Cardiovascular diseases (CVDs), including conditions like ischemic heart disease, heart failure (HF), and atherosclerosis (AS), have complex pathogenesis that involves both behavioral and metabolic factors. Nicotinamide N-methyltransferase (NNMT) is an enzyme involved in the methylation of nicotinamide (NAM), and its increased activity is associated with disruptions in the NAD+ and methionine cycles. These disruptions are considered significant risk factors for cardiovascular diseases, though the specific mechanisms of NNMT remain unclear. This review discusses the role of NNMT in cardiovascular diseases by modulating NAD+ and methionine metabolism, including mechanisms such as NAD+ depletion, mitochondrial energy crisis, SIRTs deactivation, PARP hyperactivation, as well as hyperhomocysteinemia and epigenetic dysregulation. NNMT is linked to diseases such as atherosclerosis, pulmonary arterial hypertension, heart failure, and coronary heart disease, playing a critical role in their progression. Moreover, the potential of NNMT as a therapeutic target for cardiovascular diseases is explored. RNAi therapies, NNMT small-molecule inhibitors, and exercise therapies are promising treatment approaches, but there are limitations in current research, including discrepancies between animal models and human tissue expression, the dual role of NNMT, and the dose-dependent effects of NNMT inhibitors. Future studies should further clarify NNMT's mechanisms and assess its feasibility as a therapeutic target, aiming to develop more effective treatments and enhance prevention and treatment strategies for cardiovascular diseases.

The regulation of protein levels in plants is essential for improving agricultural productivity. Recent studies have explored inducible degradation systems in plants, with some showing promising advancements. This study introduces the NRDN degradation tag as a novel tool for regulating protein stability within the nucleus in Arabidopsis thaliana. Unlike traditional gene knockout methods, NRDN offers real-time, dynamic control over protein degradation, enabling precise studies of nuclear-localized proteins. This discovery provides a valuable tool for regulating protein stability in specific cellular compartments, which presents a versatile approach for dissecting complex cellular processes and offers broad applications in functional genomics and cellular research.

Osteoarthritis (OA) is a chronic degenerative joint disease. Currently, OA is incurable. Abnormal activation of canonical Wnt/β-catenin or Indian hedgehog (Ihh) signaling could lead to OA development and progression. This study aimed to determine if targeting β-catenin and Ihh signaling could yield an effective therapeutic intervention for OA disease. CRISPR/CasRx is a new RNA interference tool that can precisely and efficiently cleave single-strand RNAs. In this study, we screened CRISPR-derived RNA (crRNA) targeting Ctnnb1 and Smo in vitro and selected two optimal crRNAs for each gene. CasRx-mediated Ctnnb1 and Smo knockdown showed high efficiency and specificity with no obvious off-target effects in vitro. We then performed intra-articular injection of selected crRNAs driven by the adeno-associated virus into an OA mouse model. Micro-CT, histological, and histomorphometric analyses were conducted to evaluate the efficacy of CasRx approach on OA treatment. We found that the knockdown of Ctnnb1 and Smo decelerated pathological damage in the keen joint of the experimental OA mouse model. Our findings suggest that CasRx-mediated Ctnnb1 and Smo knockdown could be a potential strategy for OA treatment.

Atherosclerosis, a principal cause of fatal cardiovascular diseases, is fundamentally a chronic inflammatory disease. Addressing this, the combined regulation of oxidative stress and inflammation through synergistic modalities offers an efficient therapeutic avenue. In this work, we rationally designed and engineered a highly efficient functional nanosystem, referred to as polydopamine nanoparticles doped with arginine and gadolinium ions (AGPDAR-146a), for the targeted delivery of therapeutic oligonucleotides, specifically microRNA-146a (miR-146a), to inflammatory macrophages within atherosclerotic plaques. AGPDAR-146a nanoparticles effectively load and deliver miR-146a, achieving enhanced accumulation in inflammatory macrophages through the specific interaction between miR-146a and class A scavenger receptors. Functionally, AGPDAR-146a nanoparticles excel in eliminating reactive oxygen species and exert anti-inflammatory effects, principally by modulating the nuclear factor kappa-light-chain-enhancer of activated B cells pathway and the interferon regulatory factor 5 protein, consequently helping to reduce and stabilize atherosclerotic plaques. Additionally, the intrinsic T1 magnetic resonance imaging capability of AGPDAR-146a nanoparticles enables real-time visualization of the progression of plaque inflammation. Therefore, the engineered nanosystem not only underscores the therapeutic potential of miR-146a in atherosclerosis but also illustrates a versatile microRNA delivery strategy applicable to various diseases characterized by oxidative stress and inflammation.

Hepatocellular carcinoma (HCC), the predominant form of primary liver cancer, remains a global health challenge with limited therapeutic options and high mortality rates. Despite advances in understanding its molecular pathogenesis, the role of metabolic reprogramming in HCC progression and therapy resistance demands further exploration. Nicotinamide N-methyltransferase (NNMT), a metabolic enzyme central to NAD+ and methionine cycles, has emerged as a critical regulator of tumorigenesis across cancers. However, its tissue-specific mechanisms in HCC-particularly in the context of viral hepatitis and methionine cycle dependency-remain understudied. This review systematically synthesizes current evidence on NNMT's dual role in HCC: (1) driving NAD+ depletion and homocysteine (Hcy) accumulation via metabolic dysregulation, (2) promoting malignant phenotypes (proliferation, invasion, metastasis, and drug resistance), and (3) serving as a prognostic biomarker and therapeutic target. We highlight how NNMT intersects with epigenetic modifications, immune evasion, and metabolic vulnerabilities unique to HCC. Additionally, we critically evaluate NNMT inhibitors, RNA-based therapies, and non-pharmacological strategies (e.g., exercise) as novel interventions. By bridging gaps between NNMT's molecular mechanisms and clinical relevance, this review provides a roadmap for advancing NNMT-targeted therapies and underscores the urgency of addressing challenges in biomarker validation, inhibitor specificity, and translational efficacy. Our work positions NNMT not only as a metabolic linchpin in HCC but also as a promising candidate for precision oncology.

