FOCAS: A Comprehensive Transcriptome-Wide Screening Method for Functional m6A Sites [RNA-Seq]

ABSTRACT: While N6-methyladenosine (m6A) is a widespread RNA modification with profound vital roles, functional dissection of individual m6A sites remains challenging limitated by conventional approaches. To address this gap, we developed FOCAS, a CRISPR-dCas13b-based screening platform for high-throughput, site-specific m6A demethylation. Applying FOCAS to four human cancer cell lines identified 4,475 m6A-regulated genes affecting cell fitness through both mRNA and non-coding RNAs, including newly identified cancer-associated genes with dynamic developmental expression. FOCAS revealed context-dependent and reader-specific effects of m6A within the same gene, underscoring its regulatory complexity. We uncovered both universal and cell type-specific m6A patterns, with unique sites enriched in non-coding RNAs and universal ones in transcription-related genes. In SMMC-7721 cells, we identified an m6A-regulated transcriptional network uncovered extensive epitranscriptome–transcriptome crosstalk. Collectively, FOCAS enables unbiased dissection of m6A function and provides insights into its intricate regulatory logic and therapeutic potential.

ORGANISM(S): Homo sapiens

PROVIDER: GSE273719 | GEO | 2025/10/21

REPOSITORIES: GEO