Complex genetic architecture is the major cause of heterogeneity in epilepsy, which poses challenges for accurate diagnosis and precise treatment. A large number of epilepsy candidate genes have been identified from clinical studies, particularly with the widespread use of next-generation sequencing. Validating these candidate genes is emerging as a valuable yet challenging task. Drosophila serves as an ideal animal model for validating candidate genes associated with neurogenetic disorders such as epilepsy, due to its rapid reproduction rate, powerful genetic tools, and efficient use of ethological and electrophysiological assays. Here, we systematically summarize the advantageous techniques of the Drosophila model used to investigate epilepsy genes, including genetic tools for manipulating target gene expression, ethological assays for seizure-like behaviors, electrophysiological techniques, and functional imaging for recording neural activity. We then introduce several typical strategies for identifying epilepsy genes and provide new insights into gene‒gene interactions in epilepsy with polygenic causes. We summarize well-established precision medicine strategies for epilepsy and discuss prospective treatment options, including drug therapy and gene therapy for genetic epilepsy based on the Drosophila model. Finally, we also address genetic counseling and assisted reproductive technology as potential approaches for the prevention of genetic epilepsy.

Maize (Zea mays L.) is a widely grown food crop around the world. Drought stress seriously affects the growth and development process of plants and causes serious damage to maize yield. In the early stage, our research group conducted transcriptome sequencing analysis on the drought-resistant maize inbred line H8186 and screened out a gene with significantly down-regulated expression, Phylloplanin-like (ZmPL1). The ZmPL1 gee expression pattern was analyzed under various abiotic stresses, and the results showed that this gene was greatly affected by drought stress. Subcellular localization analysis showed that the protein was localized on the cell membrane. In order to verify the role of ZmPL1 in drought stress, we overexpressed ZmPL1 in yeast and found that the expression of ZmPL1 could significantly increase the drought sensitivity of yeast. Next, ZmPL1 transgenic plants were obtained by infecting maize callus using Agrobacterium-mediated method. Under drought stress, compared with overexpression lines, gene-edited lines had higher germination rate and seedling survival rate, lower accumulation of MDA, relative conductivity and ROS, higher antioxidant enzyme activity, and the expression levels of stress-related genes and ROS scavenging-related genes were significantly increased. Exogenous application of ABA to each lines under drought stress attenuated the damage caused by drought stress on ZmPL overexpressing plants. In summary, ZmPL1 negatively regulates drought tolerance in maize.

Apoptosis is vital for improving the efficacy of lung cancer (LC) immunotherapy by targeting cancer cell elimination. Despite its importance, there is a lack of comprehensive bibliometric studies analyzing global research on apoptosis in LC immunotherapy. This analysis aims to address this gap by highlighting key trends, contributors, and future directions. A total of 969 publications from 1996 to 2024 were extracted from the Web of Science Core Collection. Analysis was conducted using VOSviewer, CiteSpace, and the R package 'bibliometrix.' The study included contributions from 6,894 researchers across 1,469 institutions in 61 countries, with research published in 356 journals. The volume of publications has steadily increased, led by China and the United States, with Sichuan University as the top contributor. The journal Cancers published the most articles, while Cancer Research had the highest co-citations. Yu-Quan Wei was the leading author, and Jemal, A. was the most frequently co-cited. Key research themes include "cell death mechanisms," "immune regulation," "combination therapies," "gene and nanomedicine applications," and "traditional Chinese medicine (TCM)." Future research is likely to focus on "coordinated regulation of multiple cell death pathways," "modulation of the tumor immune microenvironment," "optimization of combination therapies," "novel strategies in gene regulation," and the "integration of TCM" for personalized treatment. This is the first bibliometric analysis on the role of apoptosis in LC immunotherapy, providing an landscape of global research patterns and emerging therapeutic strategies. The findings offer insights to guide future research and optimize treatment approaches.

Antimicrobial resistance (AMR) poses a growing global threat. This review examines AMR from diverse angles, tracing the story of antibiotic resistance from its origins to today's crisis. It explores the rise of AMR, from its historical roots to the urgent need to counter this escalating menace. The review explores antibiotic classes, mechanisms, resistance profiles, and genetics. It details bacterial resistance mechanisms with illustrative examples. Multidrug-resistant bacteria spotlight AMR's resilience. Modern AMR control offers hope through precision medicine, stewardship, combination therapy, surveillance, and international cooperation. Converging traditional and innovative treatments presents an exciting frontier as novel compounds seek to enhance antibiotic efficacy. This review calls for global unity and proactive engagement to address AMR collectively, emphasizing the quest for innovative solutions and responsible antibiotic use. It underscores the interconnectedness of science, responsibility, and action in combatting AMR. Humanity faces a choice between antibiotic efficacy and obsolescence. The call is clear: unite, innovate, and prevail against AMR.

CRISPR-Cas9 and CRISPR-Cas12a are endonuclease systems widely used for genome editing, but their mechanisms of DNA cleavage, particularly in the presence of nucleotide mismatches, remain incompletely understood. This study deals with thermodynamic parameters governing the cleavage of DNA substrates-containing a mismatch in the region complementary to RNA-by Cas9 and Cas12a. Using a series of 55 bp DNA substrates with various mismatches, we investigated the cleavage efficiency, reaction kinetics, and thermodynamic stability of the Cas12a-crRNA complex and compared it with Cas9-sgRNA on the same substrates. Cas12a manifested strict specificity, with a mismatch leading to a significant reduction in cleavage efficiency or to nonspecific trans-cleavage, whereas Cas9 showed higher tolerance to each mismatch, especially in internal and distal regions. Thermodynamic calculations indicated that Cas12a-crRNA complexes are generally stabler with fully complementary DNA but are more destabilized by a mismatch than Cas9-sgRNA complexes are. Molecular dynamics simulations revealed that a mismatch causes greater structural destabilization in Cas12a than in Cas9, correlating with reduced cleavage efficiency. These findings highlight distinct mechanisms of mismatch recognition by Cas9 and Cas12a, provide insights into their enzymatic behavior, and inform the design of more precise genome-editing tools.