Pooled CRISPR screens are vital in the unbiased interrogation of gene function and are instrumental in uncovering therapeutic targets and biological processes. However, follow-up hit validation is critical to confirm observed results. Researchers need a simple and robust approach to rapidly verify putative hits and test resulting observations. Thus, we developed a CRISPR-based method for hit validation that tests the effect of a genetic perturbation on cell fitness. By editing target loci and monitoring the indel profiles over time, we have created a Cellular Fitness (CelFi) assay that can elucidate cellular vulnerabilities and verify hits from pooled CRISPR knockout screens. Unlike traditional cellular fitness assays that evaluate viability over time, the CelFi assay correlates changes in the indel profile at the target gene with a selective growth advantage or disadvantage in individual cells over time. Moreover, the CelFi assay can be utilized to evaluate gene dependencies and test new hypotheses, regardless of variations in single guide RNA optimization, ribonucleoprotein concentration, and gene copy number.

Oligonucleotides have emerged as a formidable new class of nucleic acid therapeutics. Fully modified oligonucleotides exhibit enhanced metabolic stability and display successful clinical applicability for targets formerly considered "undruggable". Accumulating studies show that conjugation to targeting modalities of stabilized oligonucleotides, especially small interfering RNAs (siRNAs), has enabled robust delivery to intended cells/tissues. However, the major challenge in the field has been the stability and targeted delivery of oligonucleotides (siRNAs and antisense oligonucleotides (ASOs)) to extrahepatic tissues. In this Perspective, we review chemistry innovations and emerging delivery approaches that have revolutionized oligonucleotide drug discovery and development. We explore findings from both academia and industry that highlight the potential of oligonucleotides for indications involving different extrahepatic organs─including skeletal muscles, brain, lungs, skin, heart, adipose tissue, and eyes. In all, continued advances in chemistry coupled with conjugation-based approaches or novel administration routes will further advance the delivery of oligonucleotides to extrahepatic tissues.

Short hairpin RNAs (shRNAs) are potent tools for gene silencing, offering therapeutic potential for gene and cell therapy applications. However, their efficacy and safety depend on precise processing by the RNA interference machinery and the generation of minimal by-products. In this protocol, we describe how to systematically analyze the processing of therapeutic small RNAs by DROSHA and DICER1 and their incorporation into functional AGO complexes. Using standard small RNA sequencing and tailored bioinformatic analysis (QuagmiR), we evaluate the different steps of shRNA maturation that influence processing efficiency and specificity. We provide guidelines for troubleshooting common design pitfalls and off-target effects in transcriptome-wide profiling to identify unintended mRNA targeting via the miRNA-like effect. We provide examples of the bioinformatic analysis that can be performed to characterize therapeutic shRNA. Finally, we provide guidelines for troubleshooting shRNA designs that result in suboptimal processing or undesired off-target effects. This protocol underscores the importance of rational shRNA design to enhance specificity and reduce biogenesis by-products that can lead to off-target effects, providing a framework for optimizing the use of small RNAs in gene and cell therapies.

Currently, no drugs directly treat liver fibrosis. Previously, we have shown that treatment with miR-29a-3p improved liver fibrosis in a mouse model. To investigate the effectiveness of nucleic acid therapy at a lower dose, a modified nucleic acid was prepared based on miR-29a-3p. The original microRNA was changed to an RNA-DNA hybrid structure: the 2' position of the RNA was modified with a fluorine base, and locked nucleic acid and phosphorothioate were crosslinked (hereafter called modified nucleic acid). In a mouse model of chronic liver disease treated with carbon tetrachloride (CCl4), the inhibitory effect on liver fibrosis was evaluated with oral administration of the modified nucleic acid. The modified nucleic acid was detected in the liver and gastrointestinal tract within 15 min of oral administration. After 5 weeks of stimulation with CCl4, oral administration of the modified nucleic acid for 2 weeks improved liver fibrosis; CCl4 stimulation was continued during this period as well. This treatment also suppressed the worsening of liver fibrosis. We developed a method to improve liver fibrosis orally using nuclease-resistant nucleic acids without using a drug delivery system. This method may be used as a new treatment for inhibiting the progression of liver fibrosis.

Nucleic-acid-based therapies have emerged as a pivotal domain within contemporary biomedical science, marked by significant advancements in recent years. These innovative treatments primarily operate through the precise binding of DNA or RNA molecules to discrete target genes, subsequently suppressing the expression of the target proteins. The spectrum of nucleic-acid-based therapies encompasses antisense oligonucleotides (ASOs), small interfering RNAs (siRNAs), microRNAs (miRNAs), and messenger RNAs (mRNAs), etc. Compared to more traditional medicinal approaches, nucleic-acid-based therapies stand out for their highly targeted action on specific genes, as well as their potential for chemical modification to improve resistance to nucleases, ensuring sustained therapeutic activity and mitigating immunogenicity concerns. Nevertheless, these molecules' limited cellular permeability necessitates the deployment of delivery vectors to enhance their intracellular uptake and stability. As nucleic-acid-based therapies progressively display promising pharmacodynamic profiles, there has been a burgeoning interest in these treatments for applications in clinical research. This review aims to summarize the variety of nucleic acid drugs and their mechanisms, evaluate the present status in research and application, discourse on prospective trends, and potential challenges ahead. These innovative therapeutics are anticipated to assume a pivotal role in the management of a wide array of diseases.

