🧬 PubMed RNA Editing Daily Digest

RNA oxidation is pervasive in stress and disease biology and has also been harnessed in chemical biology through proximity labeling, ligand-directed photochemistry, and oxidative RNA targeting. In most settings, the regioselectivity is specified by spatial proximity or a specialized RNA-binding ligand, and the resulting modification is typically distributed across a local region rather than at a designated nucleotide. Here we show that, with selected oxidants, RNA secondary structure can itself serve as a programmable handle to control RNA oxidation site-selectively. We define practical structure-reactivity rules that determine which guanosine is oxidized and which lesions predominate, based on loop geometry and oxidant identity. Guided by these rules, we develop LOCAL (Localized Oxidation Constrained at Loops), a DNA-programmed, postsynthetic method that directs guanosine oxidation to predetermined sites in transcribed RNAs with nucleotide-resolved selectivity. LOCAL enables oxidative base editing to modulate RNA interactions and stability, provides a plug-and-play tool for site-specific bioconjugation via nucleophile trapping of oxidized guanosines, and serves as a blue-light-gated, guanosine-focused structural readout of RNA single-strandedness and ligand-induced structural changes. Together, these results position RNA oxidation as a nucleotide-resolved, structure-encoded reaction manifold that complements existing ligand-encoded oxidative targeting chemistry, and 2´-OH- and enzyme-based RNA chemistries.

Although Cas12f (Cas14) is among the smallest Class 2 CRISPR (clustered regularly interspaced short palindromic repeats) effectors, it assembles into dimeric ribonucleoprotein (RNP) complexes with guide RNA, substantially increasing its functional size and limiting its suitability for gene editing and biosensing applications. To overcome this limitation, we systematically investigate the structural and functional roles of Cas12f dimerization using a combination of computational modeling and experimental validation. Structural analysis using Protein Data Bank data and AlphaFold-3 predictions revealed that the 5'-end sequence of tracrRNA is essential for dimer formation but dispensable for substrate cleavage. Based on this, we designed a truncated tracrRNA by removing 70 nucleotides from its 5'-end. This shortened tracrRNA successfully loaded into Cas12f to form a one guide RNA-one Cas12f monomer RNP. This functionally monomeric RNP demonstrated substantially enhanced trans-cleavage activity: 4.5-fold for ssDNA, 3.5-fold for dsDNA, and 2.5-fold for RNA, resulting in markedly improved detection sensitivity: 10-fold for ssDNA and dsDNA, and 4-fold for RNA. In addition, the functionally monomeric RNP exhibits cis-cleavage activity and gene editing efficiency comparable to that of the dimeric RNP, thereby restoring the advantage of Cas12f as a compact enzyme for in vivo gene editing. These results highlight that the functionally monomeric Cas12f RNP combines enhanced biosensing performance with retention of its uniquely compact size, benefiting gene editing applications.

The CRISPR-Cas13 system enables programmable RNA targeting with potential applications in therapeutics and research. However, while PspCas13b mediates efficient RNA knockdown following transient transfection, stable lentiviral delivery results in minimal activity, limiting its utility. Here, we performed a genome-wide CRISPR-Cas9 knockout screen to identify mammalian factors that restrict PspCas13b activity. We discovered that DUSP11, an RNA triphosphatase, suppresses PspCas13b function by dephosphorylating the 5'-triphosphate of Pol III-transcribed guide RNAs (gRNAs), triggering their degradation. DUSP11 knockout increased gRNA levels 2.5-4-fold and enhanced PspCas13b-mediated knockdown across multiple cell lines. This enhancement was sustained for at least 27 days and enabled targeting of endogenous transcripts previously refractory to PspCas13b. Our findings reveal an unexpected host restriction of bacterial CRISPR systems and demonstrate that gRNA levels are a limiting factor. We provide a simple strategy to improve PspCas13b activity in mammalian cells. These results have implications for developing PspCas13b-based therapeutics and suggest that systematic identification of host factors regulating CRISPR components could enhance genome editing technologies.

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A hybrid approach combining Oxford Nanopore long reads and Illumina short reads was used. The mitogenome was assembled via iterative seed-based mapping and annotated via GeSeq and tRNAscan-SE. Repeats were identified via MISA, TRF, and REPuter. The RNA editing sites were predicted with the PREP suite. Phylogenetic analysis was performed on 14 conserved protein-coding genes from 13 species via maximum likelihood and Bayesian inference. The mitogenome is a 554,078 bp circular molecule (GC 45.27%) encoding 51 genes (32 PCGs, 16 tRNAs, 3 rRNAs). It contains 202 simple sequence repeats (37.1% tetrameric). We predicted 53 C-to-U RNA editing sites, most frequently in This study provides the first comprehensive characterization of the