🧬 PubMed RNA Editing Daily Digest

RNA-targeting CRISPR-Cas13 enzymes are robust RNA knockdown tools with both on-target and collateral cleavage activities. However, to date, the in vivo RNA cleavage mechanisms remain poorly understood. Here, we combine in vitro and in vivo methods to elucidate the exact cleavage sites of Cas13. We reveal that some subtypes of Cas13, including Cas13b and Cas13bt, cleave the target RNA at predominant positions, and rational engineering of Cas13 further improves precision. Building on these findings, we develop RNA segment editing (RSE), a targeted RNA cleavage and repair method, to restore dysfunctional RNA in cells. We anticipate that RSE will enable precision RNA engineering for therapeutics and basic research.

Cellular transitions between states are fundamental to development, adaptation, and pathological processes, but monitoring and guiding these transitions using endogenous signals remain challenging. MicroRNAs (miRNAs) represent a powerful modality, as distinct cell states are characterized by unique miRNA expression signatures. Here, we introduce a state-specific miRNA-directed CRISPR system for detecting and responding to epithelial-to-mesenchymal transition (EMT), a critical process in development, wound healing, and cancer metastasis. This system leverages EMT-specific miRNAs to regulate activation of type II polymerase-driven ribozyme-single-guide RNA (sgRNA) constructs, which direct CRISPR-based effectors to modulate gene expression. Using this approach, we demonstrate selective elimination of cells that have undergone mesenchymal transition and dynamic filtering of cell populations. This system provides a versatile platform for precise activation of CRISPR-Cas9 effectors using endogenous, state-specific cues. Integrating miRNA signatures with CRISPR technology to monitor, modulate, and reprogram cell-state transitions paves the way for applications in regenerative medicine, cancer therapy, and beyond.

Metastasis accounts for the vast majority of tumor-related mortality. Certain populations of tumor cells exhibit organotropism by preferentially colonizing specific distant organs. The organ specificity of metastatic cells is determined by unique interactions between tumor cells and the microenvironment in target organs. Tumor extracellular vesicles (EVs), particularly exosomes, delivering tumor cell components including nucleic acid complexes, proteins, and lipids, play a crucial role in mediating intercellular communication between tumor cells and their microenvironment. ADAR-mediated microRNA (miRNA) editing has emerged as a crucial mechanism influencing miRNA stability, processing, and target specificity. Although EVs are increasingly recognized as important vehicles of intercellular signaling and promising biomarkers for cancer, the landscape of miRNA editing within EVs remains largely unexplored. Here, we present EVmiRED (Extracellular Vesicle miRNA Editing Database), a resource that integrates miRNA expression and editing profiles from tumor-derived EVs. The current release includes data from 683 samples across 12 tumor types and cell lines. EVmiRED provides detailed information on miRNA abundance, editing frequency, and the predicted functional impact of specific editing events. EVmiRED enables users to query individual miRNAs, visualize expression and editing patterns, and access raw datasets for customized analyses. Together, EVmiRED offers a valuable platform to advance our understanding of RNA editing-mediated regulation in intercellular communication, tumor progression, and cancer immunology.

Single guide RNA (sgRNA) is a critical component of the clustered, regularly interspaced short palindromic repeats (CRISPR)-Cas9 genome-editing system, guiding Cas9 to specific genomic loci for precise DNA modification. With its growing clinical potential, sgRNAs have emerged as a promising modality for gene editing-based therapeutics, underscoring the need for robust analytical characterization to ensure quality, safety, efficacy, and regulatory compliance. Here, we present an integrated strategy for deep profiling of sgRNA impurities, combining high-resolution ion-pairing reversed-phase liquid chromatography (IP-RPLC) and size-exclusion chromatography (SEC) with advanced technologies, namely, native mass spectrometry and nanopore direct RNA sequencing. The isolation and characterization of isolated chromatographic peaks revealed truncated, chemically modified, and deletion-prone species. Notably, early eluting fractions in IP-RPLC exhibited elevated deletion frequencies in the target-specific region, correlating with reduced gene-editing efficiency and increased variability in T cells when tested using the Cas-CLOVER system. In contrast, late-eluting fractions in IP-RPLC revealed polyphosphorylated variants with minimal functional impact, while off-target analyses suggested that early eluting impurities may paradoxically reduce off-target editing. Lastly, even at high levels, sgRNA aggregates showed only a limited impact on activity: fully aggregated preparations displayed ∼10% lower knockout efficiency; however, an increase in cumulative off-target frequencies was observed. Overall, this study highlights the value of combining advanced analytical tools to achieve deep profiling of sgRNA impurities. By linking specific impurity profiles to functional outcomes, these findings provide actionable insights for improving sgRNA quality control and advancing the development of safe and effective gene editing-based therapeutics.

The initial steps of the human immunodeficiency virus type 1 (HIV-1) life cycle are regulated by cellular RNA-binding proteins, but only a few have been identified. Here, we developed in virion RNA interactome capture (ivRIC) to comprehensively profile the direct protein interactors of the HIV-1 genomic (g)RNA inside the viral particles. We identified 104 cellular RNA-binding proteins in virions (ivRBPs), many of which are nuclear. We determined the interactome of the viral RBP Rev and discovered that nuclear ivRBPs may associate gRNA in the nucleus and continue bound after the genesis of the viral particles. We also observed that ivRBPs are not incorporated into viral particles based on their abundance, but likely through selective mechanisms. Moreover, we show that the ivRBPs PURA and its homolog PURB control HIV-1 particle infectivity and engage with several viral proteins and key elements within HIV-1 gRNA, showcasing the importance of ivRBPs for HIV-1 infection.