Targeted therapy has emerged as a transformative breakthrough in modern medicine. Oligonucleotide drugs, such as antisense oligonucleotides (ASOs) and small interfering RNAs (siRNAs), have made significant advancements in targeted therapy. Other oligonucleotide-based therapeutics like clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems are also leading a revolution in targeted gene therapy. However, hybridisation-dependent off-target effects, arising from imperfect base pairing, remain a significant and growing concern for the clinical translation of oligonucleotide-based therapeutics. These mismatches in base pairing can lead to unintended steric blocking or cleavage events in non-pathological genes, affecting the efficacy and safety of the oligonucleotide drugs. In this review, we examine recent developments in oligonucleotide-based targeted therapeutics, explore the factors influencing sequence-dependent targeting specificity, and discuss the current approaches employed to reduce the off-target side effects. The existing strategies, such as chemical modifications and oligonucleotide length optimisation, often require a trade-off between specificity and binding affinity. To further address the challenge of hybridisation-dependent off-target effects, we discuss DNA nanotechnology-based strategies that leverage the collaborative effects of nucleic acid assembly in the design of oligonucleotide-based therapies. In DNA nanotechnology, collaborative effects refer to the cooperative interactions between individual strands or nanostructures, where multiple bindings result in more stable and specific hybridisation behaviour. By requiring multiple complementary interactions to occur simultaneously, the likelihood of unintended partially complementary binding events in nucleic acid hybridisation should be reduced. And thus, with the aid of collaborative effects, DNA nanotechnology has great promise in achieving both high binding affinity and high specificity to minimise the hybridisation-dependent off-target effects of oligonucleotide-based therapeutics.

Cardiovascular diseases (CVDs) are the leading cause of morbidity and mortality worldwide. Over the past decade, studies have demonstrated that circulating long non-coding RNAs (lncRNAs)-recognised for their stability and ease of detection-serve as crucial regulators and potential biomarkers in multiple diseases. LncRNAs regulate key processes, including endothelial function, vascular remodelling, and myocardial hypertrophy, all of which influence CVD progression. Additionally, lncRNAs display cell-, tissue-, and disease-specific expression patterns, making them ideal therapeutic targets or tools. This review presents a comprehensive overview of the current understanding of lncRNAs in CVDs, examining their mechanisms of action and recent research advances. It also addresses the use of lncRNAs as diagnostic and prognostic markers, as well as potential applications of RNA therapeutics in novel treatment strategies.

Gene context-essentiality assessment supports precision oncology opportunities. The variability of gene effects inference from loss-of-function screenings across models and technologies limits identifying robust hits. We propose a computational framework named PRODE that integrates gene effects with protein-protein interactions to generate neighborhood-informed essential (NIE) and neighborhood-informed context essential (NICE) scores. It outperforms the canonical gene effect approach in recovering missed essential genes in shRNA screens and prioritizing context-essential hits from CRISPR-KO screens, as supported by in vitro validations. Applied to Her2 + breast cancer tumor samples, PRODE identifies oxidative phosphorylation genes as vulnerabilities with prognostic value, highlighting new therapeutic opportunities.

Synthetic trans-acting small interfering RNAs (syn-tasiRNAs) are 21-nucleotide (nt) small RNAs designed to silence plant transcripts with high specificity. Their use as biotechnological tools for functional genomics and crop improvement is limited by the need to transgenically express long TAS precursors to produce syn-tasiRNAs in vivo. Here, we show that authentic and highly effective syn-tasiRNAs can be produced from minimal, non-TAS precursors consisting of a 22-nt endogenous microRNA target site, an 11-nt spacer, and the 21 nt syn-tasiRNA sequence(s). These minimal precursors, when transgenically expressed in Arabidopsis thaliana and Nicotiana benthamiana, generated highly phased syn-tasiRNAs that silenced one or multiple plant genes with high efficacy. Remarkably, minimal but not full-length TAS precursors produced authentic syn-tasiRNAs and induced widespread gene silencing in N. benthamiana when expressed from an RNA virus, which can be applied by spraying infectious crude extracts onto leaves in a transgene-free manner. This strategy, named syn-tasiRNA-based virus-induced gene silencing (syn-tasiR-VIGS), was further used to vaccinate plants against a pathogenic virus, resulting in complete plant immunization. Our results reveal that syn-tasiRNA precursors can be significantly shortened without compromising silencing efficacy, and that syn-tasiR-VIGS represents a versatile, scalable, and nontransgenic platform for precision RNA interference and antiviral vaccination in plants.

Schistosomiasis affects over 250 million people in 78 countries. Despite praziquantel as the primary treatment, concerns about resistance in schistosomes underscore the need for alternative therapies. The success of RNA interference (RNAi) in schistosomes shows promise for identifying potential drug targets to facilitate drug discovery. Meanwhile, double-stranded RNA (dsRNA) is commonly used in functional gene analysis via RNAi, with double-stranded green fluorescent protein (ds-GFP) widely employed as a control in schistosome-related studies. However, the potential for off-target effects of dsRNAs in various biological systems raises concerns about the reliability of conventional controls in schistosome RNAi experiments. Therefore, this study aims to evaluate the safety and suitability of ds-GFP as an RNAi negative control in Schistosoma japonicum. Our data indicate that ds-GFP is innocuous and exerts no discernible impact on the host's physiology and immune responses. Comprehensive evaluations conducted in mice showed no significant alterations in body and organ weights. While a splenic immune response was observed, histopathological examinations of multiple organs confirmed the absence of significant lesions following ds-GFP treatment. Additionally, S. japonicum morphology, reproductive capacity, and host responses to parasite eggs showed no significant variations. Taken together, these findings bolster the endorsement of ds-GFP as an appropriate negative control in S. japonicum RNAi experiments, offering reliable outcomes crucial for advancing research on schistosomiasis and related parasitic diseases.

RNA interference (RNAi) and autophagy are two pivotal biological processes that regulate virus replication. This study explored the complex relationship between autophagy and RNAi in controlling influenza virus replication. Initially, we reported that influenza virus (H9N2) infection increases the viral load and the expression of autophagy markers while inhibiting the RNAi pathway. Subsequent studies employing autophagy enhancer and inhibitor treatments confirmed that avian influenza virus (AIV, H9N2) promotes viral replication by enhancing autophagy pathways. Further analysis revealed that ATG7, an autophagy protein, can interact with dicer to affect its antiviral functions. Finally, we discovered that infection with other avian RNA viruses, including infectious bursal disease virus (IBDV) and infectious bronchitis virus (IBV), induced the upregulation of ATG7, which blocked the RNAi pathway to facilitate virus replication. Our findings suggested that virus infection might trigger the upregulation of autophagy and downregulation of the RNAi pathway, revealing a complex interaction between these two biological processes in the defence against viral replication.

