Multimodal CRISPR screens uncover DDX39B as a global repressor of A-to-I RNA editing

Adenosine-to-Inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes. Despite its significance, the regulatory mechanisms controlling A-to-I editing remain largely unknown. In this study, we present two complementary CRISPR-based genetic screening platforms: CREDITS (CRISPR-based RNA EDITing regulator Screening), which enables genome-scale identification of editing regulators using an RNA recorder-based reporter system, and scCREDIT-seq (single-cell CRISPR-based RNA EDITing sequencing), which provides multiplexed single-cell characterization of transcriptome and editome changes for pooled perturbations on dozens of selected genes. Through screening 1,350 RNA-binding proteins, we identified a series of known and novel A-to-I editing regulators. Detailed mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase and ATPase activities. We demonstrate that targeting DDX39B significantly enhances the efficiency of RNA editing-based tools like CellREADR and LEAPER, and represents a promising strategy for anti-HDV therapy by modulating viral genome editing. These technological advances not only expand our understanding of RNA editing regulation but also provide powerful tools for exploring tissue-specific and context-dependent RNA modification mechanisms, with broad implications for therapeutic development.