The CRISPR-Cas9 system provides adaptive immunity against invading genetic elements through a dual-RNA-guided DNA cleavage mechanism. This system relies on the precise assembly of a ribonucleoprotein (RNP) complex composed of the Cas9 endonuclease, a CRISPR-derived RNA (crRNA), and a trans-activating CRISPR RNA (tracrRNA). Around 100 anti-CRISPR proteins that inhibit CRISPR-Cas systems have been identified, and the mechanisms by which they act are increasingly being elucidated. However, the inhibitory mechanisms of many Acrs, including AcrIIA7, remain poorly understood. Here, we present the structure of AcrIIA7 and uncover a previously unrecognized mechanism by which it inhibits Cas9 function. Structural and biochemical analyses reveal that AcrIIA7 specifically binds to tracrRNA, preventing its association with crRNA and thereby blocking formation of the active Cas9 RNP complex. This tracrRNA hijacking mechanism represents a unique strategy for CRISPR inhibition, in which an anti-CRISPR protein targets an RNA scaffold essential for Cas9 activation rather than interacting directly with the Cas9 protein. Our findings provide the first structural insight into tracrRNA-targeted anti-CRISPR activity and highlight RNA-RNA interaction interfaces as vulnerable nodes in CRISPR-Cas immunity.

Photothermal therapy (PTT), a non-invasive tumor treatment, shows promise but is limited in solid tumors by restricted tissue penetration, thermotolerance, anti-apoptotic and immunosuppressive effects. In this study, tumor microenvironment-responsive nanoplatform VARH was constructed based on MXene. Under NIR-II laser irradiation, VARH achieves a high photothermal conversion efficiency of 44.21%. Loaded 2,2'-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride decomposes at high temperatures to generate alkyl radicals, synergizing with hydroxyl radicals from V4+-catalyzed endogenous H2O2 decomposition, enabling chemodynamic therapy (CDT) and thermal dynamic therapy to enhance tumor cell oxidative damage. Triggered by high glutathione, VARH releases ribonucleoprotein (RNP) complexes to knockout heat shock protein 90 (HSP90), attenuating cellular heat resistance and promoting apoptosis. It also enhances T cell-mediated anti-tumor immunity and, with free radicals, promotes tumor cell immunogenic cell death (ICD), achieving immunotherapeutic multi-effect synergy. Integrating nanotechnology with precise gene editing, this study develops a novel multimodal synergistic therapy system, providing new insights for multi-modal treatment R&D and advancing PTT and free radical-based cancer therapies.

The genomic RNA of negative-strand RNA viruses is encapsidated by nucleocapsid proteins and associates with RNA polymerase to form a ribonucleoprotein (RNP) complex. Lacking both a 5' cap and a 3' poly (A) tail, viral RNAs are highly unstable and prone to degradation by cellular nucleases. Therefore, newly synthesized genomic and complementary-strand RNAs must be rapidly protected through RNP formation. However, the molecular mechanisms governing RNP assembly in cytoplasm-replicating negative-strand RNA viruses remain largely unknown. Here, we screened a yeast knockout library and isolated mutants in several components of the endosomal sorting complexes required for transport (ESCRT) genes that affected RNA replication of tomato spotted wilt virus (TSWV). In wild-type (WT) yeast cells, TSWV nucleocapsid (N) and RNA polymerase (L) proteins colocalize at the trans-Golgi network (TGN) in a replicon-RNA-dependent manner, suggesting that TSWV RNPs accumulate at the TGN. However, in the snf7Δ, bro1Δ, and doa4Δ mutant cells, N localization to TGN and RNP formation were impaired. Another RNA replication-defective mutant, vps36Δ, showed normal N localization, and SNF7, BRO1, and DOA4 were recruited to the TGN by TSWV N or L proteins, implying that the ESCRT components have additional roles in TSWV RNA replication beyond facilitating N transport. These findings suggest that ESCRT components play multifaceted roles in TSWV RNA replication, including the intracellular transport of N to the TGN-where RNA replication takes place-thereby ensuring accurate and efficient RNP assembly.