Genome and transcriptome engineering have emerged as powerful tools in modern biotechnology, driving advancements in precision medicine and novel therapeutics. In this review, we provide a comprehensive overview of the current methodologies, applications, and future directions in genome and transcriptome engineering. Through this, we aim to provide a guide for tool selection, critically analyzing the strengths, weaknesses, and best use cases of these tools to provide context on their suitability for various applications. We explore standard and recent developments in genome engineering, such as base editors and prime editing, and provide insight into tool selection for change of function (knockout, deletion, insertion, substitution) and change of expression (repression, activation) contexts. Advancements in transcriptome engineering are also explored, focusing on established technologies like antisense oligonucleotides (ASOs) and RNA interference (RNAi), as well as recent developments such as CRISPR-Cas13 and adenosine deaminases acting on RNA (ADAR). This review offers a comparison of different approaches to achieve similar biological goals, and consideration of high-throughput applications that enable the probing of a variety of targets. This review elucidates the transformative impact of genome and transcriptome engineering on biological research and clinical applications that will pave the way for future innovations in the field.

The exogenous application of RNAi technology offers new promises for crops improvement. Cell-based or synthetically produced strands are economical, non-transgenic and could induce the same responses. The substantial population growth demands novel strategies to produce crops without further damaging the environment. RNA interference mechanism is one of the promising technologies to biologically control pests and pathogens in crops, suppressing them by cancelling protein synthesis related to parasitism/pathogenesis. The transgenic approach to generate host-induced gene silencing demonstrated high efficacy in controlling pests or pathogens by RNAi mechanism. However, transgenic technology is tightly regulated and still negatively perceived by global consumers. This review presents the basic biology of small RNA, the main actor of the RNAi mechanism, and tested non-transgenic approaches to induce RNAi exogenously. Novel avenues are offered by the discovery of cross-kingdom RNAi, that naturally, plants also deliver small RNA to suppress the growth of their threats. Future applications of non-transgenic RNAi-based biocontrol will involve the production of dsRNA on an industrial scale. Here, the attempts to provide dsRNA for routine application in farms are also discussed, emphasizing that the technology must be accessible by the countries with the greatest population which mostly are poorer ones.

Severe fever with thrombocytopenia syndrome (SFTS) caused by the SFTS virus (SFTSV) is an emerging tick-borne disease with a high mortality rate. Haemaphysalis longicornis is the primary reservoir and vector of SFTSV. Here, we found that targeting subolesin (SUB), an anti-tick vaccine candidate, affects the infection and transmission of SFTSV in H. longicornis. RNAi-mediated knockdown of SUB repressed SFTSV infection in the salivary glands but not in the gut of H. longicornis, which may be associated with the modulation of protein processing in endoplasmic reticulum revealed by transcriptomic analysis. Knockdown of SUB decreased the survival and engorgement rates of ticks and impaired the horizontal and co-feeding transmission of SFTSV. Furthermore, active immunization with recombinant SUB inhibited the co-feeding transmission of SFTSV, although it had no significant effect on the blood-feeding behavior of infected ticks. Collectively, these results provide a potential target for controlling SFTS and other tick-borne viral diseases.

Phosphatidylinositol 5-phosphate 4-kinases (PI5P4K), also known as type II PIPKs or PIPKIIs, convert the lipid second messenger PI5P to PI(4,5)P2. The PI5P4K family consists of three isozymes in mammals-PI5P4Kα, β, and γ-which notably utilize both GTP and ATP as phosphodonors. Unlike the other two isozymes, which can utilize both ATP and GTP, PI5P4Kβ exhibits a marked preference for GTP over ATP, acting as an intracellular GTP sensor that alters its kinase activity in response to physiological changes in GTP concentration. Knockout studies have demonstrated a critical role for PI5P4Kα and β in tumorigenesis, while PI5P4Kγ has been implicated in regulating immune and neural systems. Pharmacological targeting of PI5P4K holds promise for the development of new therapeutic approaches against cancer, immune dysfunction, and neurodegenerative diseases. Although several PI5P4K inhibitors have already been developed, challenges remain in PI5P4K inhibitor development, including a discrepancy between in vitro and cellular efficacy. This discrepancy is attributable to mainly three factors. (a) Most PI5P4K inhibitors were developed at low ATP levels, where these enzymes exhibit minimal activity. (b) Non-catalytic functions of PI5P4K require careful interpretation of PI5P4K depletion studies, as their scaffolding roles suppress class I PI3K signaling. (c) The lack of pharmacodynamic markers for in vivo assessment complicates efficacy assessment. To address these issues and promote the development of effective and targeted therapeutic strategies, this review provides an analytical overview of the distinct roles of individual isozymes and recent developments in PI5P4K inhibitors, emphasizing structural insights and the importance of pharmacodynamic marker identification.

Hypertension (HTN), often dubbed the "silent killer," poses a significant global health challenge, affecting over 1.3 billion individuals. Despite advances in treatment, effective long-term blood pressure (BP) control remains elusive, necessitating novel therapeutic approaches. Poor control of BP remains a leading cause of cardiovascular morbidity and mortality worldwide and is becoming an even larger global health problem due to the aging population, rising rates of obesity, poorer dietary patterns and overall cardiometabolic health, and suboptimal rates of patient adherence and optimal BP control. Ribonucleic acid interference (RNAi) technology, which leverages the body's natural gene-silencing mechanism, has emerged as a promising strategy for several diseases and has recently been tested for its antihypertensive effects. We systematically reviewed peer-reviewed articles from databases including PubMed, EMBASE, and Scopus for studies examining RNAi's role in managing HTN, focusing on mechanisms, clinical utility, and safety profile. Key early-phase trials of some RNAi-leading candidate drugs are detailed. Also highlighted are challenges such as target specificity, delivery mechanisms, durability of effect, and immunogenicity. We conclude by summarizing how RNAi has a significant potential role in HTN therapy due to their unique benefits, such as long-term duration of action, infrequent dosing, and lack of major side effects.