Nucleic acid drugs represent the latest generation of precision therapeutics, holding significant promise for the treatment of a wide range of intractable diseases. Delivery technology is crucial for the clinical application of nucleic acid drugs. However, extrahepatic delivery of nucleic acid drugs remains a significant challenge. Systemic administration often fails to achieve sufficient drug enrichment in target tissues. Localized administration has emerged as the predominant approach to facilitate extrahepatic delivery. While localized administration can significantly enhance drug accumulation at the injection sites, nucleic acid drugs still face biological barriers in reaching the target lesions. This review focuses on non-viral nucleic acid drug delivery techniques utilized in local administration for the treatment of extrahepatic diseases. First, the classification of nucleic acid drugs is described. Second, the current major non-viral delivery technologies for nucleic acid drugs are discussed. Third, the bio-barriers, administration approaches, and recent research advances in the local delivery of nucleic acid drugs for treating lung, brain, eye, skin, joint, and heart-related diseases are highlighted. Finally, the challenges associated with the localized therapeutic application of nucleic acid drugs are addressed.

Gene therapy for clinical applications necessitates a comprehensive, accurate, and precise measurement of gene-edited drug products. State-of-the-art pipelines for evaluating editing outcomes rely primarily on bulk sequencing approaches, which are limited to population-level assessment. Here, we leveraged Tapestri, a single-cell sequencing technology for an in-depth analysis of editing outcomes. Using this platform, we characterized the genotype of triple-edited cells simultaneously at more than 100 loci, including editing zygosity, structural variations, and cell clonality. Our findings revealed a unique editing pattern in nearly every edited cell, highlighting the importance of single-cell resolution measurement to ensure the highest safety standards.

CRISPR-based genetically modified scaffold-free biomaterials, including extracellular vehicles, cell sheets, cell aggregates, organoids and organs, have attracted significant attention in the fields of regenerative medicine and tissue engineering in recent years. With a wide range of applications in gene therapy, modeling disease, tissue regeneration, organ xenotransplantation, modeling organogenesis as well as gene and drug screening, they are at a critical juncture from clinical trials to therapeutic applications. Xenografts have already been tested on non-human primates and humans. However, we have to admit that a series of obstacles still need to be addressed, such as immune response, viral infection, off-target effects, difficulty in mass production, and ethical issues. Therefore, future research should pay more attention to improving their safety, accuracy of gene editing, flexibility of production, and ethical rationality. This review summarizes various types of CRISPR-based genetically modified scaffold-free biomaterials, including their preparation procedures, applications, and possible improvements.

CRISPR-Cas systems have advanced many domains in life sciences, enabling diverse applications in gene editing, diagnostics, and biosensing. Here, we introduce a platform that leverages transcription factors (TFs) to regulate CRISPR-Cas12a trans-cleavage activity via engineered DNA translators. These dynamic DNA structures respond to TF binding by switching conformations, modulating Cas12a activity. Using TATA-binding protein and Myc-Max as TF models, we optimized DNA translators for precise and tunable control with rapid response kinetics. We demonstrated the platform's specificity and versatility by integrating TF-induced regulation into synthetic biology networks, including the activation of a fluorogenic RNA aptamer (Mango III) and the creation of an artificial multimolecular communication pathway between Cas12a and Cas13a. This work establishes TFs as effective regulators of CRISPR-Cas systems, enabling novel protein-nucleic acid communication channels, showing potential for novel synthetic biology applications.

Plant viruses are among the most serious threats to global agriculture, causing significant yield losses and jeopardizing food security. Identifying these viruses is crucial to prevent widespread crop damage and ensure effective management. CRISPR-Cas13a, a subtype of the RNA-targeting Cas13 family, has emerged as a transformative tool in molecular diagnostics, specifically tailored to detect these plant RNA viruses with unparalleled precision. Unlike traditional methods such as ELISA and RT-PCR, which are often limited by sensitivity, equipment dependency, and long processing times, Cas13a offers exceptional specificity and attomolar-level sensitivity. Its RNA-guided collateral cleavage mechanism allows signal amplification, making it particularly suitable for field-deployable diagnostics. Recent advances in Cas13 engineering, including compact variants such as Cas13bt3 and Cas13Y, have further improved its delivery efficiency and minimized immune responses, enhancing its agricultural applications. Integration with amplification methods like LAMP and innovative biosensor platforms like graphene-based and electrochemical systems further enhances its diagnostic potential. While challenges remain, including off-target effects, reagent stability, and scalability, innovations in CRISPR RNA (crRNA) design, reagent encapsulation, and microfluidic technologies are actively addressing these barriers. CRISPR-Cas13a represents a cutting-edge solution for rapid, accurate, and accessible plant virus diagnostics, providing a powerful safeguard for crop yields and global food security.

Developments in basic stem cell biology have paved the way for technology translation in human medicine. An exciting prospective use of stem cells is the ex vivo generation of hepatic and pancreatic endocrine cells for biomedical applications. This includes creating novel models 'in a dish' and developing therapeutic strategies for complex diseases, such as metabolic dysfunction-associated steatotic liver disease (MASLD) and diabetes. In this review, we explore recent advances in the generation of stem cell-derived hepatocyte-like cells and insulin-producing β-like cells. We cover the different differentiation strategies, new discoveries, and the caveats that still exist regarding their routine use. Finally, we discuss the challenges and limitations of stem cell-derived therapies as a clinical strategy to manage metabolic diseases in humans.

The increasing changes in the climate patterns across the globe have deeply affected food systems where unparalleled and unmatched challenges are created. This jeopardizes food security due to an ever-increasing population. The extreme efficiency of C4 crops as compared to C3 crops makes them incredibly significant in securing food safety. C4 crops, maize (Zea mays L.) and sorghum (Sorghum bicolor L. Moench) in particular, have the ability to withstand osmotic stress induced by oxidative stress. Osmotic stress causes a series of physical changes in a plant thus facilitating reduced water uptake and photosynthesis inhibition, such as membrane tension, cell wall stiffness, and turgor changes. There has been a great advancement in plant breeding brought by introduction of clustered regularly interspaced short palindromic repeats (CRISPR) gene editing technology. This technology offers precise alterations to an organism's DNA through targeting specific genes for desired traits in a wide number of crop species. Despite its immense opportunities in plant breeding, it faces limitations such as effective delivery systems, editing efficiency, regulatory concerns, and off-target effects. Future prospects lie in optimizing next-generation techniques, such as prime editing, and developing novel genotype-independent delivery methods. Overall, the transformative role of CRISPR/Cas9 in sorghum and maize breeding underscores the need for responsible and sustainable utilization to address global food security challenges.

