gene editing

Comparative performance of CRISPR epigenome gene activation: a systematic review on potency and temporal dynamics in mammalian models

Epigenome editing refers to the targeted alteration of gene regulatory states without altering the underlying DNA sequence in a heritable or not manner, directly or indirectly. Specifically, CRISPR activation (CRISPRa) is a epigenome editing system based on the activity of a Cas9 that lacks endonuclease activity (dCas9) associated with transcriptional or epigenetic effector domains to promote gene overexpression, without permanent DNA modifications. A single-guide RNA directs the CRISPRa complex to a specific genomic region to enhance transcription of a target gene. Despite the rapid development of diverse CRISPRa effector systems, no systematic review has comprehensively compared their activation efficiency and kinetic profiles. This gap limits evidence-based selection of effector domains for experimental or therapeutic applications. Therefore, this systematic review aims to compare the performance of CRISPR-dCas9-based effector domains for gene upregulation across eukaryotic and mammalian experimental models, focusing on activation magnitude and stability over time. By doing so, this review is expected to provide a framework to guide experimental design and translational applications.

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This paper introduces a novel system for personalized gene editing based on dynamic gene expression modulation, leveraging biological information to tailor gene expression to individual characteristics and environmental factors. Traditional gene editing methods primarily focus on modifying genes, while this system aims to optimize individual gene function through a bio-informed approach. The core mechanism involves constructing a "bio-gene map" and dynamically adjusting gene expression to achieve improved health outcomes. This represents a significant advancement in precision medicine, offering the potential for more effective and targeted therapeutic interventions.

Beyond Viral Suppression: Biological, Clinical, and Social Challenges in HIV Control

While antiretroviral therapy (ART) has transformed HIV into a manageable chronic condition, the virus remains a persistent global health challenge. This review connects the biological foundations of HIV, including its zoonotic origins, the pandemic expansion of HIV-1 group M, and the genetic diversity generated by rapid replication, mutation, and recombination, with its clinical, epidemiological, and social consequences. We summarize the main routes of transmission, the HIV life cycle, current diagnostic approaches, and the mechanisms underlying modern antiretroviral therapy, while examining why epidemic control remains uneven across populations and regions. Regional disparities, delayed diagnosis, incomplete treatment access, and HIV-related stigma continue to disrupt prevention and long-term care. Finally, we discuss emerging strategies, including long-acting treatment and prevention, broadly neutralizing antibody-based vaccine approaches, cure research, and gene-editing technologies. Sustained HIV control will require not only biomedical innovation, but also equitable healthcare access, stigma reduction, and structural interventions that address persistent barriers to care.

Comprehensive Comparative Evaluation and Consensus Strategy for CRISPR/Cas9 gRNA Design Targeting the TaDREB1 Gene in Bread Wheat (Triticum aestivum)

SAVI: molecular mechanisms, clinical spectrum and precision medicine approaches beyond type-I IFN

Type I interferonopathies (TI-IFN) represent a heterogeneous group of autoinflammatory disorders characterized by upregulated type I interferon (IFN) signaling. Among them, STING-associated vasculopathy with onset in infancy (SAVI) is a rare, severe autoinflammatory disease caused by gain-of-function mutations in the STING1 gene. Mechanistically, these mutations lead to a constitutive activation of the STING protein, resulting in excessive type I interferon production, which drives chronic inflammation and damage to various organs, primarily as cutaneous vasculopathy and progressive interstitial lung disease (ILD). Emerging clinical data indicate that current therapeutic options – including Janus kinase inhibitors (JAKi), biological agents as anifrolumab and solid organ transplantation – for SAVI face limited and often inconsistent long-term efficacy, highlighting a significant unmet need. Consistently, preclinical models highlight that SAVI pathogenesis is not only driven by the interferon storm, but relies also on non-canonical IFN-independent cellular pathways across distinct hematopoietic and non-hematopoietic compartments. This review provides a comprehensive overview of SAVI pathogenesis, from mutational mechanisms and comparative phenotypes to the clinical challenges of advanced interventions. Finally, we discuss future therapeutic prospects and clinical implications, exploring next-generation frontiers such as patient-derived induced pluripotent stem cell (iPSCs) models, hematopoietic stem cell transplantation (allo-HSCT) and gene editing (GE) technologies. The transition from immune - to a mutational-perspective, provides a foundational framework to optimize diagnostic and therapeutic strategies for precision medicine in SAVI patients.

