Genome editing technologies have revolutionized biomedical sciences and biotechnology. However, their delivery in vivo remains one of the major obstacles for clinical translation. Here, we introduce various emerging genome editing systems and review different delivery systems have been developed to realize the promise of in vivo gene editing therapies. In particular, we focus on virus-like particles (VLPs), an emerging delivery platform and provide in depth analysis on recent advancements to improve VLPs delivery potential and highlight opportunities for future improvements. To this end, we also provide detail workflows for engineered VLP (eVLP) selection, production, and purification, along with methods for characterization and validation.

Microbial engineering has made a significant breakthrough in pharmaceutical biotechnology, greatly expanding the production of biologically active compounds, therapeutic proteins, and novel drug candidates. Recent advancements in genetic engineering, synthetic biology, and adaptive evolution have contributed to the optimization of microbial strains for pharmaceutical applications, playing a crucial role in enhancing their productivity and stability. The CRISPR-Cas system is widely utilized as a precise genome modification tool, enabling the enhancement of metabolite biosynthesis and the activation of synthetic biological pathways. Additionally, synthetic biology approaches allow for the targeted design of microorganisms with improved metabolic efficiency and therapeutic potential, thereby accelerating the development of new pharmaceutical products. The integration of artificial intelligence (AI) and machine learning (ML) plays a vital role in further advancing microbial engineering by predicting metabolic network interactions, optimizing bioprocesses, and accelerating the drug discovery process. However, challenges such as the efficient optimization of metabolic pathways, ensuring sustainable industrial-scale production, and meeting international regulatory requirements remain critical barriers in the field. Furthermore, to mitigate potential risks, it is essential to develop stringent biocontainment strategies and implement appropriate regulatory oversight. This review comprehensively examines recent innovations in microbial engineering, analyzing key technological advancements, regulatory challenges, and future development perspectives.

Stem cell research is a dynamic and fast-advancing discipline with great promise for the treatment of diverse human disorders. The incorporation of gene editing technologies, including ZFNs, TALENs, and the CRISPR/Cas system, in conjunction with progress in nanotechnology, is fundamentally transforming stem cell therapy and research. These innovations not only provide a glimmer of optimism for patients and healthcare practitioners but also possess the capacity to radically reshape medical treatment paradigms. Gene editing and nanotechnology synergistically enhance stem cell-based therapies' precision, efficiency, and applicability, offering transformative potential for treating complex diseases and advancing regenerative medicine. Nevertheless, it is important to acknowledge that these technologies also give rise to ethical considerations and possible hazards, such as inadvertent genetic modifications and the development of genetically modified organisms, therefore creating a new age of designer infants. This review emphasizes the crucial significance of gene editing technologies and nanotechnology in the progress of stem cell treatments, particularly for degenerative pathologies and injuries. It emphasizes their capacity to restructure and comprehensively revolutionize medical treatment paradigms, providing fresh hope and optimism for patients and healthcare practitioners.

Wiskott-Aldrich syndrome (WAS) is a severe X-linked disorder caused by loss-of-function mutations in the WAS gene, responsible for encoding WAS protein (WASP), a key regulator of the actin cytoskeleton in all hematopoietic cells, except red blood cells. The mechanism underlying microthrombocytopenia, a distinctive feature of WAS and a major contributor to mortality, remains not fully elucidated. In this study, using different gene-editing strategies, we corrected mutations in patient-derived WAS-induced pluripotent stem cell (iPSC) lines, generating isogeneic WAS-iPSC lines. These included lines with direct mutation-specific correction and lines incorporating a WASP transgene cassette regulated by the MND or WAS1.6 kb promoter integrated at the safe harbor AAV1 site. Our results demonstrated that direct mutation correction successfully restored WASP levels to the equivalent of the wild-type in iPSC-derived megakaryocytes (MKs). In contrast, the AAV1-targeted strategy using the MND and WAS1.6 promoters yielded a lower level of WASP. Notably, only the mutation-specific correction lines exhibited improvements in proplatelet structures and generated larger-sized platelets. Our findings underscore the crucial roles of WASP during human thrombopoiesis and suggest that therapeutic approaches, such as direct gene correction, which can achieve physiologic levels of WASP in MKs, hold promise for ameliorating platelet defects in individuals with WAS.