Psychiatric genetics has evolved from candidate-gene studies to whole-genome sequencing efforts. With hundreds of disease-associated loci now identified, functional interpretation of the associated loci becomes the critical next step toward translational applications. The article discusses achievements, challenges, and opportunities in psychiatric genomics associated with complexity and heterogeneity. Brain expression quantitative trait loci, single-cell ribonucleic acid-sequence, and functional genomics technologies are highlighted. It also covers newly developed techniques with improved spatiotemporal resolution, quality and sensitivity, coupled with advanced analytical methods and artificial intelligence. The power of collaborative research and inclusion of diverse populations will ensure a bright future for precision psychiatry.

Alternative splicing (AS) is a vital and highly dynamic RNA regulatory mechanism that allows a single gene to generate multiple mRNA and protein isoforms. Dysregulation of AS has been identified as a key contributor to the pathogenesis of autism spectrum disorders (ASD). A comprehensive understanding of aberrant splicing mechanisms and their functional consequences in ASD can help uncover the molecular basis of the disorder and facilitate the development of therapeutic strategies. This review focuses on the major aberrant splicing events and key splicing regulators associated with ASD, highlighting their roles in linking defective splicing to ASD pathogenesis. In addition, a discussion of how emerging technologies, such as long-read sequencing, single-cell sequencing, spatial transcriptomics and CRISPR-Cas systems are offering novel insights into the role and mechanisms of AS in ASD is presented. Finally, the RNA splicing-based therapeutic strategies are evaluated, emphasizing their potential to address unmet clinical needs in ASD treatment.

Artificial DNA nanostructures, with their sequence programmability, precise molecular recognition and tunable stimuli responsiveness, bridge material chemistry and biomedicine. Here we detail the design and construction of hybridization chain reaction (HCR)-based DNA nanoframeworks, a class of DNA nanostructures with programmable sequences and customizable functions. HCR is an efficient, enzyme-free amplification strategy that isothermally produces nicked double-stranded DNA with periodically repeated modules via the assembly of two DNA hairpins, triggered by a DNA initiator. In contrast to other available assembly methods for the synthesis of DNA nanostructures, such as tile-mediated assembly, DNA origami and rolling circle amplification, the HCR method offers improved stability and efficiency under mild conditions, without reliance on enzymatic activity. The procedure uses radical polymerization to integrate DNA initiator into nanoframeworks, with overhangs complementary to functional sequences - termed linkers -which are amplified and incorporated through HCR. The linkers enable the incorporation of functional nucleic acid sequences. The HCR-based DNA nanoframeworks facilitate the loading capability of the delivered molecules, showing notable therapeutic efficacy and biosensing sensitivity. Preparation time for HCR-based DNA nanoframeworks ranges from 30 h to 45 h, depending on the payload.

Gene editing technology based on clustered regularly interspaced short palindromic repeats/associated protein (CRISPR/Cas) systems serves as an efficient tool in cancer therapy. Tracking the gene editing process can help identify the progress of cancer treatment. However, existing techniques for monitoring the gene editing process rely on lysed cells, which can not reflect the dynamic changes of nucleic acid in living cells. It urgently needs in situ and real-time imaging technologies to track the gene editing process at a living single-cell level more effectively and precisely. Here, we reported a highly efficient nuclear-targeted material, phenylboronic acid modified linear PEI (LPBA), for loading gene editing plasmids and fluorescent probes to track gene editing processes of microRNA. Based on LPBA, we achieved efficient intranuclear microRNA imaging at the living cell level, reaching 32.4-fold higher than the linear PEI (LPEI) delivery system, which facilitated further sensitive monitoring of the gene editing process both in living cells and in vivo. Meanwhile, this efficient gene-editing and real-time detection technique could be extended to screening effective gene-editing plasmids. Such LPBA-based imaging technology extended the imaging area of microRNA and offered new insight in the field of gene editing and nucleic acid detection.

Fungi represent a broad and evolutionarily unique group within the eukaryotic domain, characterized by extensive ecological adaptability and metabolic versatility. Their inherent biological intricacy is evident in the diverse and dynamic relationships they establish with various hosts and environmental niches. Notably, fungi are integral to disease processes and a wide array of biotechnological innovations, highlighting their significance in medical, agricultural, and industrial domains. Recent advances in genetic engineering have revolutionized fungal research, with CRISPR/Cas emerging as the most potent and versatile genome editing platform. This technology enables precise manipulation of fungal genomes, from silencing efflux pump genes in Candida albicans (enhancing antifungal susceptibility) to targeting virulence-associated sirtuins in Aspergillus fumigatus (attenuating pathogenicity). Its applications span gene overexpression, multiplexed mutagenesis, and secondary metabolite induction, proving transformative for disease management and biotechnological innovation. CRISPR/Cas9's advantages-unmatched precision, cost-effectiveness, and therapeutic potential-are tempered by challenges like off-target effects, ethical dilemmas, and regulatory gaps. Integrating nanoparticle delivery systems and multi-omics approaches may overcome technical barriers, but responsible innovation requires addressing these limitations. CRISPR-driven fungal genome editing promises to redefine solutions for drug-resistant infections, sustainable bioproduction, and beyond as the field evolves. In conclusion, genome editing technologies have enhanced our capacity to dissect fungal biology and expanded fungi's practical applications across various scientific and industrial domains. Continued innovation in this field promises to unlock the vast potential of fungal systems further, enabling more profound understanding and transformative biotechnological progress.