From Design to Single-Cell Cloning: A Complete RNP-Based CRISPR-Cas9 Protocol for Precision Gene Correction in Human iPSCs.

Precise genome editing of induced pluripotent stem cells (iPSCs) using clustered regularly interspaced short palindromic repeats and CRISPR-associated protein 9 (CRISPR-Cas9) has opened unprecedented avenues for advancements in regenerative medicine and disease modelling. However, the establishment of isogenic single-cell-derived iPSC populations, particularly upon CRISPR-Cas9 gene editing modifications, is one of the major challenges still associated with these advanced methodologies relying on low-rate editing events and requiring defined clonogenicity. In response, we have developed a systematic, comprehensive and efficient workflow combining generation, genotyping and expansion of high-quality monoclonal iPSC lines following CRISPR-Cas9 genome editing. In particular, the protocol incorporates optimized single-cell cloning procedures for two commercially available dispensing platforms, one based on microfluidic imaging and the other on impedance technology, together with rapid droplet digital PCR (ddPCR)-based screening of non-homologous end joining (NHEJ) and homology-directed repair (HDR) outcomes. By combining gentle single-cell handling with advanced genotyping methodologies, the protocol enables efficient early assessment of editing outcomes before commitment to labour-intensive clonal derivation, thereby accelerating project timelines, minimising cell stress and loss, preserving genetic fidelity and supporting scalability. Coupled with precisely defined culture conditions tailored for post-seeding recovery, these approaches aim to improve iPSC viability and clonal outgrowth, achieving at least 60% in 96-well plate format within 10 days. Importantly, the protocol goes beyond step-by-step experimental instructions by providing comprehensive design strategies, decision-making criteria, and practical advice for avoiding and addressing the common pitfalls and unintended consequences of these advanced methods. Collectively, this integrated end-to-end approach provides a robust and high-throughput framework for the reliable production of monoclonal genome-edited iPSCs, thereby advancing translational research and the development of iPSC-based regenerative therapies and disease models.

Molecular Scissors With a Search Bar: A Historical Development Review of CRISPR and the Editing of the Genome

This article presents a narrative review of CRISPR and the Editing of the Genome in the context of Biology. The literature on this topic has expanded substantially over recent decades, yet it remains fragmented across subfields, methods, and national research traditions. Drawing on an interpretive synthesis of representative contributions, the review reconstructs the historical development of the area, examines the conceptual foundations and definitional disputes that organize its debates, and maps the contemporary landscape of research, including the methodological shift toward data-intensive approaches and the institutional pressures that shape publication practice. Particular attention is given to the role of CRISPR and gene editing as organizing themes, and to the conditions under which findings from different research traditions can be brought into productive comparison. The review identifies three synthetic conclusions: the literature is cumulatively strong but organizationally weak; methodological pluralism is better understood as a resource than as a defect; and the growing practical salience of the topic raises the stakes of its unresolved conceptual questions. An agenda for future work is proposed, emphasizing integrative research designs, transparent synthesis practices, and the protection of definitional and infrastructural work on which cumulative progress depends. The article is intended as both a reference map for newcomers and a provocation for specialists in Biology.

Next-generation vascular organoids: multi-lineage and immune integration for disease modeling and regenerative medicine

Traditional human blood vessel organoids, built primarily around endothelial monocultures, fail to replicate the multicellular architecture and dynamic immunological functions of native microvasculature. This review synthesizes an emerging paradigm shift toward third-generation, multi-lineage, and immune-competent vascular organoids. Multicellular integration, combining perivascular mural lineages (pericytes and vascular smooth muscle cells) with functional immune populations (macrophages, microglia, and lymphocytes), is delineated. Its role in reinstating baseline barrier tightness, contractility, and tissue-level immunosurveillance is highlighted. Critical bioengineering workflows are examined, with emphasis on fluidic shear stress in microfluidic platforms, the spatial precision afforded by three-dimensional (3D) bioprinting, and multiplex CRISPR gene editing. These technologies resolve lineage-specific media conflicts and enable off-the-shelf, hypoimmunogenic vascular constructs. Furthermore, the capacity of these systems to recapitulate complex pathophysiology is evaluated. Such pathophysiology includes complement-driven immunothrombosis in SARS-CoV-2 infection, neurovascular degeneration in Alzheimer’s disease, genetic small-vessel disorders, and tumor-immune barriers that govern chimeric antigen receptor T (CAR-T) cell infiltration. Finally, persistent translational hurdles are outlined, including metabolic bottlenecks, diffusion limits, scale‑up challenges, and the lack of large‑animal efficacy and safety data. Strategies that may help transition these models from research tools toward clinically relevant platforms are also discussed.

