🧬 PubMed RNA Editing Daily Digest

CRISPR-based gene editing holds promise for treating genetic diseases, yet its application to lung disorders has been hindered by the challenges of pulmonary delivery. Inspired by the modularity and biocompatibility of amino acid-derived chemistries, we report the combinatorial synthesis of 960 ionizable lipids incorporating chemically diverse backbones from both proteinogenic and non-proteinogenic α-amino acids. Through high-throughput screening and structure-function analysis, we identify CHCha-10, a cyclohexyl amino acid-derived lipid that forms biodegradable nanoparticles capable of efficiently delivering mRNA-based gene editors to lung epithelial cells. Following intratracheal administration, CHCha-10 nanoparticles exhibit enhanced mucus penetration and epithelial-specific transfection in both mice and ferrets. Here, as a functional application, we demonstrate in vivo base editing in the lung via inhalation. Delivery of adenine base editor mRNA and guide RNA targeting the CFTR G542X mutation restores CFTR expression and chloride channel function in G542X human airway epithelial cells, mouse-derived intestinal organoids and the lungs of cystic fibrosis mice. This work establishes a chemically modular design framework for ionizable lipids and a translatable platform for RNA-based pulmonary gene correction.

RNA regulation offers fast, energy-efficient, and highly programmable control over prokaryotic gene expression and is emerging as a necessary complement to DNA-level engineering for building complex and responsive bacterial systems. We review cis- and trans-acting RNA regulators through an engineer-facing framework organized around three actionable control knobs: transcription termination, translation initiation, and mRNA stability. Cis-Encoded strategies including 5' UTR and RBS engineering, riboswitches, and ribozymes often act early on nascent transcripts and can achieve low leak and high dynamic range. Trans-Acting systems such as synthetic small RNAs and CRISPR-based RNA-targeting tools provide an orthogonal capability that is especially valuable in bacteria: operon-resolved, gene-specific regulation within polycistronic transcripts with minimal polar effects. Across both classes, we highlight practical design determinants and failure modes shaped by target accessibility, co-transcriptional folding, RNase and RNA-chaperone context, and expression burden, and discuss how these constraints govern composability in multigene networks. We further outline emerging design workflows that integrate computation and AI-assisted modeling with screening and benchmarking to improve predictability and portability beyond E. coli-centric implementations. Together, this review aims to make RNA regulation a routine, engineerable layer for next-generation prokaryotic synthetic biology.

The HIV-1 genome [genomic RNA (gRNA)] has an unusually biased nucleotide content and is rich in adenosines. Selective packaging of the gRNA is thought to be driven by specific binding of the nucleocapsid (NC) domain of the viral Gag protein to the packaging signal (Ψ) in the host cell cytosol. However, deletion of regions within Ψ reduces-but does not completely abolish-genome packaging. To probe whether another feature of the gRNA may contribute to the selective gRNA packaging process, we replaced NC with heterologous RNA-binding domains (RBDs) with distinct RNA-binding properties. Surprisingly, despite disparate RNA binding specificities, all Gag-RBD chimeras successfully recruited the gRNA to the plasma membrane, suggesting that the initial gRNA recognition in the cytosol is not rate limiting. Notwithstanding, many chimeras exhibiting G/C binding specificity were arrested at the assembly stage. Only the Gag-SRSF5 chimera, which multimerized efficiently on adenosine-rich sequences on the gRNA, assembled efficiently and packaged gRNA at near wild-type levels. Importantly, rationally designed mutations that altered the A/G-rich binding specificity of Gag-SRSF5 decreased genome encapsidation efficiency. Furthermore, many Gag chimeras displayed potent dominant negative activities, highlighting NC functions as a targetable step in virus replication. Together, our findings reveal an unexpected aspect of the HIV-1 gRNA, its biased nucleotide content, as a key driver of selective genome packaging.

APOBEC3A catalyzes cytosine-to-uracil deamination in single-stranded DNA and RNA. Physiologically, APOBEC3A functions in innate immunity and aberrant deamination is associated with cytosine mutations in enzymatically preferred YTCW substrate motifs in multiple cancers. Much less is known about the potential contribution of APOBEC3A-catalyzed RNA editing to virus and cancer evolution. Here, we present HAMMER (hairpin-based APOBEC3A-mediated messenger RNA editing reporter), a rapid luminescence-based cellular assay for measuring RNA editing by APOBEC3A. HAMMER reports APOBEC3A activity as a reduction in the ratio of firefly to renilla luciferase activity. Briefly, tandem renilla and firefly luciferase open reading frames are separated by an optimal APOBEC3A hairpin substrate, in which C-to-U editing of a CGA motif yields a UGA stop codon thus preventing translation of the downstream firefly luciferase reporter, without impacting the upstream renilla reporter. HAMMER activation is dose-responsive, catalytic activity-dependent, and specific to human APOBEC3A. A panel of herpesviral ribonucleotide reductase constructs was used to show that direct inhibition of APOBEC3A results in a dose-responsive recovery of firefly luciferase expression. HAMMER is therefore a scalable and easy-to-use method for quantifying cellular APOBEC3A RNA editing activity and characterizing inhibitors.

Tissues rely on unique landscapes of gene regulation to allow the organism to correctly develop, function, and respond to changes. One component of these gene regulatory networks is RNA Binding Proteins (RBPs) which bind and modify RNA molecules leading to changes in the cellular fate of transcripts. The Adenosine DeAminase acting on RNA (ADAR) family of RBPs modify RNAs by catalyzing the deamination of adenosine (A) to inosine (I), known as A-to-I RNA editing. Prompted by recent evidence that ADARs play important roles in germline biology, we profiled editing activity of the A-to-I editing enzyme ADR-2 on transcripts in the Caenorhabditis elegans germline. These analyses revealed that many germline editing events are distinct from editing events in other tissues; however, the previously described role of the inactive deaminase ADR-1 in regulating editing activity by ADR-2 is conserved in the germline. We find that complete loss or misregulation of editing has little effect on the expression of edited transcripts within the germline; however, loss of ADARs results in the misexpression of several unedited germline transcripts. Intriguingly, further investigation suggests that these expression changes are buffered at the translational level. In all, the results of this study suggest that ADARs show unique activity in the C. elegans germline and that compensatory mechanisms exist to lessen the immediate consequences of loss of ADAR function within the germline.