Cervical and other anogenital malignancies are largely caused by E6 and E7 oncogenes of high-risk human papillomaviruses (HPVs), which inhibit important tumor suppressors like p53 and pRb when they are persistently activated. The main goal of traditional treatments is to physically or chemically kill cancer cells, but they frequently only offer temporary relief, have serious side effects, and have a high risk of recurrence. Exploring the efficacy and accuracy of CRISPR-Cas9 gene editing in both inducing death in HPV-infected cancer cells and restoring the activity of tumor suppressors is our main goal. In this study, we propose a novel precision oncology strategy that targets and inhibits the detrimental effects of the E6 and E7 oncogenes using the CRISPR-Cas9 gene editing system. In order to do this, we create unique guide RNAs that target the integrated HPV DNA and reactivate p53 and pRb. Reactivation is meant to halt aberrant cell development and restart the cell's natural dying pathways. This review discusses the potential of CRISPR/Cas9 in targeting HPV oncogenes, with a focus on studies that have demonstrated its promise in cancer treatment. Given the absence of a definitive treatment for papillomavirus infection and its subsequent association with various cancers, future clinical trials and experimental investigations appear essential to establish and evaluate the therapeutic potential of CRISPR-based approaches. This approach provides a less invasive alternative to conventional treatments and opens the door to personalized care that considers the genetic makeup of each patient's tumor.

Gold-based nanomaterials have marked the last few decades with the emergence of new medical technologies presenting unique features. For instance, the conjugation of gold nanoparticles (AuNPs) and nucleic acids has allowed the creation of nanocarriers with immense promise for gene therapy applications. Although the use of lipid particles as RNA delivery vectors has been broadly explored, this review aims to focus on the limited models reported for the conjugation of RNA with AuNPs. This is nonetheless unexpected regarding the manifold strategies existing to conjugate DNA to gold nanoparticles, which are exhaustively listed in this paper. Furthermore, new processes such as fast microwave and freezing methods have been described very recently, and it therefore seemed necessary to review these recent but promising conjugation pathways and to pick out those applicable to RNA. Indeed, RNA is considerably more attractive than DNA for therapeutic purposes, but its low stability involves numerous difficulties in the construction of effective nanodevices. However, from the many approaches developed for DNA, it turns out that just two of them are frequently used for the building of RNA delivery platforms based on gold: the salt-aging method with thiolated RNA strands and physisorption. However, both approaches present strong limitations such as the low stability of the Au-S bond and the potential cytotoxicity of polycations. To conclude, this general assessment highlights that the exploration of innovating approaches implying different chemistries is needed for the creation of more robust and shapeable AuNPs-RNA conjugates.

The poultry industry faces multifaceted challenges, including escalating demand for poultry products, climate change impacting feed availability, emergence of novel avian pathogens, and antimicrobial resistance. Traditional disease control measures are costly and not always effective, prompting the need for complementary methods. Gene editing (GE, also called genome editing) technologies, particularly CRISPR/Cas9, offer promising solutions. This article summarizes recent advancements in utilizing CRISPR/Cas GE to enhance infectious disease control in poultry. It begins with an overview of modern GE techniques, highlighting CRISPR/Cas9's advantages over other methods. The potential applications of CRISPR/Cas in poultry infectious disease prevention and control are explored, including the engineering of innovative vaccines, the generation of disease-resilient birds, and in vivo pathogen targeting. Additionally, insights are provided regarding regulatory frameworks and future perspectives in this rapidly evolving field.

The intuitive manipulation of specific amino acids to alter the activity or specificity of CRISPR-Cas9 has been a topic of great interest. As a large multi-domain RNA-guided endonuclease, the intricate molecular crosstalk within the Cas9 protein hinges on its conformational dynamics, but a comprehensive understanding of the extent and timescale of the motions that drive its allosteric function and association with nucleic acids remains elusive. Here, we investigated the structure and multi-timescale molecular motions of the recognition (Rec) lobe of GeoCas9, a thermophilic Cas9 from Geobacillus stearothermophilus. Our results provide new atomic details about the GeoRec subdomains (GeoRec1, GeoRec2) and the full-length domain in solution. Two rationally designed mutants, K267E and R332A, enhanced and redistributed micro-millisecond flexibility throughout GeoRec, and NMR studies of the interaction between GeoRec and its guide RNA showed that mutations reduced this affinity and the stability of the ribonucleoprotein complex. Despite measured biophysical differences due to the mutations, DNA cleavage assays reveal no functional differences in on-target activity, and similar specificity. These data suggest that guide RNA interactions can be tuned at the biophysical level in the absence of major functional losses but also raise questions about the underlying mechanism of GeoCas9, since analogous single-point mutations have significantly impacted on- and off-target DNA editing in mesophilic Streptococcus pyogenes Cas9. A K267E/R332A double mutant did also did not enhance GeoCas9 specificity, highlighting the robust tolerance of mutations to the Rec lobe of GeoCas9 and species-dependent complexity of Rec across Cas9 paralogs. Ultimately, this work provides an avenue by which to modulate the structure, motion, and guide RNA interactions at the level of the Rec lobe of GeoCas9, setting the stage for future studies of GeoCas9 variants and their effect on its allosteric mechanism.

Functional genomics using CRISPR (Clustered Regulatory Interspaced Short Palindromic Repeats)/Cas (CRISPR-associated endonuclease)-based approaches has revolutionized biomedical sciences. Gene editing is also widespread in parasitology generally and its use is increasing in studies on helminths including flatworm and roundworm parasites. Here, we survey the progress, specifically with experimental CRISPR-facilitated functional genomics to investigate helminth biology and pathogenesis, and also with the burgeoning use of CRISPR-based methods to assist in diagnosis of helminth infections. We also provide an historical timeline of the introduction and uses of CRISPR in helminth species to date.

This study aims to explore the molecules that affect the survival of Human Umbilical Vein Endothelial Cells (HUVECs) under hypoxia and their mechanisms of action. In hypoxia, plasmolipin (PLLP) was identified through the screening of CRISPR/Cas9 and small guide RNA (sgRNA) library. Functionally, PLLP knockout led to increase cell proliferation, cellular metabolism, tight junction formation, angiogenesis ability, migration and invasion in hypoxic HUVECs. Furthermore, PLLP knockout countered the inhibitory effects of bevacizumab on HUVECs angiogenesis and cell survival in hypoxic conditions. PLLP knockout was found to modulate the survival of HUVECs in hypoxia by enhancing the phosphorylation of AKT and ERK1/2 proteins. In conclusion, inhibiting the expression of PLLP in HUVECs promotes cell survival and maintenance of cellular functions under hypoxic condition. PLLP plays a crucial role in regulating cell survival in hypoxia through the activation of AKT and ERK1/2 pathways. This study identifies novel molecules that affect HUVECs survival under hypoxic conditions and provides a new possibility for future studies on cell survival under hypoxic conditions.