Structured RNAs play many roles in cells and emerging biotechnology. While large RNAs and ribonucleoprotein complexes often benefit from high-resolution structural analysis through cryogenic-sample electron microscopy (cryoEM), single-domain RNAs, particularly those smaller than ~100 nt (33 kDa), have proven challenging. Here we address this methodological gap by engineering two- and fourfold symmetric scaffolds that enable de novo structure solution of covalently attached RNA guests to beyond 3 Å overall resolution for the best resolved guests. We apply C2 and D2-symmetric scaffolds to post-transcriptionally unmodified tRNAAsp, the fluorogenic aptamer Mango-III, and previously uncharacterized quinine- and 8-oxoguanine-binding aptamers. Experimental Coulomb potential maps with quality sufficient for small-molecule ligand, cation and water molecule placement reveal the molecular basis for specificity and suggest routes for structure-guided RNA engineering. Optimized scaffolds with intrinsic quaternary structure are a new general tool to interrogate the atomistic architecture of natural and designed compact RNA folds by single-particle cryoEM.

Translation is carried out by the most conserved assemblies in biology. Among these assemblies, the ribosome and RNase P are central players. These ancient ribonucleoprotein complexes achieved structural and functional maturity by the last universal common ancestor (LUCA) of life. In prior work, we reconstructed the evolutionary history of the ribosome using its three-dimensional structure, based on accretion and molecular fingerprints that date back to life's earliest stages. Here, we extend our structural phylogenetic framework-based on the accretion model-to RNase P, a ribonucleoprotein responsible for processing pre-tRNAs. By sampling RNase P RNA (RPR) sequences and structures across phylogeny and partitioning them into RNA fragments based on insertion fingerprints, we characterize the state of RPR at LUCA and reconstruct the chronology of its emergence. The chronology reveals that RNase P, like the ribosome, accreted modular RNA elements over evolution, while preserving the structure of preexisting elements, thus maintaining a structural record. We used interactions with tRNA to link and unify the evolutionary trajectories of RPR and rRNA. These results support the view that RNase P and the ribosome coevolved as part of a functionally integrated system. The ancestral catalytic sites of rRNA and RPR formed by the same process, fusion of two stem-elbow-stem elements. Analysis of these two coevolving RNAs also suggests that some of their accreted elements share common ancestry. Application of the accretion model requires correct secondary structures and was successful for RPR only when the traditional secondary structure was corrected by reorganizing a pseudoknot.

SORT LNPs encapsulating Cas9 mRNA achieve efficient editing in skeletal muscle in a dystrophic mouse model.

First report of CRISPR prime editing in a globally significant non-model organism, the fall armyworm, Spodoptera frugiperda (Lepidoptera: Noctuidae).

Host cells combat avian influenza virus (AIV) infection by targeting viral polymerase PB2 for degradation, yet how the virus counteracts this remains elusive. In this study, we analyze the host proteins interacting with H5 AIV PB2 and identify USP39 as a deubiquitinase with dual functions in viral replication. Catalytically, USP39 directly deubiquitinates PB2 at lysine 660 (K660), preventing its degradation and sustaining polymerase activity. In parallel, independently of enzymatic activity, USP39 promotes PB2-PB1 association, facilitating formation of ribonucleoprotein (RNP) complexes. These complementary functions amplify viral RNA synthesis, dampen host antiviral responses, and drive efficient viral replication. Consistently, a PB2 K660R substitution enhances viral replication in vitro and increases pathogenicity in mice. Our findings reveal a mechanism by which AIV hijacks USP39 to circumvent host ubiquitination, facilitate RNP biogenesis, and identify USP39 as a promising therapeutic target for broadly effective antivirals against pandemic-prone H5 viruses.

Prime editing (PE) is a precise gene-editing technology with potential for treating genetic disorders, but efficient delivery systems remain a challenge. Viral vectors offer high efficiency but pose safety concerns related with their immunogenicity, while non-viral methods struggle with stability and scalability. Cell-penetrating peptides (CPPs) present a promising alternative due to their low immunogenicity. In this study, we explored LAH5, a histidine-rich CPP, for delivering PE ribonucleoproteins (RNPs) into PLN R14del mutant cell lines. We purified engineered SpGPEmax protein, evaluating its intracellular uptake and editing frequency in HEK293T.PLN R14del reporter cells and human-induced pluripotent stem cell-derived cardiomyocytes (iPSC-CMs). Our results demonstrate that LAH5 effectively delivers intracellularly SpGPEmax RNP components, resulting in correction of the R14del mutation, thereby offering a viable non-viral strategy for direct cellular precise genome editing.

