METTL3-based epitranscriptomic editing screening identifies functional m6A sites in cancers [CRISPR screen_22Rv1]

In this study, we used a targeted CRISPR/CasRx-METTL3 screen to identify m6A peaks that are critical for prostate and lung cancers. Overall design: The CRISPR screen employed in our pooled sgRNA library consisted of ~12,045 sgRNAs targeting m6A peaks in prostate cancer and lung cancer patients, with 5 sgRNAs per gene on average. The sgRNAs were designed using the "cas13design". Stable dCasRx-METTL3-expressing 22Rv1 cell line was generated. dCasRx-METTL3-expressing 22Rv1 cell line was infected with the library at an MOI of ~ 0.3 and coverage of 400x. 24h post-infection, cells were selected with blasticidin for 5 days and part of cells were sujected to DNA extraction as day 0. Other cells were cultured in dish for ~16 days (day 16), maintaining 400x coverage prior to our screening assay. Genomic DNA was extracted from replicate samples and sgRNA inserts were amplified by PCR. The input amount of genomic DNA was calculated to achieve 400x coverage of the library and resulting libraries were sequenced on an Illumina HiSeq500.