Gene delivery systems are essential for investigating gene regulation mechanisms and enhancing the genetic improvement of functional traits in plants. However, fewer than 0.1% of higher plant species on Earth can be genetically modified. Even for these species, the genetic modification process relies on complex tissue culture methods, which are time-consuming, costly, and often require specialized technical skills. Additionally, the efficiency of genetic modification is extremely low in some species. Notably, over the past five years, significant progress has been made in establishing non-tissue culture genetic modification systems. This advancement effectively resolved a series of previously mentioned challenges and innovated in biotechnology for the improvement of many valuable plant species. This review summarizes the research advancements in non-tissue culture genetic modification technologies and presents examples of successful species modified using various methods, including fast-treated Agrobacterium co-culture (Fast-TrACC), cut-dip-budding (CDB), particle bombardment, and nano-mediated delivery systems. Additionally, we propose a working guideline to classify, analyze, evaluate, and select non-tissue culture genetic modification systems for plant species of interest. Our review also discusses the potential for enhancing plant regeneration capacity, improving genetic modification efficiency, and the future application prospects for plant improvement.

High-temperature requirement protein A1 (HTRA1) is a secreted serine protease with diverse substrates, including extracellular matrix proteins, proteins involved in amyloid deposition, and growth factors. Accordingly, HTRA1 has been implicated in a variety of neurodegenerative diseases including a leading cause of blindness in the elderly, age-related macular degeneration (AMD). In fact, genome wide association studies have identified that the 10q26 locus which contains HTRA1 confers the strongest genetic risk factor for AMD. A recent study has suggested that AMD-associated risk alleles in HTRA1 correlate with a significant age-related defect in HTRA1 synthesis in the retinal pigmented epithelium (RPE) within the eye, possibly accounting for AMD susceptibility. Thus, we sought to identify small molecule enhancers of HTRA1 transcription and/or protein abundance using an unbiased high-throughput screening approach. To accomplish this goal, we used CRISPR/Sp.Cas9 engineering to introduce an 11 amino acid luminescent peptide tag (HiBiT) onto the C-terminus of HTRA1 in immortalized ARPE-19 cells. Editing was very efficient (~88%), verified by genomic DNA analysis, short interfering RNA (siRNA), and HiBiT blotting. Nineteen-hundred and twenty compounds from two libraries were screened. An azo compound with reported anti-amyloidogenic and cardioprotective activity, Chicago Sky Blue 6B (CSB), was identified as an enhancer of endogenous HTRA1 secretion (2.0 ± 0.3 fold) and intracellular levels (1.7 ± 0.2 fold). These results were counter-screened using HiBiT complement factor H (CFH) edited ARPE-19 cells, verified using HiBiT blotting, and were not due to HTRA1 transcriptional changes. Importantly, serine hydrolase activity-based protein profiling (SH-ABPP) demonstrated that CSB does not affect HTRA1's specific activity. However, interestingly, in follow-up studies, Congo Red, another azo compound structurally similar to CSB, also substantially increased intracellular HTRA1 levels (up to 3.6 ± 0.3 fold) but was found to significantly impair HTRA1 enzymatic reactivity (0.45 ± 0.07 fold). Computational modeling of potential azo dye interaction with HTRA1 suggests that CSB and Congo Red can bind to the non-catalytic face of the trimer interface but with different orientation tolerances and interaction energies. These studies identify select azo dyes as HTRA1 chemical probes which may serve as starting points for future HTRA1-centered small molecule therapeutics.

Non-viral DNA donor templates are commonly used for targeted genomic integration via homologous recombination (HR), with efficiency improved by CRISPR/Cas9 technology. Circular single-stranded DNA (cssDNA) has been used as a genome engineering catalyst (GATALYST) for efficient and safe gene knock-in. Here, we introduce enGager, an enhanced GATALYST associated genome editor system that increases transgene integration efficiency by tethering cssDNA donors to nuclear-localized Cas9 fused with single-stranded DNA binding peptide motifs. This approach further improves targeted integration and expression of reporter genes at multiple genomic loci in various cell types, showing up to 6-fold higher efficiency compared to unfused Cas9, especially for large transgenes in primary cells. Notably, enGager enables efficient integration of a chimeric antigen receptor (CAR) transgene in 33% of primary human T cells, enhancing anti-tumor functionality. This 'tripartite editor with ssDNA optimized genome engineering (TESOGENASE) offers a safer, more efficient alternative to viral vectors for therapeutic gene modification.

The enhancement of humans' core cognitive abilities-such as intelligence-is a frequently debated topic in scientific and public discourse. Different enhancement methods such as cognitive trainings, smart drugs, and brain stimulation techniques have been proposed and tested to enhance human's cognition. In this narrative review, we summarize the main psychological and neuroscientific findings regarding those cognitive enhancement methods. We thereby distinguish passive (e.g., smart drugs) and active enhancement methods (e.g., working memory training)-which require different levels of agency. While for both forms of enhancement there is no (or only little) empirical evidence on their effectiveness to improve overall cognitive abilities, passive methods entail severe risks. Thus, we criticize promoting an overly optimistic view on especially passive enhancement. Furthermore, we highlight which individuals might be willing to enhance themselves, related motivational aspects of cognitive enhancement, and ethical considerations thereof. To raise awareness for the (in)effectiveness and risks of passive and active enhancement, we propose a category framework that distinguishes cognitive enhancement from clinical methods to treat disorders or diagnosed deficits and introduces important dimensions thereof. Finally, we present open questions for psychological and neuroscientific research which should become part of enhancement debates. Taken together, our narrative review provides a broad overview and critical assessment of enhancement-related topics such as effectiveness and risks of enhancement methods, motivational aspects to apply enhancement, and societal implications of cognitive enhancement.