Approaches to delivering gene editing tools in the form of ribonucleoproteins may provide a safety advantage over the delivery of nucleic acids encoding ribonucleoproteins. Virus-based vectors are widely used as a delivery platform. However, the persistence of viral exogenous nucleic acids can cause increased genotoxicity. Virus-like particles (VLPs) do not contain an expression cassette and can act as a platform for the delivery of ready-made ribonucleoprotein complexes. The absence of nucleic acids in VLPs eliminates the risk of insertional mutagenesis compared to widely used lentiviruses or adeno-associated viruses. Therefore, we used VLPs to deliver the ribonucleoprotein complex MmCas12m–TadDE to disrupt the HIV-1 gag gene start codon. We detected VLP morphogenesis using electron microscopy. We confirmed the incorporation of MmCas12m–TadDE into VLPs. We achieved an editing efficiency of about 9% in some cases with minimal off-target effects, which confirms the prospect of using VLPs as a platform for delivering genomic editing tools.

Enolase 1 suppresses influenza A virus replication by blocking the nuclear import of the viral ribonucleoprotein complex.

The Arabidopsis thaliana La1 (AtLa1) protein is a member of the genuine La family of RNA biogenesis proteins, which are structurally similar to the La-related protein 7 (LARP7) family. LARP7 proteins participate in the biogenesis of the telomerase ribonucleoprotein complex in model systems, but are absent in plants. We show that AtLa1 binds to telomerase RNA in a manner reminiscent of the Tetrahymena LARP7 protein p65. Classical in vitro methods and microscale thermophoresis (MST) were used to specify the molecular structures involved in this multi-surface interaction. AtLa1 also enhances the binding of TR to the telomerase reverse transcriptase RNA binding domain. We therefore propose that biogenesis of telomerase RNA in plants and ciliates is achieved by a similar pathway, differing in the employment of genuine La or LARP7-like proteins, respectively. We also report that the domain of unknown function (DUF3223, DeCL) found in the AtLa1 protein binding partner, Domino, is an RNA binding domain with modest TR-binding capacity. This domain is also found in plant and ciliate proteins, including plant polymerases IV/V and the Tetrahymena La protein Mlp1. Together, these suggest that RNA biogenesis pathways in plants and ciliates have a conserved evolutionary relationship, with parallels between their La proteins.

Vaults are some of the largest ribonucleoprotein complexes known and are highly conserved across eukaryotes, but both their function and key details of their architecture remain unclear. While high-resolution structures of the vault shell are available, the architecture and symmetry of the cap have remained unresolved. Here, we present a 2.25-angstrom cryo-electron microscopy structure of the vault cap, revealing an unexpected 13-fold symmetric arrangement that contrasts with the 39-fold symmetry of the vault body, with each repeating module of the cap formed by an asymmetric homotrimer of adjacent subunits. The center of the cap features an unusual architecture, consisting of two concentric β barrels surrounded by an interwoven two-layer stack of α helices. The vault cap features a positively charged exterior and a negatively charged interior surface, with implications for binding partner recruitment and engineering of modified vault particles.

Gliotoxin of Aspergillus fumigatus has been extensively studied for its role in pathogenesis in animals and humans. It triggers pathogenesis by its immunosuppressive and cytotoxic effects. Biosynthetic gene cluster (BGC) consisting of 13 genes regulates its biosynthesis. We targeted gliZ, gliP and gliA genes of this BGC using CRISPR/Cas9 system in a multigene editing approach to check the pathogenesis in broilers. crRNAs were designed using EuPaGDT and 3 single guide RNAs (sgRNA) were commercially synthesized. Each sgRNA was combined with Cas9 to form ribonucleoprotein complexes which were then used for simultaneously transfecting fungal protoplasts. Thin-layer chromatography showed the absence of gliotoxin on silica plate and DNA sequencing showed various indels in target genes. These indels caused amino acid substitutions in all three gene products but, the gliP mutation, since it was synonymous, was likely not functionally relevant. Regenerated protoplasts were matured to form fungal hyphae and spore production was induced. These spores were inoculated intra-air sac in broiler chicks. During one-week infection trial, birds infected with the wild-type spores (group 1) showed morbidity and their mortality rate was 30%. Birds inoculated with RNP-treated spores (group 2) showed mild clinical signs and no mortality. No morbidity or mortality was recorded in birds in negative control group (group 3). Histopathological analysis of lungs showed necrosis and congestion, and presence of mixed population of inflammatory cells in wild-type infected birds, while no such lesions were seen in birds infected with RNP-treated spores. These results show that multigene editing approach was successful in creating indels simultaneously in 3 gliotoxin genes which resulted in amino acid substitution which negatively impacted gliotoxin biosynthesis and export. In vivo experiment results show that RNP-treated fungal spores were unable to cause A. fumigatus pathogenicity in broiler. Targeting gliotoxin biosynthesis could thus be a promising approach to develop antifungal therapy.

