A large body of evidence has highlighted the role of non-coding RNAs in neurodevelopment and neuroinflammation. This evidence has led to increasing speculation that non-coding RNAs may be involved in the pathophysiological mechanisms underlying hydrocephalus, one of the most common neurological conditions worldwide. In this review, we first outline the basic concepts and incidence of hydrocephalus along with the limitations of existing treatments for this condition. Then, we outline the definition, classification, and biological role of non-coding RNAs. Subsequently, we analyze the roles of non-coding RNAs in the formation of hydrocephalus in detail. Specifically, we have focused on the potential significance of non-coding RNAs in the pathophysiology of hydrocephalus, including glymphatic pathways, neuroinflammatory processes, and neurological dysplasia, on the basis of the existing evidence. Lastly, we review the potential of non-coding RNAs as biomarkers of hydrocephalus and for the creation of innovative treatments.

The recent approval of the first gene editing therapy for sickle cell disease and transfusion-dependent beta-thalassemia by the U.S. Food and Drug Administration (FDA) demonstrates the immense potential of CRISPR (clustered regularly interspaced short palindromic repeats) technologies to treat patients with genetic disorders that were previously considered incurable. While significant advancements have been made with ex vivo gene editing approaches, the development of in vivo CRISPR/Cas gene editing therapies has not progressed as rapidly due to significant challenges in achieving highly efficient and specific in vivo delivery. Adeno-associated viral (AAV) vectors have shown great promise in clinical trials as vehicles for delivering therapeutic transgenes and other cargos but currently face multiple limitations for effective delivery of gene editing machineries. This review elucidates these challenges and highlights the latest engineering strategies aimed at improving the efficiency, specificity, and safety profiles of AAV-packaged CRISPR/Cas systems (AAV-CRISPR) to enhance their clinical utility.

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.

A novel Cas13d-based gene-editing approach has been developed to target viral RNAs in infected cells, reducing the replication of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and Zika virus (ZIKV) by up to 90% compared with controls. Despite its potential, the use of Cas13d as an antiviral faces several challenges that limit its effectiveness before reaching target cells. This study presents a proof-of-concept strategy for constructing Cas13d with gold nanoparticles (Au_NPs) to destroy SARS-CoV-2 and ZIKV genomes into cells. The Au_NPs Cas13d complexes were administered to Huh-7 cells infected with either virus, in single or multiple doses. The study demonstrated that Au_NPs Cas13d cuts target RNAs with comparable efficiency as lipofected ribonucleoprotein (RNP). Additionally, we found that Au_NPs Cas13d can spontaneously enter cells by endocytosis or diffusion, before the first 4 h of treatment. Au_NPs Cas13d co-localized with SARS-CoV-2 virions in early endosomes and reduced SARS-CoV-2 replication after a single administration, unlike RNPs, which showed no antiviral activity. However, Au_NPs Cas13d was less efficient at reducing ZIKV replication compared with lipofected Cas13d-RNPs, likely due to different intracellular localization. These results suggest that Au_NPs can be adapted as a new antiviral strategy, highlighting an innovative delivery method of Cas13d against viruses without the need for transfecting, providing a new gene-editing-based approach against emerging RNA viruses.

Nonsense mutations in the CFTR gene are responsible for approximately 8 % of cystic fibrosis (CF) cases worldwide. The consequent premature termination of translation leads to the production of a truncated and non-functional CFTR protein. Despite the intensive research in the field, these patients cannot benefit from specific and approved therapies yet. To address this issue, in this study we evaluated a potential therapeutic strategy to overcome the nonsense G542X (UGG > UGA) mutation in the CFF-16HBEge human bronchial epithelial cells by restoring the full-length CFTR protein.

We applied the RESTORE (Recruiting endogenous ADAR to specific transcripts for oligonucleotide-mediated RNA editing) approach, based on specifically designed antisense RNA oligonucleotides (ASOs) to recruit endogenous ADAR (adenosine deaminase acting on RNA) enzymes. The ADAR's recruitment to the target CFTR mRNA is expected to promote the deamination of adenosine (A) into inosine (I) within the premature termination codon (UGA). As the ribosome reads the inosine as guanosine (G), the stop codon could be recoded as a tryptophan (UGG), thereby allowing the synthesis of a full-length CFTR protein, albeit with a different amino acid.

Our results indicate that in the CFF-16HBEge G542X cell line, the transfection of a specific ASO allows the rescue of the CFTR transcript and protein expression, compared to the untransfected mutated cells. Next generation sequencing of CFTR cDNA also confirmed the occurrence of the expected RNA editing outcome.

The obtained results suggest that the RESTORE approach might be explored as a promising strategy to treating nonsense mutations in CFTR, potentially contributing to novel therapeutic options for CF patients.