During the past decades, advances in RNA interference (RNAi) technology have paved the way for the systematic exploration of gene function, and phenotypic screening of small interfering RNA (siRNA) oligonucleotides is a strategy still commonly pursued for the identification and validation of targets, particularly in oncology drug discovery. Here we present a method for large-scale automated siRNA transfection and cell phenotypic screening using colony formation as a readout. Experimental conditions were optimized to achieve efficient and nontoxic transfection of siRNA oligonucleotides in different cell lines using liposomal reagents. For each gene, the most active and specific siRNA oligos were selected through a phenotypic prescreening in HeLa cells, selected as control cell line, and grouped in the same oligo pool. Cells were then transfected at low seeding density in 96-well plates, and after 7-14 days colony formation was analyzed. We have found this procedure to be more sensitive than standard 48-72 h proliferation assays for identifying genes essential for cell viability/proliferation, as it allows to reveal long-term consequences in slow growing cell lines, or phenotypes that occur after multiple cell divisions. This approach generated robust and reliable results through the limitation of siRNA off-target toxic effects by combining a pool of different siRNA oligos designed against the same target. Furthermore, a parallel evaluation of gene silencing phenotypes is performed against a large panel of cell lines, allowing the simultaneous identification of target related genetic dependencies in several cancer cell line models of different tumor origin.

The vast majority of the human genome codes for RNA, but RNA-targeting therapeutics account for a small fraction of approved drugs. As such, there is great incentive to improve old and develop new approaches to RNA targeting. For many RNA targeting modalities, just binding is not sufficient to exert a therapeutic effect; thus, targeted RNA degradation and induced decay emerged as powerful approaches with a pronounced biological effect. This review covers the origins and advanced use cases of targeted RNA degrader technologies grouped by the nature of the targeting modality as well as by the mode of degradation. It covers both well-established methods and clinically successful platforms such as RNA interference, as well as emerging approaches such as recruitment of RNA quality control machinery, CRISPR, and direct targeted RNA degradation. We also share our thoughts on the biggest hurdles in this field, as well as possible ways to overcome them.

The mammalian target of rapamycin (mTOR), a serine/threonine kinase, promotes cell growth and inhibits autophagy. The following two complexes contain mTOR: mTORC1 with the regulatory associated protein of mTOR (RAPTOR) and mTORC2 with the rapamycin-insensitive companion of mTOR (RICTOR). The phosphatidylinositol 3-kinase (PI3K)/Akt/mTOR signaling pathway is important in the intervertebral disk, which is the largest avascular, hypoxic, low-nutrient organ in the body. To examine gene-silencing therapeutic approaches targeting PI3K/Akt/mTOR signaling in degenerative disk cells, an in vitro comparative study was designed between small interfering RNA (siRNA)-mediated RNA interference (RNAi) and clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated protein 9 (Cas9) gene editing. Surgically obtained human disk nucleus pulposus cells were transfected with a siRNA or CRISPR-Cas9 plasmid targeting mTOR, RAPTOR, or RICTOR. Both of the approaches specifically suppressed target protein expression; however, the 24-h transfection efficiency differed by 53.8-60.3% for RNAi and 88.1-89.3% for CRISPR-Cas9 (p < 0.0001). Targeting mTOR, RAPTOR, and RICTOR all induced autophagy and inhibited apoptosis, senescence, pyroptosis, and matrix catabolism, with the most prominent effects observed with RAPTOR CRISPR-Cas9. In the time-course analysis, the 168-h suppression ratio of RAPTOR protein expression was 83.2% by CRISPR-Cas9 but only 8.8% by RNAi. While RNAi facilitates transient gene knockdown, CRISPR-Cas9 provides extensive gene knockout. Our findings suggest that RAPTOR/mTORC1 is a potential therapeutic target for degenerative disk disease.

Emerging research has positioned Nicotinamide N-methyltransferase (NNMT) as a key player in oncology, with its heightened expression frequently observed across diverse cancers. This increased presence is tightly linked to tumor initiation, proliferation, and metastasis. The enzymatic function of NNMT is centered on the methylation of nicotinamide (NAM), utilizing S-adenosylmethionine (SAM) as the methyl donor, which results in the generation of S-adenosyl-L-homocysteine (SAH) and methyl nicotinamide (MNAM). This metabolic process reduces the availability of NAM, necessary for Nicotinamide adenine dinucleotide (NAD+) synthesis, and generates SAH, precursor to homocysteine (Hcy). These alterations are theorized to foster the resilience, expansion, and invasiveness of cancer cells. Furthermore, NNMT is implicated in enhancing cancer malignancy by affecting multiple signaling pathways, such as phosphatidylinositol 3-kinase (PI3K)-protein kinase B (AKT), cancer-associated fibroblasts (CAFs) and 5-Methyladenosine (5-MA), epithelial-mesenchymal transition (EMT), and epigenetic mechanisms. Upregulation of NNMT metabolism plays a key role in the formation and maintenance of the tumour microenvironment. While the use of small molecule inhibitors and RNA interference (RNAi) to target NNMT has shown therapeutic promise, the full extent of NNMT's influence on cancer is not yet fully understood, and clinical evidence is limited. This article systematically describes the relationship between the functional metabolism of NNMT enzymes and the cancer and tumour microenvironments, describing the mechanisms by which NNMT contributes to cancer initiation, proliferation, and metastasis, as well as targeted therapies. Additionally, we discuss the future opportunities and challenges of NNMT in targeted anti-cancer treatments.

CRISPR/Cas13 system, recognized for its compact size and specificity in targeting RNA, is currently employed for RNA degradation. However, the potential of various CRISPR/Cas13 subtypes, particularly concerning the knockdown of endogenous transcripts, remains to be comprehensively characterized in plants.