Restriction-modification systems (RMS) are ubiquitous in prokaryotes and serve as primitive immune-like mechanisms that safeguard microbial genomes against foreign genetic elements. Beyond their well-known role in sequence-specific defense, RMS also contribute significantly to genomic stability, drive evolutionary processes, and mitigate the deleterious effects of mutations. This review provides a comprehensive synthesis of current insights into RMS, emphasizing their structural and functional diversity, ecological and evolutionary roles, and expanding applications in biotechnology. By integrating recent advances with an analysis of persisting challenges, we highlight the critical contributions of RMS to both fundamental microbiology and practical applications in biomedicine and industrial biotechnology. Furthermore, we discuss emerging research directions in RMS, particularly in light of novel technologies and the increasing importance of microbial genetics in addressing global health and environmental issues.

Removal of host cell components is a significant cost driver in the production of live virus vaccines. Filtration processes such as tangential flow filtration can be effective in this capacity by leveraging the relative size difference between viral particles and host proteins; however, filtration membranes can be fouled by larger proteins, particularly those of the extracellular matrix. In this study, we used CRISPR editing to insert the recognition element of the highly-selective TEV protease into various positions of the gene encoding fibronectin in the genome of the Vero cell line, a common platform for viral production. By screening edited cell lines, we identified a promising candidate line in which fibronectin could be effectively removed by treating with the protease during processing, eliminating filter fouling and allowing for viral purification without the need for costly chromatography steps.

Although there are many challenges in using nanobodies for treating various complex tumor diseases, including rapid renal clearance and the complex blood-brain barrier environment, nanobodies have shown great potential due to their high antigen affinity, excellent tumor penetration ability, and favorable safety profile. Since the discovery of the variable domain (VHH) of camelid heavy-chain antibodies in 1993, nanobodies have been progressively applied to various cancer therapy platforms, such as antagonistic drugs and targeting agents for effector domains. In recent years, several nanobody-based drugs, including Caplacizumab, KN-035, and Ozoralizumab, have been approved for clinical use. Among them, KN-035 is used for treating advanced solid tumors, and these advancements have propelled nanobody development to new heights. Currently, nanobodies are being rapidly applied to the treatment of a wide range of diseases, from viral infections to cancer, demonstrating strong advantages in areas such as targeted protein degradation, bioimaging, nanobody-drug conjugation, bispecific T-cell engagers, and vaccine applications. Bibliometric tools, including CiteSpace, HisCite Pro, and Alluvial Generator, were employed to trace the historical development of nanobodies in cancer research. The contributions of authors, countries, and institutions in this field were analyzed, and research hotspots and emerging trends were identified through keyword analysis and influential articles. Future trends were also predicted. This study provides a unique, comprehensive, and objective perspective on the use of nanobodies in tumor research, laying a foundation for future research directions and offering valuable insights for researchers in the field.

Genome editing technologies, particularly CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats), have broadened the possibilities of genetic research and molecular biology by enabling precise modifications of the genome, offering novel therapeutic potential for various disorders. Herein, we present an overview of traditional genome editing techniques and delve deeper into the CRISPR toolbox, with particular attention given to epigenetic and transcriptional regulation. In the context of the intervertebral disc (IVD), CRISPR offers an unprecedented approach to address the mechanisms underlying tissue degeneration, advancing the development of revolutionary therapies for Low Back Pain (LBP). As so, we showcase how to leverage CRISPR systems for IVD. This cutting-edge technology has been successfully used to improve our understanding of IVD biology through functional studies and disease modeling. Most relevant research prioritizes new targets associated with the extracellular matrix (ECM), pain sensing or inflammatory pathways. Promising CRISPR applications encompass IVD regeneration by recapitulation of a regenerative environment or by targeting important degenerative catalysts. In the future, priority should be given to fetal gene reactivation, multiple healthy gene expression enhancement and disease-associated polymorphisms' correction. Despite several challenges such as effective delivery, off-target effects, as well as ethical and safety concerns, exciting clinical trials are anticipated in the years to come, providing more effective and long-lasting solutions for IVD degeneration.

Realistic paths to gene therapy for the X-linked bleeding disorder hemophilia started to materialize in the mid 1990s, resulting in disease correction in small and large animal models. Out of a diversity of approaches, in vivo adeno-associated viral (AAV) gene transfer to hepatocytes emerged as the most promising strategy, eventually forming the basis for multiple advanced clinical trials and regulatory approval of two products for the treatment of hemophilia B (coagulation factor IX deficiency) and one for hemophilia A (factor VIII deficiency). Ideally, gene therapy is effective with a single administration, thus providing therapeutic factor levels over a period of years, without the need for frequent injections. Overcoming multiple obstacles, some not predicted by preclinical studies, sustained partial to complete correction of coagulation for several years to an entire decade has now been documented in patients, with observation ongoing. A hyperactive form of FIX improved efficacy in hemophilia B, and superior engineered variants of FVIII are emerging. Nonetheless, challenges remain, including pre-existing immunity to AAV capsids, toxicities, inter-patient variability in response to treatment, and difficulty in obtaining durable therapeutic expression of FVIII. In alternative approaches, in vivo gene editing and ex vivo gene therapies targeting hemopoietic cells are in development.

CRISPR/Cas12a-powered biosensors with guanine (G)-rich sequence reporters (e.g., G-quadruplex and G-triplex) are widely used in detection applications due to their simplicity and sensitivity. However, when these biosensors are employed for molecular detection in complex samples, they may encounter difficulties such as high background signal and susceptibility to interference because of the "turn-off" signal output. Herein, we explore, for the first time, a set of phosphorothioate (ps)-modified G-quadruplex (G4) and G-triplex (G3) sequences that can bind with thioflavin T (ThT) in an active split CRISPR/Cas12a system (SCas12a) to generate a "turn-on" fluorescent signal. To apply this new phenomenon, we develop a universal SCas12a-powered biosensor for "turn-on" fluorescent detection of nucleic acid (miRNA-21) and non-nucleic acid (kanamycin) targets by using ps-modified hairpin G3 as a reporter (SCas12a/psHG3). Target recognition activates SCas12a's trans-cleavage activity, leading to cleavage at the loop region of the psHG3 reporter. The released prelocked psG3 DNA binds ThT to produce a strong fluorescence signal. Without preamplification, this strategy can detect miRNA-21 with a detection limit of 100 fM. Moreover, the SCas12a/psHG3 system was further utilized for detecting kanamycin by incorporating its aptamers, enabling the detection of kanamycin at concentrations as low as 100 pM. This work is the first to develop a "turn-on" SCas12a/psHG3 system, showcasing its improved performance and wide range of applications in synthetic biology-based sensing technology.