MicroRNAs (miRNAs) are short non‑coding RNAs, which perform a key role in cellular differentiation and development. Most human diseases, particularly cancer, are linked to miRNA functional dysregulation implicated in the expression of tumor‑suppressive or oncogenic targets. Cancer hallmarks such as continued proliferative signaling, dodging growth suppressors, invasion and metastasis, triggering angiogenesis, and avoiding cell death have all been demonstrated to be affected by dysregulated miRNAs. Thus, for the treatment of different cancer types, the detection and quantification of this type of RNA is significant. The classical and current methods of RNA detection, including northern blotting, reverse transcription‑quantitative PCR, rolling circle amplification and next‑generation sequencing, may be effective but differ in efficiency and accuracy. Furthermore, these approaches are expensive, and require special instrumentation and expertise. Thus, researchers are constantly looking for more innovative approaches for miRNA detection, which can be advantageous in all aspects. In this regard, an RNA manipulation tool known as the CRISPR and CRISPR‑associated sequence 13 (CRISPR/Cas13) system has been found to be more advantageous in miRNA detection. The Cas13‑based miRNA detection approach is cost effective and requires no special instrumentation or expertise. However, more research and validation are required to confirm the growing body of CRISPR/Cas13‑based research that has identified miRNAs as possible cancer biomarkers for diagnosis and prognosis, and as targets for treatment. In the present review, current updates regarding miRNA biogenesis, structural and functional aspects, and miRNA dysregulation during cancer are described. In addition, novel approaches using the CRISPR/Cas13 system as a next‑generation tool for miRNA detection are discussed. Furthermore, challenges and prospects of CRISPR/Cas13‑based miRNA detection approaches are described.

Human induced pluripotent stem cell (hiPSC)-based disease modelling has significantly advanced the field of cardiogenetics, providing a precise, patient-specific platform for studying genetic causes of heart diseases. Coupled with genome editing technologies such as CRISPR/Cas, hiPSC-based models not only allow the creation of isogenic lines to study mutation-specific cardiac phenotypes, but also enable the targeted modulation of gene expression to explore the effects of genetic and epigenetic deficits at the cellular and molecular level. hiPSC-based models of heart disease range from two-dimensional cultures of hiPSC-derived cardiovascular cell types, such as various cardiomyocyte subtypes, endothelial cells, pericytes, vascular smooth muscle cells, cardiac fibroblasts, immune cells, etc., to cardiac tissue cultures including organoids, microtissues, engineered heart tissues, and microphysiological systems. These models are further enhanced by multi-omics approaches, integrating genomic, transcriptomic, epigenomic, proteomic, and metabolomic data to provide a comprehensive view of disease mechanisms. In particular, advances in cardiovascular tissue engineering enable the development of more physiologically relevant systems that recapitulate native heart architecture and function, allowing for more accurate modelling of cardiac disease, drug screening, and toxicity testing, with the overall goal of personalised medical approaches, where therapies can be tailored to individual genetic profiles. Despite significant progress, challenges remain in the maturation of hiPSC-derived cardiomyocytes and the complexity of reproducing adult heart conditions. Here, we provide a concise update on the most advanced methods of hiPSC-based disease modelling in cardiogenetics, with a focus on genome editing and cardiac tissue engineering.

As newer CRISPR variants have emerged and corresponding toolkits have been developed, researchers can now readily target multiple genes simultaneously for knockout, activation, or repression alongside being able to bind or cleave mRNA. However, as larger multitargeting crRNA arrays are required for these experiments, the design process becomes more complicated, taking more time and increasing risks of errors being introduced. The Array Assembler seeks to address the critical bottleneck that emerges during longer crRNA array design by providing a highly user-friendly tool to process input crRNA spacer sequences into the oligos required for efficient assembly of the corresponding crRNA array. By enabling rapid and reliable design of oligos for efficient assembly of crRNA arrays from a user-defined list of crRNA spacer sequences this tool should prove useful for a wide range of laboratories employing genomic perturbations.

Homeostatic synaptic plasticity is a negative feedback mechanism through which neurons modify their synaptic strength to counteract chronic increases or decreases in activity. In response to activity deprivation, synaptic strength is enhanced by increasing the number of AMPA receptors (AMPARs), particularly Ca2+-permeable AMPARs, at the synapse. Here, we found that this increase in Ca2+-permeable AMPARs during homeostatic upscaling was mediated by decreased posttranscriptional editing of GRIA2 mRNA encoding the AMPAR subunit GluA2. In cultured neurons, activity deprivation resulted in increases in the amount of unedited GluA2, such that its ion channel pore contains a glutamine (Q) codon instead of arginine (R), and in the number of Ca2+-permeable AMPARs at the synapse. These effects were mediated by a splicing factor-dependent decrease in ADAR2 abundance and activity in the nucleus. Overexpression of ADAR2 or CRISPR-Cas13-directed editing of GluA2 transcripts blocked homeostatic upscaling in activity-deprived primary neurons. In mice, dark rearing resulted in decreased Q-to-R editing of GluA2-encoding transcripts in the primary visual cortex (V1), and viral overexpression of ADAR2 in the V1 blocked the induction of homeostatic synaptic plasticity. The findings indicate that activity-dependent regulation of GluA2 editing contributes to homeostatic synaptic plasticity.