Here we present a full spectrum of editing profiles for seven Cas13 orthologs from five distinct subtypes: VI-A (LwaCas13a), VI-B (PbuCas13b), VI-D (RfxCas13d), VI-X (Cas13x.1 and Cas13x.2), and VI-Y (Cas13y.1 and Cas13y.2). A systematic evaluation of the knockdown effects on two endogenous transcripts (GhCLA and GhPGF in cotton) as well as an RNA virus (TMV in tobacco) reveals that RfxCas13d, Cas13x.1, and Cas13x.2 exhibit enhanced stability with editing efficiencies ranging from 58 to 80%, closely followed by Cas13y.1 and Cas13y.2. Notably, both Cas13x.1 and Cas13y.1 can simultaneously degrade two endogenous transcripts through a tRNA-crRNA cassette approach, achieving editing efficiencies of up to 50%. Furthermore, different Cas13 orthologs enable varying degrees of endogenous transcript knockdown with minimal off-target effects, generating germplasms that exhibit a diverse spectrum of mutant phenotypes. Transgenic tobacco plants show significant reductions in damage, along with mild oxidative stress and minimal accumulation of viral particles after TMV infection.

In conclusion, our study presents an efficient and reliable platform for transcriptome editing that holds promise for plant functional research and future crop improvement.

RNA sensing in vivo evaluates past or ongoing endogenous RNA disturbances, which is crucial for identifying cell types and states and diagnosing diseases. Recently, the CRISPR-driven genetic circuits have offered promising solutions to burgeoning challenges in RNA sensing. This review delves into the cutting-edge developments of CRISPR-powered RNA sensors in vivo, reclassifying these RNA sensors into four categories based on their working mechanisms, including programmable reassembly of split single-guide RNA (sgRNA), RNA-triggered RNA processing and protein cleavage, miRNA-triggered RNA interference (RNAi), and strand displacement reactions. Then, we discuss the advantages and challenges of existing methodologies in diverse application scenarios and anticipate and analyze obstacles and opportunities in forthcoming practical implementations.

RNA interference (RNAi) technology has emerged as a pivotal strategy in sustainable pest management, offering a targeted approach that significantly mitigates the environmental and health risks associated with traditional insecticides. Originally implemented through genetically modified organisms (GMOs) to produce specific RNAi constructs, the technology has evolved in response to public and regulatory concerns over GMOs. This evolution has spurred the development of non-transgenic RNAi applications such as spray-induced gene silencing (SIGS), which employs double-stranded RNA (dsRNA) to silence pest genes directly without altering the plant's genetic makeup. Despite its advantages in specificity and reduced ecological footprint, SIGS faces significant obstacles, particularly the instability of dsRNA in field conditions, which limits its practical efficacy. To overcome these limitations, innovative delivery mechanisms have been developed. These include nanotechnology-based systems, minicells, and nanovesicles, which are designed to protect dsRNA from degradation and enhance its delivery to target organisms. While these advancements have improved the stability and application efficiency of dsRNA, comprehensive assessments of their environmental safety and the potential for increased exposure risks to non-target organisms remain incomplete. This comprehensive review aims to elucidate the environmental fate of dsRNA and evaluate the potential risks associated with its widespread application on non-target organisms, encompassing soil microorganisms, beneficial insects, host plants, and mammals. The objective is to establish a more refined framework for RNAi risk assessment within environmental and ecotoxicological contexts, thereby fostering the development of safer, non-transgenic RNAi-based pest control strategies.

The development of precise RNA-editing tools is essential for the advancement of RNA therapeutics. CRISPR (clustered regularly interspaced short palindromic repeats) PspCas13b is a programmable RNA nuclease predicted to offer superior specificity because of its 30-nucleotide spacer sequence. However, its design principles and its on-target, off-target and collateral activities remain poorly characterized. Here, we present single-base tiled screening and computational analyses that identify key design principles for potent and highly selective RNA recognition and cleavage in human cells. We show that the de novo design of spacers containing guanosine bases at precise positions can greatly enhance the catalytic activity of inefficient CRISPR RNAs (crRNAs). These validated design principles (integrated into an online tool, https://cas13target.azurewebsites.net/ ) can predict highly effective crRNAs with ~90% accuracy. Furthermore, the comprehensive spacer-target mutagenesis revealed that PspCas13b can tolerate only up to four mismatches and requires ~26-nucleotide base pairing with the target to activate its nuclease domains, highlighting its superior specificity compared to other RNA or DNA interference tools. On the basis of this targeting resolution, we predict an extremely low probability of PspCas13b having off-target effects on other cellular transcripts. Proteomic analysis validated this prediction and showed that, unlike other Cas13 orthologs, PspCas13b exhibits potent on-target activity and lacks collateral effects.

Idiopathic pulmonary fibrosis (IPF) is a prevalent chronic pulmonary fibrosis disease characterized by alveolar epithelial cell damage, fibroblast proliferation and activation, excessive extracellular matrix deposition, and abnormal epithelial-mesenchymal transition (EMT), resulting in tissue remodeling and irreversible structural distortion. The mortality rate of IPF is very high, with a median survival time of 2-3 years after diagnosis. The exact cause of IPF remains unknown, but increasing evidence supports the central role of epigenetic changes, particularly microRNA (miRNA), in IPF. Approximately 10% of miRNAs in IPF lung tissue exhibit differential expression compared to normal lung tissue. Diverse miRNA phenotypes exert either a pro-fibrotic or anti-fibrotic influence on the progression of IPF. In the context of IPF, epigenetic factors such as DNA methylation and long non-coding RNAs (lncRNAs) regulate differentially expressed miRNAs, which in turn modulate various signaling pathways implicated in this process, including transforming growth factor-β1 (TGF-β1)/Smad, mitogen-activated protein kinase (MAPK), and phosphatidylinositol-3-kinase/protein kinase B (PI3K/AKT) pathways. Therefore, this review presents the epidemiology of IPF, discusses the multifaceted regulatory roles of miRNAs in IPF, and explores the impact of miRNAs on IPF through various pathways, particularly the TGF-β1/Smad pathway and its constituent structures. Consequently, we investigate the potential for targeting miRNAs as a treatment for IPF, thereby contributing to advancements in IPF research.

