🧬 PubMed RNA Editing Daily Digest

Methylmalonic acidemia (MMA) is a recessive genetic disease caused by variants in the

APOBEC-mediated cytidine deamination is a major endogenous source of mutagenesis in human cancers and has been linked to tumor evolution, clonal diversification and therapeutic resistance. Among the APOBEC family, APOBEC3A (A3A) is a potent and inducible cytidine deaminase, with dynamic and context-dependent activation. Most approaches for studying the role of A3A in cancer infer A3A activity indirectly via its expression level or retrospective mutational signatures, or through molecular assays that are limited to endpoint measurements and do not readily allow longitudinal interrogation of A3A editing dynamics. Therefore, quantifying the timing, persistence, and cellular heterogeneity of A3A activity remains challenging. Here, we describe ApoFLARE, a genetically encoded reporter that converts A3A-mediated cytidine deamination into a quantitative luminescent signal in living cells. ApoFLARE allows for scalable, ratiometric measurement of editing activity and enables time-resolved analysis of editing kinetics. Reporter activation is selectively dependent on A3A catalytic function and was absent in A3A-deficient, but not A3B-deficient cells. Under stress and targeted therapy conditions, reporter activity correlated with endogenous RNA editing measured by digital droplet PCR, including contexts in which catalytic activity persisted beyond transient A3A transcript induction. Thus, ApoFLARE offers a scalable platform to investigate the regulation, kinetics, and heterogeneity of A3A editing.

DNA-templated silver nanoclusters (DNA/AgNCs) have created a new class of non-FRET DNase substrates, termed Subak, that exhibits a color change upon DNase digestion. Although Subak substrates offer advantages such as ratiometric readouts and low manufacturing costs over traditional FRET substrates, the mechanism governing AgNC color switching remains unclear. Here, using a site-specific cleavage strategy, we identify color-switching hotspots and demonstrate that AgNC transformation can be controlled by the cleavage positions within the nucleic acid host. Our data support a cleavage-driven reorganization of the AgNC coordination environment, converting a non-emissive precursor into a red-emitting cluster, rather than direct enzyme-cluster interactions. Leveraging this insight, we engineer rSubak, an RNA-incorporated Subak that displays 95 nm red shift (530 to 625 nm) upon RNase cleavage. In amplification-free CRISPR/Cas13 assays for SARS-CoV-2, influenza A (A/H5N1), and measles viruses (MV) detection, rSubak achieved a limit of detection of 0.3 pM, superior to that of the commercial RNaseAlert (∼250 pM). Collectively, our results establish Subak as a generalizable, non-FRET platform for sensitive ratiometric reporting the activities of diverse nucleases.

The widespread use of CRISPR-Cas9 (Clustered regularly interspaced short palindromic repeats-CRISPR-associated protein 9) in plants highlights the need for compact and efficient multiplexed genome editing systems. This study optimizes single-guide RNA (sgRNA) expression in CRISPR by leveraging endogenous tRNA processing mechanisms for efficient multiplexed genome editing. Screening in Arabidopsis thaliana and Oryza sativa identified superior tRNAs that outperformed the widely used AtGly-tRgcc. Leveraging tRNA's dual functions in sgRNA processing and their intragenic RNA polymerase III promoter activity, we established a compact multiplexed system for simultaneous editing of at least ten genomic loci in rice and soybean. Moreover, we developed plant tRNA large language models that learn sequence representations to identify both canonical and noncanonical tRNAs, uncovering thousands of tRNAs missed by traditional algorithms and expanding the repertoire for genome editing. This work provides a robust tRNA-based CRISPR platform, an artificial intelligence-guided tRNA mining framework, and a comprehensive tRNA resource for advanced plant genome engineering and germplasm innovation.

Prime editing enables precise genome modifications without DNA double-strand breaks, yet bacterial applications are limited by low efficiency and small edit sizes. Here, we develop PE-STAR, Prime Editing with SOS-Triggered and RecJ-Augmented Repair, to enhance prime editing in Escherichia coli. Removing three inhibitory 3'→5' exonucleases (SbcB, ExoX, and XseA) improved edited-strand retention, and extending post-transformation outgrowth increased editing efficiency. RecJ overexpression strengthened 5'-directed processing during flap resolution and gap expansion, biasing repair toward incorporation of the reverse-transcribed edited strand. To enrich edited cells, we integrated an SOS-responsive counter-selection circuit that links PE3-associated dual nicking to LexA-dependent gRNA expression targeting a plasmid encoding the toxin CcdB, thereby eliminating unedited cells. PE-STAR achieved up to 80%-90% editing efficiency for short-fragment modifications, representing up to 16-fold improvement across loci. The platform supported insertions, deletions, and replacements of up to 46 bp with high efficiency. Furthermore, installing an attB site by prime editing, followed by Bxb1 integrase recombination, enabled chromosomal integration of 3.2 and 8.0 kb cassettes with 100% recombination efficiency among screened colonies, including GFP reporter and riboflavin biosynthetic pathway. PE-STAR expands both the efficiency and functional scope of bacterial prime editing for programmable genome engineering.