Metastatic cancer, the primary cause of cancer mortality, frequently exhibits heightened dependence on certain transcription factors (TFs), which serve as master regulators of oncogenic signaling yet are often untargetable by small molecules. Selective Cell in ORgan Targeting (SCORT) nanoparticles are developed for precise CRISPR/Cas13d mRNA and gRNA delivery to metastatic cancer cells in vivo, aiming to knock down the undruggable oncogenic TF HoxB13. In prostate cancer liver metastasis models driven by HoxB13, repeated systemic SCORT-Cas13d-gHoxB13 treatment significantly decreases HoxB13 expression, reduces metastasis, and extends mouse survival. Prolonged treatment shows no significant impact on major organ function, histology or immune markers. Mechanistically, SCORT-Cas13d-gHoxB13 treatment suppresses metastatic tumor proliferation and angiogenesis while promoting apoptosis by regulating multiple gene pathways. Unexpectedly, it inhibits the non-canonical, EMT-independent oncogenic function of Snail. These findings suggest that SCORT-Cas13d-gHoxB13 can effectively and safely target the undruggable HoxB13 in metastatic prostate cancer, positioning CRISPR/Cas13d as a potential treatment.

Gene therapy stands at the forefront of medical innovation, offering unique potential to treat the underlying causes of genetic disorders and broadly enable regenerative medicine. However, unregulated production of therapeutic genes can lead to decreased clinical utility due to various complications. Thus, many technologies for controlled gene expression are under development, including regulated transgenes, modulation of endogenous genes to leverage native biological regulation, mapping and repurposing of transcriptional regulatory networks, and engineered systems that dynamically react to cell state changes. Transformative therapies enabled by advances in tissue-specific promoters, inducible systems, and targeted delivery have already entered clinical testing and demonstrated significantly improved specificity and efficacy. This review highlights next-generation technologies under development to expand the reach of gene therapies by enabling precise modulation of gene expression. These technologies, including epigenome editing, antisense oligonucleotides, RNA editing, transcription factor-mediated reprogramming, and synthetic genetic circuits, have the potential to provide powerful control over cellular functions. Despite these remarkable achievements, challenges remain in optimizing delivery, minimizing off-target effects, and addressing regulatory hurdles. However, the ongoing integration of biological insights with engineering innovations promises to expand the potential for gene therapy, offering hope for treating not only rare genetic disorders but also complex multifactorial diseases.

Pairs of split protein segments can modify a variety of target proteins in a living cell.

Synthetic genetic circuits program the cellular input-output relationships to execute customized functions. However, efforts to scale up these circuits have been hampered by the limited number of reliable regulatory mechanisms with high programmability, performance, predictability and orthogonality. Here we report a class of split-intron-enabled trans-splicing riboregulators (SENTRs) based on de novo designed external guide sequences. SENTR libraries provide low leakage expression, wide dynamic range, high predictability with machine learning and low crosstalk at multiple component levels. SENTRs can sense RNA targets, process signals by logic computation and transduce them into various outputs, either mRNAs or noncoding RNAs. We subsequently demonstrate that digital logic operation with up to six inputs can be implemented using multiple orthogonal SENTRs to regulate a single gene simultaneously and coupling SENTRs with split intein-mediated protein trans-splicing. SENTR represents a powerful and versatile regulatory tool at the post-transcriptional level in Escherichia coli, suggesting broad biotechnological applications.

Clustered regularly interspaced short palindromic repeats (CRISPR) tools are revolutionizing the establishment of genotype-phenotype relationships and are transforming cell- and gene-based therapies. In the field of oncology, CRISPR/CRISPR-associated protein 9 (Cas9), Cas12, and Cas13 have advanced the generation of cancer models, the study of tumor evolution, the identification of target genes involved in cancer growth, and the discovery of genes involved in chemosensitivity and resistance. Moreover, preclinical therapeutic strategies employing CRISPR/Cas have emerged. These include the generation of chimeric antigen receptor T (CAR-T) cells and engineered immune cells, and the use of precision anticancer gene-editing agents to inactivate driver oncogenes, suppress tumor support genes, and cull cancer cells in response to genetic circuit output. This review summarizes the collective impact that CRISPR technology has had on basic and applied cancer research, and highlights the promises and challenges facing its clinical translation.

