🧬 PubMed RNA Editing Daily Digest

Translational regulation offers a powerful biological control axis with the potential to enable programmable control over synthetic mRNAs. Here, we introduce inducible Deaminases Acting on RNA (iDARs): deaminase domains (DDs) with conditional RNA-editing activities. Using a domain-insertion strategy, we designed autoinhibited enzymes that can be converted into active RNA editors in response to triggers based on small molecules (chemiDARs), intracellular antigens (antiDARs), protease cleavage (lysiDAR), and optical excitation (optiDAR). Coupling these domains with novel stop codon containing RNA substrates enabled conditional protein translation or transcript degradation. Mutational tuning of inositol hexaphosphate (IP

Mutations in MYOC, the most common genetic cause of glaucoma, cause misfolded myocilin to accumulate in the endoplasmic reticulum (ER), leading to trabecular meshwork (TM) dysfunction, elevated intraocular pressure, and progressive vision loss. While gene editing offers curative potential, current delivery methods rely on viral vectors, which are limited by inflammation, off-target effects, and poor translatability. Here, we report a nonviral lipid nanoparticle (LNP) platform that enables selective in vivo delivery of mRNA encoding an adenine base editor and single guide RNA (LNP-ABE) to TM cells. A direct comparison of LNP-mCherry with lentiviral GFP revealed that LNPs outperform viral vectors, achieving markedly higher efficiency and greater selectivity for the TM without inducing ocular inflammation. In a Cre-inducible Tg.CreMYOCY437H glaucoma mouse model, LNP-Cre mRNA selectively induced mutant MYOC expression in the TM, faithfully recapitulating key disease features. A single administration of LNP-ABE achieved efficient on-target editing of mutant MYOC, reducing mutant myocilin protein by approximately 46%, decreasing aggregates, alleviating ER stress, and fully rescuing the glaucomatous phenotype in Tg.CreMYOCY437H mice. Importantly, no off-target editing or ocular toxicity was detected. These findings establish LNP-based mRNA delivery as a safe, efficient, and clinically translatable approach for TM-targeted genome editing with broad therapeutic potential in glaucoma.

Leaf thickness in rice critically influences photosynthetic efficiency and yield, yet its genetic basis remains poorly understood, with few functional genes previously characterized. In this study, we employ a pangenome-wide association study (Pan-GWAS) on 302 diverse rice accessions from southern China, identifying 49 quantitative trait loci (QTLs) associated with leaf thickness. The most significant locus, qLT9, is fine-mapped to a 79-kb region on chromosome 9. Transcriptomic and genomic sequence analyses identify LOC_Os09g33480, which encodes a protein belonging to Multiple Organellar RNA Editing Factor family, as the key candidate gene. Overexpression and complementation transgenic experiments confirm LOC_Os09g33480 (OsLT9) as the functional gene underlying qLT9, demonstrating a 24-bp Indel in its promoter correlates with the expression levels and leaf thickness. Notably, OsLT9 overexpression lines show not only thicker leaf, but also significantly enhanced photosynthetic efficiency and grain yield, establishing a link between leaf thickness modulation and yield enhancement. Population genomic analyses indicate strong selection for OsLT9 during domestication and breeding, with modern cultivars favoring thick leaf haplotype of OsLT9. This study establishes OsLT9 as a key regulator controlling leaf thickness in rice, and provides a valuable genetic resource for molecular breeding of high-yielding rice through optimization of plant architecture.

RNA-guided DNA nucleases Cas9 and IscB (insertion sequences Cas9-like OrfB) are components of type II CRISPR-Cas adaptive immune systems and transposon-associated OMEGA (obligate mobile element-guided activity) systems, respectively. Sequence and structural comparisons indicate that IscB (~500 residues) evolved into Cas9 (~700-1,600 residues) through protein expansion coupled with guide RNA miniaturization. However, the specific sequence of events in this evolutionary transition remains unknown. Here, we report cryo-electron microscopy structures of four phylogenetically diverse RNA-guided nucleases-two IscBs and two Cas9s-each in complex with its cognate guide RNA and target DNA. Comparisons of these four complex structures to previously reported IscB and Cas9 structures indicate that evolution from IscB to Cas9 involved the loss of the N-terminal PLMP domain and the acquisition of the zinc-finger-containing REC3 domain, followed by bridge helix extension and REC1 domain acquisition. These structural changes led to expansion of the REC lobe, increasing the target DNA cleavage specificity. Additionally, the structural conservation of the RNA scaffolds indicates that the dual CRISPR RNA (crRNA) and trans-activating crRNA guides of CRISPR-Cas9 evolved from the single ωRNA guides of OMEGA systems. Our findings provide insights into the succession of structural changes involved in the exaptation of transposon-associated RNA-guided nucleases for the role of effector nucleases in adaptive immune systems.

CRISPR interference (CRISPRi) has emerged as a versatile approach for targeted gene repression in many organisms, including microbes and bacteria, due to the simple design of sequence-specific transcriptional silencing of gene expression. However, the strain-specific effects on repression efficiency and the host when translating a CRISPRi system from a laboratory strain to non-model strains are not well understood, yet they can present important limitations to its use. Here, we investigated the repression efficiency and toxicity of three CRISPRi systems (one dCas9 and two dCas12a variants) across four different