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[This corrects the article DOI: 10.1371/journal.ppat.1007705.].[This corrects the article DOI: 10.1371/journal.ppat.1007705.].

ObjectiveThe aim of the study was to investigate the role and regulatory mechanisms of fibroblast-like synoviocytes (FLSs) and their senescence in the progression of osteoarthritis (OA).MethodsSynovial tissues from normal patients and patients with OA were collected. Synovium FLS senescence was analysed by immunofluorescence and western blotting. The role of methyltransferase-like 3 (METTL3) in autophagy regulation was explored using N6-methyladenosine (m6A)-methylated RNA and RNA immunoprecipitation assays. Mice subjected to destabilisation of the medial meniscus (DMM) surgery were intra-articularly injected with or without pAAV9 loaded with small interfering RNA (siRNA) targeting METTL3. Histological analysis was performed to determine cartilage damage.ResultsSenescent FLSs were markedly increased with the progression of OA in patients and mouse models. We determined that impaired autophagy occurred in OA-FLS, resulting in the upregulation of senescence-associated secretory phenotype (SASP). Re-establishment of autophagy reversed the senescent phenotype by suppressing GATA4. Further, we observed for the first time that excessive m6A modification negatively regulated autophagy in OA-FLS. Mechanistically, METTL3-mediated m6A modification decreased the expression of autophagy-related 7, an E-1 enzyme crucial for the formation of autophagosomes, by attenuating its RNA stability. Silencing METTL3 enhanced autophagic flux and inhibited SASP expression in OA-FLS. Intra-articular injection of synovium-targeted METTL3 siRNA suppressed cellular senescence propagation in joints and ameliorated DMM-induced cartilage destruction.ConclusionsOur study revealed the important role of FLS senescence in OA progression. Targeted METTL3 inhibition could alleviate the senescence of FLS and limit OA development in experimental animal models, providing a potential strategy for OA therapy.

Plants can use small RNAs (sRNAs) to interfere with virulence factor gene expression in pathogens. Cai et al. show that the small mustard plant Arabidopsis shuttles defensive sRNAs into the necrotrophic fungus Botrytis cinerea via extracellular vesicles (see the Perspective by Thomma and Cook). The vesicles are associated with tetraspanin proteins, which can interact and form membrane microdomains. Several dozen different sRNAs targeting the pathogenic process were transported from Arabidopsis to B. cinerea in a selective manner. Science , this issue p. 1126 ; see also p. 1070 Exosomal vesicles shuttle defensive small RNAs from the host plant to a pathogenic fungus. Some pathogens and pests deliver small RNAs (sRNAs) into host cells to suppress host immunity. Conversely, hosts also transfer sRNAs into pathogens and pests to inhibit their virulence. Although sRNA trafficking has been observed in a wide variety of interactions, how sRNAs are transferred, especially from hosts to pathogens and pests, is still unknown. Here, we show that host Arabidopsis cells secrete exosome-like extracellular vesicles to deliver sRNAs into fungal pathogen Botrytis cinerea . These sRNA-containing vesicles accumulate at the infection sites and are taken up by the fungal cells. Transferred host sRNAs induce silencing of fungal genes critical for pathogenicity. Thus, Arabidopsis has adapted exosome-mediated cross-kingdom RNA interference as part of its immune responses during the evolutionary arms race with the pathogen.

The creation of genome-wide libraries for CRISPR knockout (CRISPRko), interference (CRISPRi), and activation (CRISPRa) has enabled the systematic interrogation of gene function. Here, we show that our recently-described CRISPRko library (Brunello) is more effective than previously published libraries at distinguishing essential and non-essential genes, providing approximately the same perturbation-level performance improvement over GeCKO libraries as GeCKO provided over RNAi. Additionally, we present genome-wide libraries for CRISPRi (Dolcetto) and CRISPRa (Calabrese), and show in negative selection screens that Dolcetto, with fewer sgRNAs per gene, outperforms existing CRISPRi libraries and achieves comparable performance to CRISPRko in detecting essential genes. We also perform positive selection CRISPRa screens and demonstrate that Calabrese outperforms the SAM approach at identifying vemurafenib resistance genes. We further compare CRISPRa to genome-scale libraries of open reading frames (ORFs). Together, these libraries represent a suite of genome-wide tools to efficiently interrogate gene function with multiple modalities. Genome-wide libraries for CRISPR knockout, interference, and activation have allowed the systemic interrogation of gene function. Here, the authors evaluate the Brunello CRISPRko library and introduce Dolcetto and Calabrese for CRISPRi and CRISPRa, respectively.

Therapeutics based on short interfering RNAs (siRNAs) delivered to hepatocytes have been approved, but new delivery solutions are needed to target additional organs. Here we show that conjugation of 2′- O -hexadecyl (C16) to siRNAs enables safe, potent and durable silencing in the central nervous system (CNS), eye and lung in rodents and non-human primates with broad cell type specificity. We show that intrathecally or intracerebroventricularly delivered C16-siRNAs were active across CNS regions and cell types, with sustained RNA interference (RNAi) activity for at least 3 months. Similarly, intravitreal administration to the eye or intranasal administration to the lung resulted in a potent and durable knockdown. The preclinical efficacy of an siRNA targeting the amyloid precursor protein was evaluated through intracerebroventricular dosing in a mouse model of Alzheimer’s disease, resulting in amelioration of physiological and behavioral deficits. Altogether, C16 conjugation of siRNAs has the potential for safe therapeutic silencing of target genes outside the liver with infrequent dosing. Lipophilic siRNA conjugates exert therapeutic activity in the mouse CNS.