Amyotrophic lateral sclerosis (ALS) is a neurodegenerative disease characterized by the selective death of motor neurons, which causes muscle atrophy. Genetic forms of ALS are recorded only in 10% of cases. However, over the past decade, studies in genetics have substantially contributed to our understanding of the molecular mechanisms underlying ALS. The identification of key mutations such as SOD1, C9orf72, FUS, and TARDBP has led to the development of targeted therapy that is gradually being introduced into clinical trials, opening up a broad range of opportunities for correcting these mutations. In this review, we aimed to present an extensive overview of the currently known mechanisms of motor neuron degeneration associated with mutations in these genes and also the gene therapy methods for inhibiting the expression of their mutant proteins. Among these, antisense oligonucleotides, RNA interference (siRNA and miRNA), and gene-editing (CRISPR/Cas9) methods are of particular interest. Each has shown its efficacy in animal models when targeting mutant genes, whereas some of them have proven to be efficient in human clinical trials.

Prompt and accurate diagnosis of infectious pathogens of livestock origin is of utmost importance for epidemiological surveillance and effective therapeutic strategy formulation. Among various methods, nucleic acid-based detection of pathogens is the most sensitive and specific; but the majority of these assays need expensive equipment and skilled workers. Due to the rapid advancement of clustered regularly interspaced short palindromic repeats-CRISPR-associated protein (CRISPR-Cas)-based nucleic acid detection methods, these are now being widely used for pathogen detection. CRISPR-Cas is a bacterial counterpart of "adaptive immunity", generally used for editing genome. Many CRISPR systems have been modified for nucleic acid detection due to their excellent selectivity in detecting DNA and RNA sequences. The combination of CRISPR with suitable isothermal amplification technologies has made it more sensitive, specific, versatile, and reproducible for the detection of pathogen nucleic acids at the point of care. Amplification of pathogen nucleic acid by isothermal amplification followed by CRISPR-Cas-based detection has several advantages, including short sample-to-answer times and no requirement for laboratory set-up. They are also significantly less expensive than the existing nucleic acid detection methods. This review focuses on the recent trends in the use of this precision diagnostic method for diagnosis of a wide range of animal pathogens with or without zoonotic potential, particularly various isothermal amplification strategies, and visualization methods for sensing bacteria, viruses, and parasites of veterinary and public health importance.

The Xenopus tropicalis (Western clawed frog) is an important amphibian model for genetics, developmental and regenerative biology, due to its diploid genetic background and short generation time. CRISPR-Cas13 and CRISPR interference (CRISPRi) systems have recently been employed to suppress mRNA expression in many organisms such as yeast, plants, and mammalian cells. However, no systematic study of these two systems has been carried out in Xenopus tropicalis. Here, we show that CRISPRi rather than CRISPR-Cas13 is an effective and suitable approach to suppress specific mRNA transcription in Xenopus tropicalis embryos. We demonstrated that CRISPRi composed of dCas9 and KRAB-MeCP2 (dCas9-KM) can efficiently target exogenous and endogenous transcripts in Xenopus tropicalis embryos. Moreover, our data suggest that the new KRAB domain from ZIM3 protein (ZIM3-KRAB, ZIM3K) alone has a comparable transcript targeting capacity in Xenopus tropicalis embryos to the traditional fusion repressor KRAB-MeCP2 in which the KRAB domain from KOX1 protein. In conclusion, our results demonstrate that CRISPRi rather than CRISPR-Cas13 is an efficient knockdown platform to explore specific gene function in Xenopus tropicalis embryos.

Esophageal cancer is an aggressively malignant neoplasm characterized by a high mortality rate. Frequently diagnosed at an advanced stage, it presents challenges for optimal therapeutic intervention due to its non-specific symptoms, resulting in lost opportunities for effective treatment, such as surgery, radiotherapy, chemotherapy and target therapy. The N6-methyladenosine (m6A) modification represents the most critical post-transcriptional modification of eukaryotic messenger RNA (mRNA). The reversible m6A modification is mediated by three regulatory factors: m6A methyltransferases, demethylating enzymes, and m6A recognition proteins. These components identify and bind to specific RNA methylation sites, thereby modulating essential biological functions such as RNA processing, nuclear export, stability, translation and degradation, which significantly influence tumorigenesis, invasion, and metastasis. Given the importance of m6A modification, this paper offers a comprehensive examination of the regulatory mechanisms, biological functions, and future therapeutic implications of m6A RNA methylation in the context of esophageal cancer.

As members of the α-proteobacteria group, Caulobacter crescentus and its relatives are wildly studied for their unique asymmetric life cycle and versatile applications in industry, agriculture, and biomedicine. However, genetic manipulation in these bacteria remains challenging, typically requiring time-consuming and labor-intensive procedures. Here, we report a practical CRISPR-SpCas9M-reporting system that overcomes the limitations of SpCas9 expression and CRISPR escape, enabling efficient, markerless, and rapid genome editing in C. crescentus. Two genes encoding for a pair of scaffold proteins were knocked out individually or iteratively, demonstrating their direct involvements in cellular signaling asymmetry. Key components, including the Cas protein, Cas inducer, sgRNA, homologous arms, and reporter, were systematically analyzed and optimized in the system, finally achieving the apparent editing efficiency up to 80% in C. crescentus. Furthermore, we applied the CRISPR-SpCas9M-reporting system to two C. crescentus relatives, Agrobacterium fabrum and Sinorhizobium meliloti, establishing it as an efficient and general editing strategy. We anticipate that this system could be applied to other CRISPR-Cas-recalcitrant organisms, accelerating both basic and applied research in α-proteobacteria.

