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) affects cell proliferation. Indeed, CRISPR- knock-out (KO) screenings identified m6A effectors (writers, erasers and readers) as essential in cancer cell lines, e.g., colorectal cancer (CRC) and acute myeloid leukaemia (AML). This opens new questions regarding the mechanisms by which these effectors contribute to the function of m6A in cancer, which may be investigated with CRISPR-cytosine base editing (CBE). In CBE, a cytidine deaminase fused to a nicking Cas9 nuclease performs C->T transitions, allowing programmable nucleotide changes. The aim of this project is to study m6A in cancer cell proliferation by means of a pooled CRISPR screening. By applying CRISPR-CBE and KO, enrichment analysis of the sgRNAs will determine the association between specific single-nucleotide mutations and gene-loss, and the proliferation phenotype. In details, we prepared the in-silico sgRNA library targeting m6A and control genes. We applied a tiling strategy (i.e., multiple sgRNAs targeting different gene locus) to perform a high-density mutagenesis of the targeted sequences in HT-29 (CRC) and AML (MOLM-13, NOMO-1) cell lines. Our screening will identify m6A genes important for cancer cell proliferation, and that might represent druggable sites. In addition, we will create a dataset of gene variants associated with the proliferative phenotype, which may be useful for assessing the function of variants of uncertain significance. The study will investigate the role of m6A writers, erasers and readers in CRC and AML proliferation and expect to provide molecular insights for the development of novel drug treatments.