Emerging evidence, however, reveals that this organelle plays a far more expansive role as a dynamic platform for cell fate determination. We propose the concept of Centriole-Associated Fate Determinants (CAFDs)—a class of biomolecules, including proteins, RNAs, and ribonucleoprotein complexes, that physically localize to the centriole or pericentriolar material and whose function in specifying cell identity is directly modulated by this association. Unlike core structural components, CAFDs are "guests" at the centriole, with primary biochemical roles in transcription, signaling, translation, or proteostasis. This review provides a comprehensive framework for understanding CAFDs, including their conceptual basis, classification by chemical nature and mechanism of action, and a detailed catalogue of key examples across model systems. We examine the regulatory mechanisms that control CAFD activity at the centriole—post-translational modifications, conformational changes, competitive binding, and signal-dependent release—and survey the advanced methodologies for their identification and study, from proximity-dependent biotinylation to super-resolution imaging and induced tethering. An evolutionary perspective reveals that primitive CAFD-like functions likely originated in unicellular eukaryotes to coordinate life cycle transitions, later co-opted in metazoans for asymmetric division and differentiation. Disruption of CAFDs or their centriolar adapters underlies a spectrum of human pathologies, including microcephaly, ciliopathies, and cancer. We conclude with an integrative model positioning the centriole as a strategic command post that integrates cytoskeletal architecture with gene regulatory programs, and pose the central question for future therapeutic exploration: Can cell fate be controlled by pharmacologically modulating specific CAFD-centriole interactions?

RNA-driven protein aggregation leads to cellular dysregulation and contributes to the development of diseases and tumorigenesis. Here, we show that double homeobox 4 (DUX4), an early embryonic transcription factor and causative gene of facioscapulohumeral muscular dystrophy (FSHD), induces the accumulation of stable intranuclear RNAs, including nucleolar RNA and human satellite II (HSATII) RNA that drive protein aggregation in muscle cells. Specifically, HSATII RNA sequesters RNA methylation factors. HSATII-YBX-1 ribonucleoprotein (RNP) complex formation is mediated by HSATII double-stranded RNA and RNA methylase NSUN2 activity. Aberrant HSATII-RNP complexes affect RNA processing pathways, including RNA splicing. Differential splicing of genes mediated by HSATII-RNP complexes is associated with pathways known to be dysregulated by DUX4 expression. These findings highlight the broader influence of DUX4 on nuclear RNA dynamics and suggest that HSATII RNA could be a critical mediator of RNA processing regulation. Understanding the impact of HSATII-RNP formation on RNA processing provides insight into the molecular mechanisms underlying FSHD.