Sclerotinia sclerotiorum is a globally widespread and vast destructive plant pathogenic fungus that causes significant yield losses in crops. Due to the lack of effective resistant germplasm resources, the control of diseases caused by S. sclerotiorum largely relies on chemical fungicides. However, excessive use of these chemicals not only causes environmental concerns but also leads to the increased development of resistance in S. sclerotiorum. In contrast, trans-kingdom sRNA silencing-based technologies, such as host-induced gene silencing (HIGS) and spray-induced gene silencing (SIGS), offer novel, effective, and environmentally friendly methods for the management of S. sclerotiorum infection. This review summarizes recent advances in the identification of S. sclerotiorum pathogenic genes, target gene selection, categories, and application of trans-kingdom RNA interference (RNAi) technologies targeting this pathogen. Although some challenges, including off-target effects and the efficiency of external sRNA uptake, exist, recent findings have proposed solutions for further improvement. Combined with the latest developments in CRISPR/Cas gene editing and other technologies, trans-kingdom RNAi has significant potential to become a crucial tool in the control of sclerotinia stem rot (SSR), mitigating the impact of S. sclerotiorum on crop production.

RNA-protein interactions are fundamental to a wide range of biological processes, and understanding these interactions in their native cellular context is both vital and challenging. Traditional methods for studying RNA-protein interactions rely on crosslinking, which can introduce artifacts. Recently, proximity labeling-based techniques have emerged as powerful alternatives, offering a crosslinking-free approach to investigate these interactions. This review highlights recent advancements in the development and application of proximity labeling methods, focusing on both RNA-centric and protein-centric strategies for profiling cellular RNA-protein interactions. By examining these innovative approaches, we aim to provide insights into their potential for enhancing our understanding of RNA-protein dynamics in various biological settings.

Metabolic engineering has a wide range of applications, spanning key sectors such as energy, pharmaceuticals, agriculture, chemicals, and environmental sustainability. Its core focus is on precisely modulating metabolic pathways to achieve efficient, sustainable, and environmentally friendly biomanufacturing processes, offering new possibilities for societal sustainable development. Gene attenuation is a critical technique within metabolic engineering, pivotal in optimizing metabolic fluxes and improving target metabolite yields. This review article discusses gene attenuation mechanisms, the applications across various biological systems, and implementation strategies. Additionally, we address potential future challenges and explore its potential to drive further advancements in the field.

The CRISPR system has transformed many research areas, including cancer and immunology, by providing a simple yet effective genome editing system. Its simplicity has facilitated large-scale experiments to assess gene functionality across diverse biological contexts, generating extensive datasets that boosted the development of computational methods and machine learning/artificial intelligence applications. Integrating CRISPR with single-cell technologies has further advanced our understanding of genome function and its role in many biological processes, providing unprecedented insights into human biology and disease mechanisms. This powerful combination has accelerated AI-driven analyses, enhancing disease diagnostics, risk prediction, and therapeutic innovations. This review provides a comprehensive overview of CRISPR-based genome editing systems, highlighting their advancements, current progress, challenges, and future opportunities, especially in cancer and immunology.

Molecular logic gates, as biomolecule-based computational systems, are highly suitable for multitarget detection due to their programmability and modularity. However, existing systems are primarily limited to nucleic acid detection and have not been widely applied to disease-related sensing, particularly for disease antigens. CD33 and CD123 are critical biomarkers for acute myeloid leukemia (AML), yet conventional detection methods rely on expensive equipment and complex procedures, limiting their accessibility and practicality. This study designs a DNA logic gate system integrating nucleic acid aptamers, catalytic hairpin assembly (CHA), and CRISPR-Cas12a, pioneering its use for AML antigen detection. The system comprises three modules: input recognition, signal amplification, and signal transduction. Nucleic acid aptamers specifically identify CD33 and CD123, while CHA enables efficient signal amplification and CRISPR-Cas12a generates a fluorescent output via trans-cleavage activity. The system operates stably at room temperature and implements multiple logic gate models, including YES, OR, AND, NOR, and INHIBIT, enabling the simultaneous detection of CD33 and CD123. Experimental results are visually distinguishable under blue light, and the system requires only standard fluorescence detection instruments. In serum samples, it exhibits excellent selectivity and stability, with a detection limit of 0.5 ng/mL. This study pioneers the application of logic gate technology for disease antigen detection, addressing a critical gap in AML biomarker sensing. Our study indicates that this logic detection platform, characterized by its simplicity in operation, high sensitivity, and versatility in logic functions, holds promise as a potent sensing system for the intelligent multiplex target detection of disease antigens, environmental pollutants, and heavy metals.