Editorial: The safety assessment of novel foods and new nutrient sources

An important theme that emerged from this Research Topic is the toxicological safety assessment of novel foods including genotoxicity and repeated dose toxicity. Schreppler et al., employed a standard battery of in vitro genotoxicity tests (bacterial reverse mutation assay, in vitro mouse lymphoma TK gene mutation assay, and in vitro micronucleus assay) to assess the genotoxic potential of short-, medium-, and longchain triacylglycerides 1 . This synthetic mixture of triacyglycerides showed no evidence of mutagenic, clastogenic, or aneugenic activity. Overall, the available data indicated that this novel food did not exhibit genotoxic potential under the tested conditions supporting its safety as a dietary fat ingredient. Mahadevan et al., demonstrated the safety of Hericium erinaceus and Trametes versicolor mushroom powders in acute and subchronic oral toxicity studies, as well as in in vitro and in vivo genotoxicity assays 2 .Long-term safety of the consumption of novel foods was further addressed by Punvittayagul et al., who evaluated formulated Thai rice instant granules containing turmeric extract and Phyllanthus emblica fruit pulp in a 6-month repeated-dose oral toxicity study and revealed no adverse ePects 3 . Given the presence of plant-based bioactive compounds, the expression profile of hepatic antioxidant genes was examined and found to be upregulated. Especially their work on molecular docking to identify binding aPinity interactions between major bioactive compounds and key antioxidant enzymes provides an example of the application of mechanistic toxicology to gain insights into the antioxidant potential of these bioactive constituents present in the granules.Mechanistic insights into the antioxidant potential of bioactive compounds in novel foods were further provided by Giambastiani et al., who investigated the ePects of dietary supplementation with the microalga Chlorella vulgaris in an animal study 4 . The findings showed elevated activity of hepatic xenobiotic-metabolizing cytochrome P450 (CYP) enzymes together with antioxidant and detoxification enzymes without signs of liver and kidney toxicity. The antioxidant potential of C. vulgaris was supported by compositional data indicating its richness in phenolic compounds and carotenoids. Additionally, their work illustrated anti-inflammatory and antioxidant ePects of C. vulgaris in a murine model of chronic pulmonary inflammation. These findings further support the need for comprehensive toxicological evaluations on the long-term ePects of novel foods containing bioactive compounds with pharmacological activities.Another important theme emerging from this Research Topic concerns the complexity of the safety assessment in cases of products of cellular agriculture and the increasing importance of the application of New Approach Methodologies (NAMs). Felicianna et al., investigated the chemical characterization and toxicity of plant cell cultures from scurvy grass (Cochlearia danica) and rowan (Sorbus aucuparia) and demonstrated that they are nutritionally comparable to other berry cell lines without toxic ePects 5 . By applying proteomics analysis to identify potential allergens, the study showcases how NAMs are incorporated in the allergenicity safety assessment of novel foods. Their findings also emphasized the need for further work to evaluate the outcomes of proteomics, and the ePects from phytohormone accumulation used in the growth media on the quality and safety of these plant cell culture foods.The allergenicity assessment of novel foods was also addressed by Calcinai et al., who performed protein profiling of chia seeds (Salvia hispanica) followed by in-silico homology analysis between identified chia peptides and sesame protein sequences 6 . The potential cross-reactivity with characterised linear epitopes from sesame allergens was further corroborated in vitro by IgE-binding assays using sera from sesame-allergic individuals. This study highlights the challenges faced in the assessment of the allergenic potential of novel proteins including the lack of comprehensive protein sequence data and the insuPicient characterization of potential allergenic epitopes.Complementing these studies, Laganaro et al. reviewed the data requirements for allergenicity safety assessment of novel foods within the EU framework, identifying key uncertainties and research needs 7 . The assessment should first consider the nature of the novel food, whether proteins are involved in its production, and whether its source is known to be allergenic. Literature findings, together with protein digestibility and stability data, also inform the evaluation of its allergenic potential. Where uncertainty remains, a tiered approach is applied to investigate potential cross-reactivity with known allergens, encompassing in silico, in vitro and, where necessary, in vivo approaches. The review underscores the value of integrating bioinformatic predictions with experimental validation to strengthen the evidence base for allergenicity assessment, while also highlighting the need for consensus on the interpretation of results, standardised and validated methods, and development of de novo sensitization assays.Addressing limitations and data gaps in the safety assessment of novel foods is crucial for food innovation without compromising public health. The next generation of safety assessment is expected to increasingly integrate NAMs and AI, while addressing the need for method standardisation and validation to support fit-for-purpose regulatory decisionmaking. Overall, the contributions assembled in this Research Topic reflect the breadth of approaches for toxicological and allergenicity assessment and illustrate the ongoing evolution towards more mechanistic, predictive and biologically relevant methodologies.Author contributions MG: Writing -original draft, Writing -review and editing, LP: Writing -review and editing