The high mortality rate associated with single-use CRISPR-Cas9 in Escherichia coli limits its application. Recently, new CRISPR-based techniques for E.coli gene editing have emerged. Research aims to develop a system for rapid, marker-free, multi-site, and multi-copy genome editing in E.coli to advance synthetic biology. ATP, essential for energy in living organisms, plays a crucial role in various metabolic processes. To reduce the cost of ATP-requiring reactions, it is crucial to identify and efficiently express genes in ATP synthesis pathway. This study identified a single ppk gene (No.8) capable of completing the cyclic reaction. Using MUCICAT technology, the ppk gene (No.8) was inserted into various positions and copy numbers in the E.coli genome, resulting in different activity levels. The findings suggest that the difficulty of inserting the ppk gene (No.8) into the genome follows this order: IS186 < 8array < IS186 + 8array < IS1. A single genome insertion can mimic plasmid expression level. This study explores promoter competition and offers solutions, inspiring researchers in constructing the AMP-ATP cycle system in E.coli. KEY POINTS: • The single ppk gene (No.8) can regenerate the AMP-ATP cycle, crucial for ATP-dependent reactions. • Inserting the ppk gene (No.8) into the cr5 site of the E.coli genome achieves expression levels comparable to the pET29a plasmid. • The expression level of the ppk gene (No.8) is not significantly affected by its copy number in the E.coli genome.

The rapid increase in antimicrobial resistance (AMR) projects a "global emergency" and necessitates a need to discover alternative resources for combating drug-resistant pathogens or "superbugs." One of the key themes in "One Health Concept" is based on the fact that the interconnected network of humans, the environment, and animal habitats majorly contribute to the rapid selection and spread of AMR. Moreover, the injudicious and overuse of antibiotics in healthcare, the environment, and associated disciplines, further aggravates the concern. The prevalence and persistence of AMR contribute to the global economic burden and are constantly witnessing an upsurge due to fewer therapeutic options, rising mortality statistics, and expensive healthcare. The present decade has witnessed the extensive exploration and utilization of bio-based resources in harnessing antibiotics of potential efficacies. The discovery and characterization of diverse chemical entities from endophytes as potent antimicrobials define an important yet less-explored area in natural product-mediated drug discovery. Endophytes-produced antimicrobials show potent efficacies in targeting microbial pathogens and synthetic biology (SB) mediated engineering of endophytes for yield enhancement, forms a prospective area of research. In keeping with the urgent requirements for new/novel antibiotics and growing concerns about pathogenic microbes and AMR, this paper comprehensively reviews emerging trends, prospects, and challenges of antimicrobials from endophytes and their effective production via SB. This literature review would serve as the platform for further exploration of novel bioactive entities from biological organisms as "novel therapeutics" to address AMR.

Cardiomyopathies, among the leading causes of heart failure and sudden cardiac death, are often driven by genetic mutations affecting the heart's structural proteins. Despite significant advancements in understanding the genetic basis of hypertrophic cardiomyopathy (HCM), dilated cardiomyopathy (DCM), and arrhythmogenic right ventricular cardiomyopathy (ARVC), effective long-term therapies remain limited. The advent of clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (Cas9) gene editing offers a promising therapeutic strategy to address these genetic disorders at their root. CRISPR-Cas9 enables precise modification of pathogenic variants (PVs) in genes encoding sarcomeric and desmosomal proteins, which are frequently implicated in cardiomyopathies. By inducing site-specific double-stranded breaks in DNA, followed by repair through nonhomologous end joining (NHEJ) or homology-directed repair (HDR), this system allows for targeted correction of mutations. In preclinical models, CRISPR-Cas9 has shown promise in correcting HCM-associated mutations in β-myosin heavy chain 7 (MYH7), preventing disease phenotypes such as ventricular hypertrophy and myocardial fibrosis. Similarly, gene editing has successfully rectified DCM-linked mutations in Titin (TTN) and LMNA, resulting in improved heart function and reduced pathological remodeling. For ARVC, CRISPR-Cas9 has demonstrated the ability to repair mutations in desmosomal genes such as plakophilin 2 (PKP2), thereby restoring normal cardiac function and cellular adhesion. Despite these successes, challenges remain, including mosaicism, delivery efficiency, and off-target effects. Nevertheless, CRISPR-Cas9 represents a transformative approach to treating genetic cardiomyopathies, potentially offering long-lasting cures by directly addressing their underlying genetic causes.

Gene editing techniques have emerged as powerful tools in biomedical research, offering precise manipulation of genetic material with the potential to revolutionize cancer treatment strategies. This review provides a comprehensive overview of the current landscape of gene editing technologies, including CRISPR-Cas systems, base editing, prime editing, and synthetic gene circuits, highlighting their applications and potential in cancer therapy. It discusses the mechanisms, advantages, and limitations of each gene editing approach, emphasizing their transformative impact on targeting oncogenes, tumor suppressor genes, and drug resistance mechanisms in various cancer types. The review delves into population-level interventions and precision prevention strategies enabled by gene editing technologies, including gene drives, synthetic gene circuits, and precision prevention tools, for controlling cancer-causing genes, targeting pre-cancerous lesions, and implementing personalized preventive measures. Ethical considerations, regulatory challenges, and future directions in gene editing research for cancer treatment are also addressed. This review highlights how gene editing could revolutionize precision medicine by enhancing patient care and advancing cancer treatments with targeted, personalized methods. For these benefits to be fully realized, collaboration among researchers, doctors, regulators, and patient advocates is crucial in fighting cancer and meeting clinical needs.