Recent advancements in molecular engineering have established RNA-based technologies as powerful tools for both fundamental research and translational applications. Among the various RNA-based technologies developed, RNA base editing has recently emerged as a groundbreaking advancement. It primarily involves the conversion of adenosine (A) to inosine (I) and cytidine (C) to uridine (U), which are mediated by ADAR and APOBEC enzymes, respectively. RNA base editing has been applied in both biological research and therapeutic contexts. It enables site-directed editing within target transcripts, offering reversible, dose-dependent effects, in contrast to the permanent or heritable changes associated with DNA base editing. Additionally, RNA editing-based profiling of RNA-binding protein (RBP) binding sites facilitates transcriptome-wide mapping of RBP-RNA interactions in specific tissues and at the single-cell level. Furthermore, RNA editing-based sensors have been utilized to express effector proteins in response to specific RNA species. As RNA base editing technologies continue to evolve, we anticipate that they will significantly drive advancements in RNA therapeutics, synthetic biology, and biological research.

CRISPR-associated endoribonucleases (Cas RNases) cleave single-stranded RNA in a highly sequence-specific manner by recognizing and binding to short RNA sequences known as direct repeats (DRs). Here, we investigate the potential of exploiting Cas RNases for the regulation of target genes with one or more DRs introduced into the 3' untranslated region, an approach we refer to as DREDGE (direct repeat-enabled downregulation of gene expression). The DNase-dead version of Cas12a (dCas12a) was identified as the most efficient among five different Cas RNases tested and was subsequently evaluated in doxycycline-regulatable systems targeting either stably expressed fluorescent proteins or an endogenous gene. DREDGE performed superbly in stable cell lines, resulting in up to 90% downregulation with rapid onset, notably in a fully reversible and highly selective manner. Successful control of an endogenous gene with DREDGE was demonstrated in two formats, including one wherein both the DR and the transgene driving expression of dCas12a were introduced in one step by CRISPR-Cas. Our results establish DREDGE as an effective method for regulating gene expression in a targeted, highly selective, and fully reversible manner, with several advantages over existing technologies.

Adenosine deaminase acting on RNA (ADAR)-mediated RNA base editing offers a safer alternative to genome editing for specific clinical applications because of nonpermanent editing of targets. Current guide RNA (gRNA) designs feature a fully complementary specificity domain with an A-C mismatch at the targeted adenosine. However, perfectly matched dsRNA is not the most effective ADAR substrate. Here we introduce MIRROR (mimicking inverted repeats to recruit ADARs using engineered oligoribonucleotides), an approach that implements structural motifs derived from highly edited inverted Alu repeats in human tissues to enable rational gRNA design for ADAR recruitment. We demonstrated that MIRROR is applicable to both short chemically synthesized gRNAs with modifications and long biologically generated gRNAs and surpasses current state-of-the-art approaches in both gRNA forms. It enhances editing efficiency by up to 5.7-fold in multiple human cell types and primary hepatocytes from an alpha-1 antitrypsin deficiency mouse model. Our findings improve programmable RNA editing in vitro and in vivo by rational design through the screening of highly edited natural substrate mimics.

Despite ongoing efforts to study CRISPR systems, the evolutionary origins giving rise to reprogrammable RNA-guided mechanisms remain poorly understood. Here, we describe an integrated sequence/structure evolutionary tracing approach to identify the ancestors of the RNA-targeting CRISPR-Cas13 system. We find that Cas13 likely evolved from AbiF, which is encoded by an abortive infection-linked gene that is stably associated with a conserved non-coding RNA (ncRNA). We further characterize a miniature Cas13, classified here as Cas13e, which serves as an evolutionary intermediate between AbiF and other known Cas13s. Despite this relationship, we show that their functions substantially differ. Whereas Cas13e is an RNA-guided RNA-targeting system, AbiF is a toxin-antitoxin (TA) system with an RNA antitoxin. We solve the structure of AbiF using cryoelectron microscopy (cryo-EM), revealing basic structural alterations that set Cas13s apart from AbiF. Finally, we map the key structural changes that enabled a non-guided TA system to evolve into an RNA-guided CRISPR system.

Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein (CRISPR/Cas) technologies have evolved rapidly over the past decade with the continuous discovery of new Cas systems. In particular, RNA-targeting CRISPR-Cas13 proteins are promising single-effector systems to regulate target mRNAs without altering genomic DNA, yet the current Cas13 systems are restrained by suboptimal efficiencies. Here, we show that U1 promoter-driven CRISPR RNAs (crRNAs) increase the efficiency of various applications, including RNA knockdown and editing, without modifying the Cas13 protein effector. We confirm that U1-driven crRNAs are exported into the cytoplasm, while conventional U6 promoter-driven crRNAs are mostly confined to the nucleus. Furthermore, we reveal that the end positions of crRNAs expressed by the U1 promoter are consistent regardless of guide sequences and lengths. We also demonstrate that U1-driven crRNAs, but not U6-driven crRNAs, can efficiently repress the translation of target genes in combination with catalytically inactive Cas13 proteins. Finally, we show that U1-driven crRNAs can counteract the inhibitory effect of miRNAs. Our simple and effective engineering enables unprecedented cytosolic RNA-targeting applications.