In this study, we report the synthesis of 2'-formamidonucleoside phosphoramidite derivatives and their incorporation into siRNA strands to reduce seed-based off-target effects of small interfering RNAs (siRNAs). Formamido derivatives of all four nucleosides (A, G, C and U) were synthesized in 5-11 steps from commercial compounds. Introducing these derivatives into double-stranded RNA slightly reduced its thermodynamic stability, but X-ray crystallography and CD spectrum analysis confirmed that the RNA maintained its natural A-form structure. Although the introduction of the 2'-formamidonucleoside derivative at the 2nd position in the guide strand of the siRNA led to a slight decrease in the on-target RNAi activity, the siRNAs with different sequences incorporating 2'-formamidonucleoside with four kinds of nucleobases into any position other than 2nd position in the seed region revealed a significant suppression of off-target activity while maintaining on-target RNAi activity. This indicates that 2'-formamidonucleosides represent a promising approach for mitigating off-target effects in siRNA therapeutics.

Cancers are increasing in prevalence and many challenges remain for their treatment, such as chemoresistance and toxicity. In this context, siRNA-based therapeutics have many potential advantages for cancer therapies as a result of their ability to reduce or prevent expression of specific cancer-related genes. However, the direct delivery of naked siRNA is hindered by issues like enzymatic degradation, insufficient cellular uptake, and poor pharmacokinetics. Hence, the discovery of a safe and efficient delivery vehicle is essential. This review explores various lipid and polymer-based delivery systems for siRNA in cancer treatment. Both polymers and lipids have garnered considerable attention as carriers for siRNA delivery. While all of these systems protect siRNA and enhance transfection efficacy, each exhibits its unique strengths. Lipid-based delivery systems, for instance, demonstrate high entrapment efficacy and utilize cost-effective materials. Conversely, polymeric-based delivery systems offer advantages through chemical modifications. Nonetheless, certain drawbacks still limit their usage. To address these limitations, combining different materials in formulations (lipid, polymer, or targeting agent) could enhance pharmaceutical properties, boost transfection efficacy, and reduce side effects. Furthermore, co-delivery of siRNA with other therapeutic agents presents a promising strategy to overcome cancer resistance. Lipid-based delivery systems have been demonstrated to encapsulate many therapeutic agents and with high efficiency, but most are limited in terms of the functionalities they display. In contrast, polymeric-based delivery systems can be chemically modified by a wide variety of routes to include multiple components, such as release or targeting elements, from the same materials backbone. Accordingly, by incorporating multiple materials such as lipids, polymers, and/or targeting agents in RNA formulations it is possible to improve the pharmaceutical properties and therapeutic efficacy while reducing side effects. This review focuses on strategies to improve siRNA cancer treatments and discusses future prospects in this important field.

Mycotoxin contamination of food and feed is a worldwide problem that needs to be addressed with highly efficient and biologically safe techniques. RNA interference (RNAi) is a natural mechanism playing an important role in different processes in eukaryotes, including the regulation of gene expression, maintenance of genome stability, protection against viruses and others. Recently, RNAi-based techniques have been widely applied for the purposes of food safety and management of plant diseases, including those caused by mycotoxin-producing fungi. In this review, we summarize the current state-of-the-art RNAi-based approaches for reducing the aggressiveness of key toxigenic fungal pathogens and mycotoxin contamination of grain and its products. The ways of improving RNAi efficiency for plant protection and future perspectives of this technique, including progress in methods of double-stranded RNA production and its delivery to the target cells, are also discussed.

Here, we present a protocol for small interfering RNA (siRNA)-mediated U1 small nuclear RNA (snRNA) knockdown using fluorinated α-helical polypeptide in macrophages and mouse lungs, providing a dependable approach to silence U1 snRNA in vitro and in vivo. We describe steps for preparing P7F7/siRNA polyplexes and silencing U1 snRNA with P7F7/siRNA polyplexes in macrophages and mouse lungs. Knockdown efficiency is validated through reverse-transcription quantitative real-time PCR analysis. This protocol is applicable for studying the physiological or pathophysiological function of U1 snRNA. For complete details on the use and execution of this protocol, please refer to Zhang et al.1.

The on-demand gene regulation is crucial for extensively exploring specific gene functions and developing personalized gene therapeutics, which shows great promise in precision medicines. Although some nucleic acid-based gene regulatory tools (antisense oligonucleotides and small interfering RNAs) are devised for achieving on-demand activation, the introduction of chemical modifications may cause undesired side effects, thereby impairing the gene regulatory efficacy. Herein, a methyl-engineered DNAzyme (MeDz) is developed for the visualization of endogenous alkyltransferase (AGT) and the simultaneous self-sufficiently on-demand gene regulation. The catalytic activity of DNAzyme can be efficiently blocked by O6-methylguanine (O6MeG) modification and specifically restored via the AGT-mediated DNA-repairing pathway. This simply designed MeDz is demonstrated to reveal AGT of varying expression levels in different cells, opening the possibility to explore the AGT-related biological processes. Moreover, the AGT-guided MeDz exhibits cell-selective regulation on the human early growth response-1 (EGR-1) gene, with efficient gene repression in breast cancer cells and low effectiveness in normal cells. The proposed MeDz offers an attractive strategy for on-demand gene regulation, displaying great potential in biomedical applications.

The clustered regularly interspaced short palindromic repeats (CRISPR)-Cas13d system was adapted as a powerful tool for targeting viral RNA sequences, making it a promising approach for antiviral strategies. Understanding the influence of template RNA structure on Cas13d binding and cleavage efficiency is crucial for optimizing its therapeutic potential. In this study, we investigated the effect of local RNA secondary structure on Cas13d activity. To do so, we varied the stability of a hairpin structure containing the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) target sequence, allowing us to determine the threshold RNA stability at which Cas13d activity is affected. Our results demonstrate that Cas13d possesses the ability to effectively bind and cleave highly stable RNA structures. Notably, we only observed a decrease in Cas13d activity in the case of exceptionally stable RNA hairpins with completely base-paired stems, which are rarely encountered in natural RNA molecules. A comparison of Cas13d and RNA interference (RNAi)-mediated cleavage of the same RNA targets demonstrated that RNAi is more sensitive for local target RNA structures than Cas13d. These results underscore the suitability of the CRISPR-Cas13d system for targeting viruses with highly structured RNA genomes.