基于生物网络的动态拓扑优化

This paper explores the application of dynamic topology optimization techniques to biological networks, aiming to enhance drug discovery and gene editing. Biological networks, complex systems of interconnected nodes and edges, are increasingly utilized in various fields. Traditional optimization methods often rely on fixed parameters, failing to fully exploit the inherent structure and dynamic properties of these networks. We propose a novel framework that leverages the topology of biological networks, employing dynamic optimization algorithms to iteratively refine network parameters and achieve optimal configurations. This research focuses on a specific example – protein-protein interaction networks – demonstrating the potential of this approach to improve network stability and functional efficiency. The core mechanism involves analyzing network topology, identifying critical nodes, and dynamically adjusting parameters to promote convergence and improve overall network performance. This work contributes to the development of more robust and effective strategies for optimizing biological networks.

基于生物网络的动态拓扑优化

This paper explores the application of dynamic topology optimization techniques to biological networks, aiming to enhance drug discovery and gene editing. Biological networks, complex systems of interconnected nodes and edges, are increasingly utilized in various fields. Traditional optimization methods often rely on fixed parameters, failing to fully exploit the inherent structure and dynamic properties of these networks. We propose a novel framework that leverages the topology of biological networks, employing dynamic optimization algorithms to iteratively refine network parameters and achieve optimal configurations. This research focuses on a specific example – protein-protein interaction networks – demonstrating the potential of this approach to improve network stability and functional efficiency. The core mechanism involves analyzing network topology, identifying critical nodes, and dynamically adjusting parameters to promote convergence and improve overall network performance. This work contributes to the development of more robust and effective strategies for optimizing biological networks.

Comparative evaluation of pKSE401 and pHSE401 gRNA constructs for CRISPR/Cas9-Mediated resistance to cotton leaf curl virus

Cotton leaf curl disease (CLCuD), caused by begomoviruses and their betasatellites, is a major threat to cotton production, especially in South Asia, where periodic viral outbreaks continue to affect cotton yields, quality, and livelihoods. The advent of CRISPR/Cas9 gene-editing technology has transformed plant biotechnology, offering efficient, accurate, and programmable methods for combating viral pathogens at the genetic level. Here, the antiviral efficacy of two most widely used CRISPR/Cas9 binary plant expression vectors, pKSE401 and pHSE401, was tested in Nicotiana benthamiana against Cotton leaf curl Kokhran virus (CLCuKoV) and Cotton leaf curl Multan betasatellite (CLCuMuB). The guide-RNAs (gRNAs) were designed to target viral genes that play significant roles in pathogenicity and replication, with pHSE401 encoding a single gRNA and pKSE401 a multiplex of two gRNAs. Agrobacterium-mediated transient transformation and viral inoculation experiments revealed that both CRISPR/Cas9 vectors effectively delayed symptom onset and reduced virus titers relative to infected controls. Remarkably, the multiplex pKSE401 system was more effective at suppressing viral infection, achieving about a 90% reduction in viral accumulation compared with a 75% reduction by the single gRNA pHSE401 construct. pKSE401-treated plants showed delayed symptom development, reduced severity, and partial recovery, demonstrating the improved efficiency of multiplex genome editing. The results demonstrate the cutting-edge potential of CRISPR/Cas9 multiplex approaches as next-generation methods for designing sustainable resistance to multifaceted plant virus diseases. This research not only contributes to our understanding of CRISPR-based antiviral response mechanisms but also provides a promising avenue for designing broad-spectrum, sustainable resistance against viral epidemics in cotton and other commercially valuable crops.