On August 7-9, 2024, the second annual dengue "endgame" summit was held in Syracuse, NY, hosted by the Global Health Institute at SUNY Upstate Medical University. The meeting brought together attendees from around the world, with talks spanning healthcare, government control programs, basic research, and medical countermeasure development efforts. The summit goal was to work toward a better understanding of what dengue control could look like and the steps required to reach such a goal. The objectives of the meeting were to discuss the current global state of dengue, what dengue "control" might look like, and to discuss actionable pathways for achieving dengue control. Topics covered throughout the meeting included DENV immunity and pathogenesis, challenges in countermeasure development, innovative vector control strategies, dengue diagnostics, addressing challenges in science communication, and vaccine hesitancy. Several fundamental knowledge gaps were repeatedly highlighted by the summit attendees and were cited as critical barriers to the development, deployment, and evaluation of effective dengue countermeasures. These gaps include (1) the lack of a broadly applicable immunologic biomarker/correlate of DENV immunity and (2) the lack of universally accepted/applicable metrics for quantifying dengue severity in the setting of countermeasure evaluations. In addition, the lack of clear and consistent international leadership in the global dengue control effort was cited as a barrier to widespread and synergistic research and countermeasure development/deployment activities. Despite these persistent roadblocks, summit attendees expressed optimism that holistic and multi-tiered approaches-incorporating optimal use of existing and nascent countermeasure technologies deployed in collaboration with local communities-could be effective in progressing toward dengue control.

Recruiting the endogenous editing enzyme adenosine deaminase acting on RNA (ADAR) with tailored guide RNAs for adenosine-to-inosine (A-to-I) RNA base editing is promising for safely manipulating genetic information at the RNA level. However, the precision and efficiency of editing are often compromised by bystander off-target editing. Here, we find that in 5'-UAN triplets, which dominate bystander editing, G•U wobble base pairs effectively mitigate off-target events while maintaining high on-target efficiency. This strategy is universally applicable to existing A-to-I RNA base-editing systems and complements other suppression methods such as G•A mismatches and uridine (U) depletion. Combining wobble base pairing with a circularized format of the CLUSTER approach achieves highly precise and efficient editing (up to 87%) of a disease-relevant mutation in the Mecp2 transcript in cell culture. Virus-mediated delivery of the guide RNA alone realizes functional MeCP2 protein restoration in the central nervous system of a murine Rett syndrome model with editing yields of up to 19% and excellent bystander control in vivo.

Tunable and reversible regulation of exogenous and endogenous gene expression would be useful for improving the safety and efficacy of gene therapy. Current chemically inducible systems are limited by the rapid diffusion and extended metabolism of small molecules, and associated side effects. Here we develop a photoactivatable RNA adenosine base editor (PA-rABE) by harnessing a compact Cas13 variant and a split ADAR2 deaminase fused with the Magnets system, which is activated through blue-light-induced dimerization. PA-rABE achieves highly efficient editing on endogenous RNA with minimal bystander editing and off-target effects. By editing a phosphorylation site of the endogenous CTNNB1 gene, PA-rABE stabilizes the β-catenin protein and activates Wnt signaling in vivo. Using adeno-associated virus vectors to deliver PA-rABE along with an hF9 variant containing a premature termination codon, we show amelioration of clotting defects in hemophilia B mice upon illumination. In summary, PA-rABE offers a controlled RNA base-editing technology for diverse biomedical applications, enabling reversible and spatiotemporally specific modulation.

Aneuploidy is increasingly recognized globally as a common cause of miscarriage among expectant mothers. The existing prenatal screening techniques for detecting aneuploidy have several limitations. The ability to diagnose aneuploidy early in a non-invasive manner is not feasible with the current screening methods, as they may produce false positive or false negative results. Recently, the widely used gene editing tool CRISPR/Cas has shown great promise in diagnostics. This review summarizes the prenatal screening tests used in the first trimester to assess aneuploidy conditions. Additionally, we discuss the advantages and disadvantages of molecular diagnostic tests, including the benefits and challenges of CRISPR/Cas-based trisomy detection. Thus, the proposed prenatal screening using CRISPR/Cas could provide significant benefits to expectant mothers by potentially enabling the early diagnosis of trisomy, helping to prevent miscarriage and birth defects. Furthermore, it opens new avenues for research, allowing clinicians and researchers to develop, optimize, and implement CRISPR/Cas-based prenatal screening assays in the future.

