🧬 PubMed RNA Editing Daily Digest

Argonautes are an evolutionarily conserved family of proteins that use guide oligonucleotides for specific recognition of nucleic acid targets. While eukaryotic Argonautes share a conserved structure and act in RNA interference, prokaryotic Argonautes (pAgos) display remarkable structural and functional variations, including various guide and target specificities. Most studied catalytically active pAgos use 5'-phosphorylated DNA guides to recognize and cleave DNA targets. Here, we describe a new group of pAgos from mesophilic bacteria that use RNA guides to cleave DNA targets and are active at physiological temperatures. In contrast to most characterized pAgo nucleases, these proteins can utilize guides of varying lengths containing either a 5'-phosphate or a 5'-hydroxyl group with similar efficiencies. Measurements of the kinetics of target DNA binding show that the annealing of the guide-target duplex in pAgo occurs in an ordered way and that pAgo increases the rate of guide-target interaction in the seed region. We show that the analyzed pAgos can produce alternative DNA products depending on the structure of guide RNA and the cleavage conditions. The results demonstrate that RNA-guided pAgo nucleases are more widespread than previously anticipated and that these pAgos have a relaxed specificity for target DNA cleavage.

Bacteriophages have evolved anti-CRISPR (Acr) proteins to combat the adaptive immunity provided by bacterial CRISPR-Cas systems. Here, we report the cryo-electron microscopy structure of an anti-Cas9 protein AcrIIA27 bound to SpyCas9-sgRNA (single guide RNA) complex. Our structure reveals that AcrIIA27 binds the solvent-exposed phosphate backbone of the sgRNA, acting as a potent inhibitor of diverse Cas9 orthologs. AcrIIA27 in the structure is positioned near the protospacer-adjacent motif DNA-binding pocket on SpyCas9, causing steric hindrance that prevents substrate DNA recognition. This mechanism suggests solvent-exposed regions of sgRNAs (PTP RNAs), prone to nonspecific binding of positively charged components, may compromise CRISPR-Cas genome-editing efficiency. Indeed, truncations of the PTP RNAs in different editing systems significantly enhance genome-editing efficiency in human cells. Overall, our findings reveal a previously uncharacterized inhibition mechanism of an anti-Cas protein and offers a general strategy for developing more efficient genome-editing tools.

The pathogenesis of liver fibrosis centres on the activation of hepatic stellate cells (HSCs). Adenosine-to-inosine RNA editing, primarily catalysed by adenosine deaminase acting on RNA1 (ADAR1), is the most prevalent post-transcriptional modification that increases transcriptome diversity. This study aims to elucidate the role of ADAR1-imposed RNA editome in HSC activation and to determine the therapeutic potential of targeting ADAR1 for liver fibrosis. ADAR1 expression was measured in fibrotic human and mouse livers, as well as in primary human and mouse HSCs. Adar1 loss-of-function effect was evaluated in ADAR1 is decreased in human and mouse fibrotic livers and activated HSCs. HSC-specific ablation or pharmacological inhibition of ADAR1 ameliorated HSC activation and liver fibrosis. In contrast, forced expression of ADAR1, but not its editing-deficient mutant, exacerbated HSC activation. Mechanistically, ADAR1 ablation accumulated double-stranded RNA and activated HSC-intrinsic innate immunity in a melanoma differentiation-associated gene 5-dependent manner. Interferon-β was identified as a key antifibrotic effector via the activation of the JAK1/2 pathway. RNA editome analysis revealed the ADAR1-imposed RNA editome suppresses HSC-intrinsic innate immunity and promotes collagen production, leading to aggravated HSC activation and liver fibrosis. Targeting ADAR1 with its pharmacological inhibitor or HSC-selective RNAi shows great promise in treating liver fibrosis.

RNA editing by adenosine deaminases acting on RNA (ADARs) is an essential cellular process performed by three enzymes in mammals: ADAR1-p150, ADAR1-p110, and ADAR2, demonstrating different target specificity and selectivity. Here we describe TSniffer, a novel tool to analyze RNA editing in RNA-sequencing datasets. TSniffer uses a rolling window approach to identify editing sites and operates in two modes allowing identification and quantification in single samples, and quantification in predefined regions across multiple datasets. Using wild type and ADAR-deficient datasets, we provide strategies for identification of ADAR editing sites and verify the accuracy and biological relevance of our findings.

Single-nucleotide variants (SNVs) and small insertions or deletions (indels) underlie most rare monogenic disorders, yet therapeutic strategies to precisely correct these mutations remain limited. Prime editing enables the repair of such pathogenic variants without introducing double-stranded breaks. Here, we applied CRISPR prime editing to model and correct a de novo GDF11 nonsense mutation (Tyr336∗) identified in a participant from the Undiagnosed Diseases Network with growth delay and multisystem abnormalities. Using HEK293T cells, we generated heterozygous (HET) GDF11 Tyr336∗ clones, which exhibited reduced GDF11 protein levels due to post-translational degradation likely mediated by endoplasmic reticulum- and Golgi-associated quality control pathways. These cells displayed marked Golgi abnormalities, including an increased number of compact, irregularly shaped Golgi structures, findings consistent with Golgi fragmentation and stress. Transcriptomic profiling of HET cells revealed a broad dysregulation of gene networks, including downregulation of metabolic and Golgi-linked biosynthetic genes, and upregulation of cell-adhesion and extracellular matrix genes. These transcriptional shifts paralleled the participant's developmental, neural, and cardiovascular phenotypes. To correct the mutation, we tested multiple bespoke prime editing strategies and identified PE7, in combination with a prime editing guide RNA designed by Pridict, as the most effective ribonucleoprotein complex for rescue. Editing efficiency was further enhanced by introducing an additional silent protospacer-adjacent motif-disrupting mutation, likely preventing both Cas9 re-binding and mismatch repair. Together, these findings support a haploinsufficiency mechanism for the GDF11 Tyr336∗ allele and establish a generalizable framework for disease modeling and allele-specific correction of pathogenic variants in human cells.