Evaluation of the N6-methyladenosine (m6A) RNA modification pathway as a driver of tumor proliferation via high-throughput CRISPR screening

The methylation of RNA adenosines into N6-methyladenosine (m6A) is the most abundant internal mRNA modification in mammalian cells. It is regulated by writers (e.g., methyltransferase-like 3 (METTL3)) and erasers, while readers mediate its molecular effects at the level of different biological processes (e.g., RNA translation and decay). Consequently, m6A affects multiple cellular functions. Recently, a CRISPR-Cas9 knock-out (KO) screening determined that AML proliferation depends selectively on the enzymatic activity of METTL3. This recent discovery opens new questions regarding the mechanisms by which readers and eraser proteins contribute to the function of m6A in AML, which may be investigated with CRISPR-base editing. In this technology, cytidine or adenine deaminases fused to a nicking Cas9-UGI (uracil glycosylase inhibitor) nuclease perform C->T and A->G transitions respectively, allowing programmable nucleotide changes. These enable the study of m6A at increased resolution and precision, if compared to classic genetic knockouts. The aim of this project is to study m6A in AML cell proliferation by means of a pooled CRISPR- base editing screening. We will develop a CRISPR-base editing platform for the high-density mutagenesis of the genes that are active in the m6A-pathway as well as AML-driver genes. Enrichment analysis of the sgRNAs will determine the association between specific single-nucleotide mutations and the proliferation phenotype. Our project will 1) study the m6A pathway without the confounding factors related to the removal of whole genes; 2) highlight domains associated with proliferation phenotype and that might represent druggable sites; 3) assess the function of variants of uncertain significance (VUS). The study will investigate the role of m6A writers, erasers and readers in AML proliferation and expect to provide molecular insights for the development of novel drug treatments.