Fibrillarin (FBL), a core component of the C/D box small nucleolar ribonucleoprotein (snoRNP) complex, catalyzes the 2'-O-methylation (Nm) of the ribose 2'-hydroxyl moiety in ribosomal RNA (rRNA). Distinct Nm patterns contribute to ribosome heterogeneity, which is linked to selective translation of oncogenes. FBL dysregulation generates an aberrant Nm signature in triple-negative breast cancer (TNBC), the most aggressive breast cancer subtype. This study investigated the role of FBL in TNBC via translation-driven mechanisms. Our findings show that FBL knockdown impairs oncogenic traits, triggers metabolic stress, and reduces the translation efficiency of oncogenes, such as metastasis-associated protein 1 (MTA1), interleukin-1 receptor-associated kinase 1 (IRAK1), and thymosin beta 10 (TMSB10). RiboMethSeq confirmed that the rRNA Nm sites exhibited differential sensitivity to FBL depletion. Additionally, FBL knockdown led to alterations in 18S ribosome structure confirmed by SHAPE and specifically reduced RPS28 incorporation into ribosomes. Notably, silencing RPS28 also disrupted both the oncogenic phenotype and downregulated MTA1, IRAK1, and TMSB10 expression. These findings reveal a complex interplay between FBL, rRNA Nm modifications, and RPS28 in shaping oncogenic protein pools and ribosomal composition in TNBC, offering promising insights into therapeutic approaches targeting this aggressive cancer subtype.

Phytoene desaturase (PDS; EC 1.3.5.5) is a key enzyme of the carotenoid biosynthetic pathway, catalyzing the desaturation of phytoene, precursor of all carotenoids. In this study, several PDS-knockout (PDS-KO) transformants of the chlorophyte microalga Chlamydomonas reinhardtii were generated using a reverse genetics strategy. Two single guide RNAs (sgRNA) were designed to target the first exon of the PDS gene, and pre-assembled Cas9 ribonucleoprotein (RNPs) complexes were delivered into microalgal nuclei by electroporation. Multiple white PDS-KO transformants were successfully obtained by this approach, and three independent transformant lines were subsequently characterized. By integrating ultrastructural, pigment and transcriptomic analyses of dark-grown cells of several PDS-KO carotenoid-deficient mutants in comparison with the parental strain, it was demonstrated that carotenoids are indispensable components of multiple cellular architectures. Chromatographic analysis confirmed that the only carotenoid accumulated in these transformants was phytoene, which lacks the critical structural and photoprotective functions of its colored derivatives. Transmission Electron Microscopy (TEM) observations revealed profound ultrastructure alterations, including poorly developed chloroplasts and effects on other cellular structures that were either absent or severely disorganized. Consistently, clustering differentially expressed genes into functional groups revealed downregulation of pathways associated with photosynthesis, chlorophyll and carotenoid biosynthesis, ribosome biogenesis, and vesicle and membrane trafficking in the PDS-KO lines. Conversely, upregulation of regulatory and retrotransposon-inducing genes was observed. These findings underscore the central metabolic role of colored carotenoids in plant cells, highlighting their essential contribution to cellular homeostasis and photosynthetic competence.

The molecular basis of photoperiodism, by which insects use photoperiodic cues to anticipate seasonal changes and regulate key life-history events such as development, diapause, and reproduction, remains poorly understood. Studies on the molecular mechanisms of photoperiodism in hemimetabolous insects are limited compared with those in holometabolous insects, largely due to the lack of appropriate model organisms. The band-legged ground cricket Dianemobius nigrofasciatus represents a valuable model system because it exhibits clear photoperiodic responses in the maternal induction of embryonic diapause, the wing morph, and the rate of nymphal development. With the recent availability of the D. nigrofasciatus genome sequence, the establishment of effective genome-editing methods and reliable marker genes is expected to promote functional genomic analyses. In this study, we aimed to establish a direct parental (DIPA)-CRISPR genome-editing approach and evaluate the utility of vermilion (Dn-v), a gene involved in ommochrome synthesis, as a visible eye color marker for mutant screening. Cas9 ribonucleoprotein complexes were injected into females 3-5 days after adult emergence, during the vitellogenic stage, successfully yielding Dn-v knockout mutants. These mutants had white compound eyes throughout development, with pigmentation reaching a vermilion color about 20 days after adult emergence. We further examined the photoperiodic response associated with maternal diapause induction in knockout mutants. Similar to the wild-type, knockout mutants exhibited low and high diapause incidence under long-day and short-day conditions, respectively. Our results demonstrate that DIPA-CRISPR is an effective genome-editing method in D. nigrofasciatus and that Dn-v serves as a practical and reliable marker gene. The establishment of these genomic tools provides a foundation for future functional analyses aimed at elucidating the molecular basis of photoperiodism in hemimetabolous insects.

MicroRNA-mediated regulation of hypoxic kidney adaptation in naked mole-rats (Heterocephalus glaber).