 3 (METTL3) and methyltransferase-like 14 (METTL14) that form a heterodimer regulated by the METTL-associated complex (MACOM). Its removal is mediated by “erasers”, including fat-mass and obesity-associated protein (FTO), while the methylated adenosines of RNAs are detected by “readers”, which mediate in the regulation of target RNAs, for instance by promoting splicing, nuclear export, stability and decay. Altered m6A deposition, removal and identification affect gene expression resulting in pathogenic disease phenotypes such as cancer proliferation. For instance, METTL3 promotes proliferation in colorectal cancer (CRC) and acute myeloid leukaemia (AML), and its downregulation by small molecule inhibitor affects proliferation in AML and is currently in phase I-II clinical trial. Therefore, expanding the study to all m6A effectors in cancer cell proliferation might be relevant for the discovery of novel molecular therapeutic targets for tumors, including CRC and AML. Furthermore, the possible clinical use of METTL3 inhibitor arises the need to identify mutations of m6A-pathway effectors that might cause resistance or sensitivity to this treatment in AML. In this work, we used Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) Cytosine base editing (CBE) screening to perform saturating mutagenesis of m6A-pathway effectors. In details, we assessed the effect of single nucleotide variants (SNVs) in CRC proliferation, highlighting domains of m6A effectors that are critical for promoting, or downregulating, tumor growth. In AML, the proliferative effect of m6A-effectors mutagenesis was determined upon METTL3 inhibition, enabling to identify mutations that might confer resistance or sensitivity to this treatment. Validation of these variants will 1) identify domains of m6A effectors that could represent putative novel therapeutical targets and 2) allow to study the molecular effects that link edited mutations to the proliferative phenotype observed in CRC and AML. In the second part of this work, we explored technological innovations of prime editing for precise generation of genetic disease models and therapy. This enabled to develop expertise in the use of Prime editing technology as a valuable tool for validation of sgRNAs resulted from our screen.