MicroRNAs (miRNAs) are short non-coding RNAs that inhibit the expression of target genes by directly binding to their mRNAs. miRNAs are transcribed as precursor molecules, which are subsequently cleaved by the endoribonucleases Drosha and Dicer. Mature miRNAs are bound by a member of the Argonaute (AGO) protein family to form the RNA-induced silencing complex (RISC) in a process termed RISC loading. Advances in structural analyses of Drosha and Dicer complexes enabled elucidation of the mechanisms that drive these molecular machines. Transcription of miRNAs, their processing by Drosha and Dicer and RISC loading are key steps in miRNA biogenesis, and various additional factors facilitate, support or inhibit these processes. Recent work has revealed that regulatory factors not only coordinate individual miRNA processing steps but also connect miRNA biogenesis with other cellular processes. Protein phosphorylation, for example, links miRNA biogenesis to various signalling pathways, and such modifications are often associated with disease. Furthermore, not all miRNAs follow canonical processing routes, and many non-canonical miRNA biogenesis pathways have recently been characterized.

Two decades of research on RNA interference (RNAi) have transformed a breakthrough discovery in biology into a robust platform for a new class of medicines that modulate mRNA expression. Here we provide an overview of the trajectory of small-interfering RNA (siRNA) drug development, including the first approval in 2018 of a liver-targeted siRNA interference (RNAi) therapeutic in lipid nanoparticles and subsequent approvals of five more RNAi drugs, which used metabolically stable siRNAs combined with N -acetylgalactosamine ligands for conjugate-based liver delivery. We also consider the remaining challenges in the field, such as delivery to muscle, brain and other extrahepatic organs. Today’s RNAi therapeutics exhibit high specificity, potency and durability, and are transitioning from applications in rare diseases to widespread, chronic conditions. With six approved drugs, siRNA is now an established therapeutic modality poised for expansion.

Despite the great potential of CRISPR-based detection, it has not been competitive with other market diagnostics for on-site and in-home testing. Here we dissect the rate-limiting factors that undermine the performance of Cas12b- and Cas13a-mediated detection. In one-pot testing, Cas12b interferes with loop-mediated isothermal amplification by binding to and cleaving the amplicon, while Cas13a directly degrades the viral RNA, reducing its amplification. We found that the protospacer-adjacent motif-interacting domain engineered Cas12b accelerated one-pot testing with 10–10,000-fold improved sensitivity, and detected 85 out of 85 SARS-CoV-2 clinical samples with a sensitivity of 0.5 cp μl −1 , making it superior to wild-type Cas12b. In parallel, by diminishing the interference of Cas13a with viral RNA, the optimized Cas13a-based assay detected 86 out of 87 SARS-CoV-2 clinical samples at room temperature in 30 min with a sensitivity of 0.5 cp μl −1 . The relaxed reaction conditions and improved performance of CRISPR-based assays make them competitive for widespread use in pathogen detection. Tong et al. engineered Cas12b and redesigned the target sequence of Cas13a to minimize the interference caused by the cleavage mediated by Cas effectors in one-pot testing, which enables sensitive nucleic acid detection in a simple way at room temperature.

Honey bees are essential pollinators threatened by colony losses linked to the spread of parasites and pathogens. Here, we report a new approach for manipulating bee gene expression and protecting bee health. We engineered a symbiotic bee gut bacterium, Snodgrassella alvi, to induce eukaryotic RNA interference (RNAi) immune responses. We show that engineered S. alvi can stably recolonize bees and produce double-stranded RNA to activate RNAi and repress host gene expression, thereby altering bee physiology, behavior, and growth. We used this approach to improve bee survival after a viral challenge, and we show that engineered S. alvi can kill parasitic Varroa mites by triggering the mite RNAi response. This symbiont-mediated RNAi approach is a tool for studying bee functional genomics and potentially for safeguarding bee health.

CRISPR–Cas interference is mediated by Cas effector nucleases that are either components of multisubunit complexes—in class 1 CRISPR–Cas systems—or domains of a single protein—in class 2 systems 1 – 3 . Here we show that the subtype III-E effector Cas7-11 is a single-protein effector in the class 1 CRISPR–Cas systems originating from the fusion of a putative Cas11 domain and multiple Cas7 subunits that are derived from subtype III-D. Cas7-11 from Desulfonema ishimotonii ( Di Cas7-11), when expressed in Escherichia coli , has substantial RNA interference effectivity against mRNAs and bacteriophages. Similar to many class 2 effectors—and unique among class 1 systems— Di Cas7-11 processes pre-CRISPR RNA into mature CRISPR RNA (crRNA) and cleaves RNA at positions defined by the target:spacer duplex, without detectable non-specific activity. We engineered Cas7-11 for RNA knockdown and editing in mammalian cells. We show that Cas7-11 has no effects on cell viability, whereas other RNA-targeting tools (such as short hairpin RNAs and Cas13) show substantial cell toxicity 4 , 5 . This study illustrates the evolution of a single-protein effector from multisubunit class 1 effector complexes, expanding our understanding of the diversity of CRISPR systems. Cas7-11 provides the basis for new programmable RNA-targeting tools that are free of collateral activity and cell toxicity. Cas7-11—the fusion of a putative Cas11 domain and four Cas7 subunits—cleaves RNA without detectable non-specific activity and, when optimized for RNA knockdown and editing in mammalian cells, has no effects on cell viability.