FOCAS: Transcriptome-wide screening of individual m⁶A sites functionally dissects epitranscriptomic control of gene expression in cancer.

Abstract

Although N⁶-methyladenosine (m⁶A) is a pervasive RNA modification essential for gene regulation, dissecting the functions of individual m⁶A sites remains technically challenging. To overcome this, we developed functional m⁶A sites detection by CRISPR-dCas13b-FTO screening (FOCAS), a CRISPR-dCas13b-based platform enabling high-throughput, site-specific functional screening of m⁶A. Applying FOCAS to four human cancer cell lines identified 4,475 m⁶A-regulated genes influencing cell fitness via both mRNAs and non-coding RNAs (ncRNAs), many of which are newly linked to cancer and exhibit dynamic developmental expression. FOCAS uncovered context-dependent and reader-specific effects of m⁶A within the same gene, revealing its intricate regulatory logic. We further uncovered universal and cell-type-specific m⁶A patterns, with unique sites enriched in ncRNAs and universal ones in transcription-related genes. In SMMC-7721 cells, we identified m⁶A-regulated transcriptional networks that demonstrated extensive epitranscriptome-transcriptome crosstalk. Overall, this study established a powerful, unbiased approach for the functional dissection of m⁶A, advancing the understanding of its complexity and therapeutic relevance in cancers.