Adenosine-to-inosine (A-to-I) RNA editing is a critical post-transcriptional modification that diversifies the transcriptome and influences various cellular processes, yet its regulatory mechanisms remain largely unknown. Here, 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. By screening 1,350 RNA-binding proteins, we identified a series of A-to-I editing regulators. Mechanistic investigation revealed DDX39B as a global repressor of A-to-I editing, which functions by preventing double-stranded RNA accumulation through its helicase activity. Targeting DDX39B significantly enhances the efficiency of RNA-editing-based tools, such as CellREADR (cell access through RNA sensing by endogenous ADAR) and LEAPER (leveraging endogenous ADAR for programmable editing of RNA), and disrupts hepatitis D virus (HDV) RNA editing homeostasis. 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.
Epithelial-mesenchymal transition (EMT) is a fundamental cellular process frequently hijacked by cancer cells to promote tumor progression, especially metastasis. EMT is orchestrated by a complex molecular network acting at different layers of gene regulation. In addition to transcriptional regulation, post-transcriptional mechanisms may also play a role in EMT. Here, we performed a pooled CRISPR screen analyzing the influence of 1547 RNA binding proteins (RBPs) on cell motility in colon cancer cells and identified multiple core components of P-bodies (PBs) as negative modulators of cancer cell migration. Further experiments demonstrated that PB depletion by silencing DDX6 or EDC4 could activate hallmarks of EMT thereby enhancing cell migration in vitro as well as metastasis formation in vivo. Integrative multi-omics analysis revealed that PBs could repress the translation of the EMT-driver gene HMGA2, which contributed to PB-meditated regulation of EMT. This mechanism is conserved in other cancer types. Furthermore, endoplasmic reticulum (ER) stress was an intrinsic signal that induced PB disassembly and translational derepression of HMGA2. Taken together, this study has identified a function of PBs in regulation of EMT in cancer.
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