Gene silencing is crucial in crop breeding for desired trait development. RNA interference (RNAi) has been used widely but is limited by ectopic expression of transgenes and genetic instability. Introducing an upstream start codon (uATG) into the 5'untranslated region (5'UTR) of a target gene may 'silence' the target gene by inhibiting protein translation from the primary start codon (pATG). Here, we report an efficient gene silencing method by introducing a tailor-designed uATG-containing element (ATGE) into the 5'UTR of genes in plants, occupying the original start site to act as a new pATG. Using base editing to introduce new uATGs failed to silence two of the tested three rice genes, indicating complex regulatory mechanisms. Precisely inserting an ATGE adjacent to pATG achieved significant target protein downregulation. Through extensive optimization, we demonstrated this strategy substantially and consistently downregulated target protein expression. By designing a bidirectional multifunctional ATGE4, we enabled tunable knockdown from 19% to 89% and observed expected phenotypes. Introducing ATGE into Waxy, which regulates starch synthesis, generated grains with lower amylose, revealing the value for crop breeding. Together, we have developed a programmable and robust method to knock down gene expression in plants, with potential for biological mechanism exploration and crop enhancement.

The E6 protein is a known oncogene in cervical cancer and plays a key role in the development and progression of cervical cancer by reducing the expression level of the tumor suppressor protein P53 and ultimately leading to enhanced cell proliferation and reduced apoptosis. Therefore, antiviral agents that inhibit the expression of E6 oncoprotein are expected to be potential therapies for human cervical cancer. Here we developed CRISPR/Cas13a: crRNA dual plasmid system and demonstrated that CRISPR/Cas13a could effectively and specifically knock down human papillomavirus 18 E6 mRNA, downregulate the expression level of E6 protein, and restore the expression of the tumor suppressor gene P53 protein, thereby inhibiting the growth of cervical cancer cells and increasing their apoptosis, the E6-2, E6-3, and E6-5 groups resulted in apoptosis rates of 25.4%, 22.4%, and 22.2% in HeLa cells. Moreover, CRISPR/Cas13a enhances the proliferation inhibition and apoptosis induction of cisplatin in cervical cancer HeLa cells. The CRISPR/Cas13a system targeting HPV E6 mRNA may be a promising therapeutic approach for the treatment of human papillomavirus-associated cervical cancer.

Colorectal cancer (CRC) ranks as the third most common cancer and the second leading cause of cancer-related deaths. According to clinical diagnosis and treatment, liver metastasis occurs in approximately 50 % of CRC patients, indicating a poor prognosis. The unique immune tolerance of the liver fosters an immunosuppressive tumor microenvironment (TME). In the context of tumors, numerous membrane and secreted proteins have been linked to tumor immune evasion as immunomodulatory molecules, but much remains unknown about how these proteins contribute to immune evasion in colorectal cancer liver metastasis (CRLM). This article reviews recently discovered membrane and secreted proteins with roles as both immunostimulatory and immunosuppressive molecules within the TME that influence immune evasion in CRC primary and metastatic lesions, particularly their mechanisms in promoting CRLM. This article also addresses screening strategies for identifying proteins involved in immune evasion in CRLM and provides insights into potential protein targets for treating CRLM.

The GPS-Nanoconveyor (MA-NV@DOX-Cas13a) is a targeted nanoplatform designed for the imaging and gene/chemotherapy synergistic treatment of melanoma. It utilizes rolling circle amplification (RCA) products as a scaffold to construct a DNA "Nanoconveyor" (NV), which incorporates a multivalent aptamer (MA) as a "GPS", encapsulates doxorubicin (DOX) in the transporter, and equips it with CRISPR/Cas13a ribonucleoproteins (Cas13a RNP). Carrying MA enhances the ability to recognize the overexpressed receptor nucleolin on B16 cells, enabling targeted imaging and precise delivery of MA-NV@DOX-Cas13a through receptor-mediated endocytosis. The activation of signal transducer and activator of transcription 3 (STAT3) in cancer cells triggers cis-cleavage of CRISPR/Cas13a, initiating its trans-cleavage function. Additionally, deoxyribonuclease I (DNase I) degrades MA-NV, releasing DOX for intracellular imaging and as a chemotherapeutic agent. Experiments demonstrate the superior capabilities of this versatile nanoplatform for cellular imaging and co-treatment while highlighting the advantages of these nanodrug delivery systems in mitigating DOX side effects.

The increasing emergence and re-emergence of RNA virus outbreaks underlines the urgent need to develop effective antivirals. RNA interference (RNAi) is a sequence-specific gene silencing mechanism that is triggered by small interfering RNAs (siRNAs) or short hairpin RNAs (shRNAs), which exhibits significant promise for antiviral therapy. AGO2-dependent shRNA (agshRNA) generates a single-stranded guide RNA and presents significant advantages over traditional siRNA and shRNA. In this study, we applied a logistic regression algorithm to a previously published chemically siRNA efficacy dataset and built a machine learning-based model with high predictive power. Using this model, we designed siRNA sequences targeting diverse RNA viruses, including human enterovirus A71 (EV71), Zika virus (ZIKV), dengue virus 2 (DENV2), mouse hepatitis virus (MHV) and severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), and transformed them into agshRNAs. We validated the performance of our agshRNA design by evaluating antiviral efficacies of agshRNAs in cells infected with different viruses. Using the agshRNA targeting EV71 as an example, we showed that the anti-EV71 effect of agshRNA was more potent compared with the corresponding siRNA and shRNA. Moreover, the antiviral effect of agshRNA is dependent on AGO2-processed guide RNA, which can load into the RNA-induced silencing complex (RISC). We also confirmed the antiviral effect of agshRNA in vivo. Together, this work develops a novel antiviral strategy that combines machine learning-based algorithm with agshRNA design to custom design antiviral agshRNAs with high efficiency.