RNA editing can be a promising therapeutic approach. However, ectopic expression of RNA editing enzymes has been shown to trigger off-target editing. Here we identified adenosine deaminase acting on RNA (ADAR) inhibitors (ADIs) that suppress the activity of the fused ADAR2 deamination domain (ADAR2DD). Using these specific ADIs, we develop an RNA transformer adenosine base editor (RtABE) with high specificity. Fusing ADI to ADAR2DD, RtABE remains inactive until it binds to its target site. After binding to the target site, ADI is cleaved from ADAR2DD, and RtABE becomes active. RtABE can induce efficient editing in broad sequence contexts, including UAN, AAN, CAN and GAN. Using an adeno-associated virus for delivery of RtABE enables therapeutic RNA correction and restoration of α-L-iduronidase activity in Hurler syndrome mice with no substantial off-target editing. RtABE is a specific and efficient RNA editing system with a broad scope that may be a better alternative to existing RNA editing tools.

The CRISPR-Cas12a system has been extensively utilized in nucleic acid detection owing to its remarkable sensitivity and specificity. Nonetheless, its strict dependency on the presence of a protospacer adjacent motif (PAM) within double-stranded DNA (dsDNA) introduces considerable limitations, thereby constraining its applicability, flexibility, and broader accessibility in molecular diagnostics. Here, we communicate a universal, robust, and high-fidelity method for a PAM-independent nucleic acid assay based on the CRISPR-Cas12a system, named TRACER (mutant target-recognized PAM-independent CRISPR-Cas12a enzyme reporting system). TRACER can effectively distinguish target nucleic acids at concentrations as low as 0.5 aM, thereby enabling it to identify the presence of a 0.1% single nucleotide variant (SNV)-included mutant-type gene in heterozygotes. Thus, TRACER exhibits comparable sensitivity, specificity, and accuracy to Sanger sequencing in analyzing the SNV-related clinical tumor samples. Overall, TRACER introduces a brand-new perspective for SNV assays by eliminating the dependency on PAM sites and significantly expands the application range of the CRISPR-Cas12a system, thus holding immense potential for clinical diagnostics, biomedical research, and drug discovery.

Anti-CRISPR proteins (Acrs) inhibit CRISPR-Cas immune defenses, with almost all known Acrs acting on the Cas nuclease-CRISPR (cr)RNA ribonucleoprotein (RNP) complex. Here, we show that AcrVIB1 from Riemerella anatipestifer, the only known Acr against Cas13b, principally acts upstream of RNP complex formation by promoting unproductive crRNA binding followed by crRNA degradation. AcrVIB1 tightly binds to Cas13b but not to the Cas13b-crRNA complex, resulting in enhanced rather than blocked crRNA binding. However, the more tightly bound crRNA does not undergo processing and fails to activate collateral RNA cleavage even with target RNA. The bound crRNA is also accessible to RNases, leading to crRNA turnover in vivo even in the presence of Cas13b. Finally, cryoelectron microscopy (cryo-EM) structures reveal that AcrVIB1 binds a helical domain of Cas13b responsible for securing the crRNA, keeping the domain untethered. These findings reveal an Acr that converts an effector nuclease into a crRNA sink to suppress CRISPR-Cas defense.

Nucleic acid therapeutics have become increasingly recognized in recent years for their capability to target both coding and non-coding sequences. Several types of nucleic acid modalities, including siRNA, mRNA, aptamer, along with antisense oligo, have been approved by regulatory bodies for therapeutic use. The field of nucleic acid therapeutics has been brought to the forefront by the rapid development of vaccines against COVID-19, followed by a number of approvals for clinical use including much anticipated CRISPR-Cas9. However, obstacles such as the difficulty of achieving efficient and targeted delivery to diseased sites remain. This review provides an overview of nucleic acid therapeutics and highlights substantial advancements, including critical engineering, conjugation, and delivery strategies, that are paving the way for their growing role in modern medicine.

Viruses have evolved unique strategies to circumvent host control of protein synthesis and enable viral protein synthesis in the face of the host response. Defining the factors that regulate viral messenger RNA (mRNA) translation is thus critical to understand how viruses replicate and cause disease. To identify factors that might regulate viral mRNA translation, we developed a technique for identifying proteins associated with a native RNA expressed from its endogenous promoter and genomic locus. This approach uses a guide RNA to target dCas13b fused to a biotin ligase domain to a specific RNA, where it covalently labels proteins in close proximity. Using this approach, we identified multiple proteins associated with transcripts encoding the human cytomegalovirus (HCMV) IE1 and IE2 proteins and found that several associated proteins positively or negatively regulate HCMV replication. We confirmed that one such protein, the cellular Y-box binding protein 1 (YBX1), binds to HCMV immediate early mRNAs and is required for efficient viral protein expression and virus replication. Ablating YBX1 expression reduced the association of HCMV immediate early mRNAs with polysomes, demonstrating a role for YBX1 as a positive regulator of viral RNA translation. These results provide a powerful tool for unraveling RNA-protein interactions that can be used in a wide range of biological processes and reveal a role for YBX1 as a critical regulator of HCMV immediate early gene expression.