CRISPR/Cas9 is widely recognized as the most effective, efficient, and precise genome editing tool, inspiring numerous applications in basic science, medicine, and biotechnology. In the CRISPR/Cas9 system, single guide RNA (sgRNA) and Cas9 enzyme form a ribonucleoprotein complex that specifically and effectively cleaves target DNA. Accurate sequencing of sgRNA, particularly identifying the target sequence within the first 20 nucleotides (nt) at the 5'-end, is crucial for quality assurance and regulatory compliance. In this study, we used site-directed cleavage using ribonuclease H (RNase H) and DNAzyme for the first time to digest 100 nt sgRNA, achieving full sequencing with 100% coverage by analyzing the two cleaved fragments separately via LC MS/MS. We evaluated four different DNA-RNA chimeras as capture probes for the RNase H site-directed cleavage approach, finding that the chimera with four deoxynucleotides provided the most specific cleavage. Compared to RNase H, the DNAzyme demonstrated higher specificity and stability for 100 nt sgRNA digestion, successfully identifying up to 200 nucleotides of large RNA with 100% sequence coverage by fully sequencing the four short cleaved fragments. Due to the high specificity of DNAzyme cleavage, we used this method to study the designed 5'-end N-X truncated impurities of 100 nt sgRNA, demonstrating accurate identification and relative quantification. For 100 nt sgRNA, the limited available cleavage site was set on the scaffold sequence for both site-directed cleavage approaches, and the captured probes designed for RNase H and DNAzyme can be universally applied to sequence all 100 nt sgRNAs because of the conserved scaffold sequence.

Germline gene editing refers to altering the DNA in the reproductive cells (gametes or embryos). Germline gene editing experiments in human embryos have primarily focused on correcting genetic mutations linked to inherited diseases. This technology has the potential to prevent genetic disease before birth and in future generations. Advances in CRISPR-Cas9 and other gene-editing tools have accelerated scientific progress, raising both promise and safety and ethical concerns. A translational pathway for human heritable genome editing will require an approach that integrates scientific validation, ethical oversight, regulatory frameworks, and public engagement.

RNA switches regulated by specific inducer molecules have become a powerful synthetic biology tool for precise gene regulation in mammalian systems. The engineered RNA switches can be integrated with natural RNA-mediated gene regulatory functions as a modular and customizable approach to probe and control cellular behavior. RNA switches have been used to advance synthetic biology applications, including gene therapy, bio-production, and cellular reprogramming. This review explores recent progress in the design and functional implementation of synthetic riboswitches in mammalian cells based on diverse RNA regulation mechanisms by highlighting recent studies and emerging technologies. We also discuss challenges such as off-target effects, system stability, and ligand delivery in complex biological environments. In conclusion, this review emphasizes the potential of synthetic riboswitches as a platform for customizable gene regulation in diverse biomedical applications.

Advances in precision gene editing have enabled the rapid domestication of wild crop relatives, a process known as neo-domestication. During domestication, breeding rice for maximum productivity under optimal growth conditions reduced genetic diversity, eliminating variants for stress tolerance and grain nutrients. Wild rice varieties have rich genetic diversity, including variants for disease resistance, stress tolerance, and grain nutritional quality. For example, the grain of pigmented wild rice has abundant antioxidants (anthocyanins, proanthocyanidins, and flavonoids), but low yield, poor plant architecture, and long life cycle limit its cultivation. In this review, we address the neo-domestication of wild pigmented rice, focusing on recent progress, CRISPR-Cas editing toolboxes, selection of key candidate genes for domestication, identifying species with superior potential via generating genomic and multi-omics resources, efficient crop transformation methods and highlight strategies for the promotion and application pigmented rice. We also address critical outstanding questions and potential solutions to enable efficient neo-domestication of wild pigmented rice and thus enhance food security and nutrition.

Lipid nanoparticle-messenger RNA formulations have garnered significant attention for their therapeutic potential in infectious diseases, cancer and genetic disorders. However, effective mRNA delivery to the central nervous system (CNS) remains a formidable challenge. To overcome this limitation, a class of brain-targeting lipids (BLs) is developed by incorporating brain-targeting small molecules with amino lipids and formulated them with helper lipids to generate brain-targeting lipid nanoparticles (BLNPs) for mRNA delivery. Screening studies led to a lead formulation, TD5 BLNPs, outperforming FDA-approved DLin-MC3-DMA LNPs in delivering mRNA to the brain upon intrathecal injection. Specifically, a single intrathecal injection of TD5 BLNP-GFP mRNA led to GFP expression in 29.6% of neurons and 38.1% of astrocytes across the brain. In an Ai14 mouse model, TD5 BLNP-Cre recombinase mRNA treatment induced tdTomato expression in ≈30% of neurons and 40% of astrocytes across major brain regions. Notably, delivery of Cas9 mRNA/sgRNA complex using TD5 BLNPs achieved effective genome editing in the brain. Additionally, TD5 BLNPs showed comparable safety profiles to MC3 LNPs, indicating promising biocompatibility. Overall, this TD5 BLNP formulation effectively delivers mRNA to brain tissues via intrathecal injection and facilitates efficient expression in both neurons and astrocytes, presenting a potential strategy for treating CNS diseases.

The preference for a particular thermal range is a key determinant of the distribution of animal species. However, we know little on how temperature preference behaviour evolves during the colonization of new environments. Here we show that at least two distinct neurobiological mechanisms drive the evolution of temperature preference in flies of the genus Drosophila. Fly species from mild climates (D. melanogaster and D. persimilis) avoid both innocuous and noxious heat, and we show that the thermal activation threshold of the molecular heat receptor Gr28b.d precisely matches species-specific thresholds of behavioural heat avoidance. We find that desert-dwelling D. mojavensis are instead actively attracted to innocuous heat. Notably, heat attraction is also mediated by Gr28b.d (and by the antennal neurons that express it) and matches its threshold of heat activation. Rather, the switch in valence from heat aversion to attraction correlates with specific changes in thermosensory input to the lateral horn, the main target of central thermosensory pathways and a region of the fly brain implicated in the processing of innate valence1-5. Together, our results demonstrate that, in Drosophila, the adaptation to different thermal niches involves changes in thermal preference behaviour, and that this can be accomplished using distinct neurobiological solutions, ranging from shifts in the activation threshold of peripheral thermosensory receptor proteins to a substantial change in the way temperature valence is processed in the brain.

