N6-methyladenosine in DNA promotes genome stability

DNA base lesions, such as incorporation of uracil into DNA or base mismatches, can be mutagenic and toxic to replicating cells. To discover factors in repair of genomic uracil, we performed a CRISPR knockout screen in the presence of floxuridine, a chemotherapeutic agent that incorporates uracil and fluoro-uracil into DNA. We identified known factors, such as uracil DNA N-glycosylase (UNG), but also unknown factors, such as the N6-adenosine methyltransferase, METTL3, as required to overcome floxuridine-driven cytotoxicity. Visualized with immunofluorescence, the product of METTL3 activity, N6-methyladenosine, formed nuclear foci in cells treated with floxuridine. The observed N6-methyladenosine was embedded in DNA, called 6mA, which was confirmed using mass spectrometry. METTL3 and 6mA were required for repair of lesions driven by additional base damaging agents, including raltitrexed, gemcitabine, and hydroxyurea. Our results establish a role for METTL3 and 6mA to promote genome stability in mammalian cells, specially in response to base damage. Overall design: Cas9-expressing HT-29 cells were transduced with a lentiviral sgRNA library, split into four pools. Each pool was transduced at a MOI of 0.3 and 1 µg/mL puromycin-containing medium was added the next day. Selection was continued until 4 days post transduction, which was considered the initial time point, t0. At this point the transduced cells were divided into two populations and split into technical triplicates. One population was untreated and 2.2 nM floxuridine was added to the other. Cells were grown with or without floxuridine until t11 or t15, subculturing every three to four days. Cell pellets were frozen at each time point for genomic DNA (gDNA) isolation. A library coverage of =500 cells/sgRNA was maintained throughout the screen. gDNA from cell pellets was isolated using QIAGEN Gentra Puregene kit and genome-integrated sgRNA sequences were amplified by PCR using KOD Hot Start Polymerases. i5 and i7 multiplexing barcodes (Ilumina) were added in a second round of PCR and final gel-purified products were sequenced on Illumina HiSeq2500 or NextSeq500 systems to determine sgRNA representation in each sample. Gene knockouts enriched at t11 or t15 as compared to t0 were identified using Model-based Analysis of Genome-wide CRISPR-Cas9 Knockout (MAGeCK) analysis.