CRISPR-Cas genome editing technology has transformed genetic research, by enabling unprecedented precision in modifying DNA sequences across various organisms, including fish. This review explores the significant advancements and potential uses of CRISPR-Cas technology in the study and management of fish diseases, which pose serious challenges to aquaculture and wild fish populations. Fish diseases cause significant economic losses and environmental impacts, therefore effective disease control a top priority. The review highlights the pivotal role of CRISPR-Cas in identifying disease-associated genes, which is critical to comprehending the genetic causes of disease susceptibility and resistance. Some studies have reported key genetic factors that influence disease outcomes, using targeted gene knockouts and modifications to pave the way for the development of disease-resistant fish strains. The creation of such genetically engineered fish holds great promise for enhancing aquaculture sustainability by reducing the reliance on antibiotics and other conventional disease control measures. In addition, CRISPR-Cas has facilitated in-depth studies of pathogen-host interactions, offering new insights into the mechanisms by which pathogens infect and proliferate within their hosts. By manipulating both host and pathogen genes, this technology provides a powerful tool for uncovering the molecular underpinnings of these interactions, leading to the development of more effective treatment strategies. While CRISPR-Cas has shown great promise in fish research, its application remains limited to a few species, primarily model organisms and some freshwater fish. In addition, challenges such as off-target effects, ecological risks, and ethical concerns regarding the release of genetically modified organisms into the environment must be carefully addressed. This review also discusses these challenges and emphasizes the need for robust regulatory frameworks and ongoing research to mitigate risks. Looking forward, the integration of CRISPR-Cas with other emerging technologies, such as multi-omics approaches, promises to further advance our understanding and management of fish diseases. This review concludes by envisioning the future directions of CRISPR-Cas applications in fish health, underscoring its potential to its growing in the field.

Gene therapy is a rapidly developing field, finally yielding clinical benefits. Genetic engineering of organs for transplantation may soon be an option, thanks to convergence with another breakthrough technology, ex vivo machine perfusion (EVMP). EVMP allows access to the functioning organ for genetic manipulation prior to transplant. EVMP has the potential to enhance genetic engineering efficiency, improve graft survival, and reduce posttransplant complications. This will enable genetic modifications with a vast variety of applications, while raising questions on the ethics and regulation of this emerging technology. This review provides an in-depth discussion of current methodologies for delivering genetic vectors to transplantable organs, particularly focusing on the enabling role of EVMP. Organ-by-organ analysis and key characteristics of various vector and treatment options are assessed. We offer a road map for research and clinical translation, arguing that achieving scientific benchmarks while creating anticipatory governance is necessary to secure societal benefit from this technology.

The combination of pharmacogenomics and nanotechnology science of pharmacogenomics into a highly advanced single entity has given birth to personalized medicine known as nano-enabled pharmacogenomics. This review article covers all aspects starting from pharmacogenomics to gene editing tools, how these have evolved or are likely to be evolved for pharmacogenomic application, and how these can be delivered using nanoparticle delivery systems. In this prior work, we explore the evolution of pharmacogenomics over the years, as well as new achievements in the field of genomic sciences, the challenges in drug creation, and application of the strategy of personalized medicine. Particular attention is paid to how nanotechnology helps avoid the problems that accompanied the development of pharmacogenomics earlier, for example, the question of drug resistance and targeted delivery. We also review the latest developments in nano-enabled pharmacogenomics, such as the coupling with other nanobio-technologies, artificial intelligence, and machine learning in pharmacogenomics, and the ethical and regulatory aspects of these developing technologies. The possible uses of nanotechnology in improving the chances of pated and treating drug-resistant cancers are exemplified by case studies together with the current clinical uses of nanotechnology. In the last section, we discuss the future trends and research prospects in this dynamically growing area, stressing the importance of further advancements and collaborations which will advance the nano-enabled pharmacogenomics to their maximum potential.

Lactic acid bacteria (LABs) have a long history of use in food and beverages fermentation. Recently, several LABs have gained attention as starter or non-starter cultures and probiotics for making functional fermented foods, which have the potential to enhance human health. In addition, certain LABs show great potential as microbial cell factories for producing food-related chemicals. However, enhancing the outcomes of starter and non-starter cultures, exploring the complicated probiotic mechanism of LABs, and engineering strains to enhance the yields of high-value compounds for precision fermentation remains challenging due to the time-consuming and labor-intensive current genome editing tools. The clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated proteins (Cas) system, originally an adaptive immune system in bacteria, has revolutionized genome editing, metabolic engineering and synthetic biology. Its versatility has resulted in extensive applications across diverse organisms. The widespread distribution of CRISPR-Cas systems and the diversity of CRISPR arrays in LAB genomes highlight their potential for studying the evolution of LABs. This review discusses the current advancement of CRISPR-Cas systems in engineering LABs for food application. Moreover, it outlines future research directions aimed at harnessing CRISPR-Cas systems to advance lactic acid bacterial research and drive innovation in food science.

Following the discovery of the CRISPR-Cas technology in 2012, there has been a growing global call for public engagement regarding the potential use of human germline gene editing (HGGE). In this systematic scoping review, we aim to evaluate public engagement studies considering the following questions based on three points of attention: 1) Inclusion of underrepresented groups: who have been engaged? 2) Gathering values: what output has been reported? 3) Reaching societal impact: what objectives of public engagement have been reported? A systematic literature search from 2012 to 2023 identified 3464 articles reporting on public engagement studies regarding HGGE retrieved from 12 databases. After screening, 52 full-text articles were assessed for eligibility, resulting in 36 articles that cover 31 public engagement studies. We conclude that co-created efforts are needed to engage underrepresented groups as well as to yield values rather than acceptance levels, and to concretise how engagement might result in societal impact.