Cell and gene therapy (CGT) products are emerging and innovative biopharmaceuticals that hold promise for treating diseases that are otherwise beyond the scope of conventional medicines. The evolution of CGT from a research idea to a promising therapeutic product is due to the complementary advancements across various scientific disciplines. First, the innovations and advancements in gene editing and delivery technology have provided fundamental tools to manipulate genes and cells for therapeutic pursuits. Second, advancements in applied and translational research, including how clinical trials are designed, performed, evaluated, and analyzed, have transformed the technology into a potential therapeutic product. Third, advancements in scaling up the production of CGT products have been critical in delivering the product for preclinical studies, clinical trials, and approved treatments. In parallel, regulatory requirements have continuously evolved, with lessons learned from translational studies and biomanufacturing. These combined efforts have transformed CGT products from a promising concept into a reality with the potential to treat a wide range of diseases. However, continued R&D and regulatory oversight are crucial to further improve the safety, efficacy, and accessibility of CGT products.

Transgenic mice, including those created using Bacterial Artificial Chromosomes (BACs), are artificial manipulations that have become critical tools for studying gene function. While conventional transgenic techniques face challenges in achieving precise expression of foreign genes in specific cells and tissues, BAC transgenic mice offer a solution by incorporating large DNA segments that can include entire expression units with tissue-specific enhancers. This review provides a thorough examination of BAC transgenic mouse technology, encompassing both traditional and humanized models. We explore the benefits and drawbacks of BAC transgenesis compared to other techniques such as knock-in and CRISPR/Cas9 technologies. The review emphasizes the applications of BAC transgenic mice in various disciplines, including neuroscience, immunology, drug metabolism, and disease modeling. Additionally, we address crucial aspects of generating and analyzing BAC transgenic mice, such as position effects, copy number variations, and strategies to mitigate these challenges. Despite certain limitations, humanized BAC transgenic mice have proven to be invaluable tools for studying the pathogenesis of human diseases, drug development, and understanding intricate gene regulatory mechanisms. This review discusses current topics on BAC transgenic mice and their evolving significance in biomedical research.

Genome engineering is extensively utilized in diverse scientific disciplines, advancing human welfare and addressing various challenges. Numerous genome engineering tools have been developed to modify genomic sequences. Among these, the CRISPR-Cas system has transformed the field and remains the most commonly employed genome-editing tool. However, the CRISPR-Cas system relies on induced double-strand breaks, with editing efficiency often limited by factors such as cell type and homologous recombination, impeding further progress. CRISPR-associated transposons (CASTs) represent programmable mobile genetic elements. CASTs identified as active were developed as CAST systems, which can perform RNA-guided DNA integration and are featured by high precision, programmability, and kilobase-level payload capacity. Moreover, CAST system allows for precise genome modifications independent of host DNA repair mechanisms, addressing the constraints of conventional CRISPR-Cas systems. It expands the genome engineering toolkit and is poised to become a representative of next-generation genome editing tools. This review thoroughly examines the research progress on CASTs, highlighting the current challenges faced in genome engineering based on CASTs, and offering insights into the ongoing development of this transformative technology.

Cotton (Gossypium hirsutum L.), a vital global cash crop, significantly impacts both the agricultural and industrial sectors, providing essential fiber for textiles and valuable byproducts such as cottonseed oil and animal feed. The cultivation of cotton supports millions of livelihoods worldwide, particularly in developing regions, making it a cornerstone of rural economies. Despite its importance, cotton production faces numerous challenges, including biotic stresses from pests and diseases, and abiotic stresses like drought, salinity, and extreme temperatures. These challenges necessitate innovative solutions to ensure sustainable production. Genome editing technologies, particularly CRISPR/Cas9, have revolutionized cotton breeding by enabling precise genetic modifications. These advancements hold promise for developing cotton varieties with enhanced resistance to pests, diseases, and environmental stresses. Early genome editing tools like ZFNs and TALENs paved the way for more precise modifications but were limited by complexity and cost. The introduction of CRISPR/Cas-based technology with its simplicity and efficiency, has dramatically transformed the field, making it the preferred tool for genome editing in crops. Improved version of the technology like CRISPR/Cas12a, CRISPR/Cas13, base and prime editing, developed from CRISPR/Cas systems, provide additional tools with distinct mechanisms, further expanding their potential applications in crop improvement. This comprehensive review explores the impact of genome editing on cotton breeding and production. It discusses the technical challenges, including off-target effects and delivery methods for genome editing components, and highlights ongoing research efforts to overcome these hurdles. The review underscores the potential of genome editing technologies to revolutionize cotton cultivation, enhancing yield, quality, and resilience, ultimately contributing to a sustainable future for the cotton industry.

The transformative potential of genetic engineering in ophthalmology is remarkable, promising new treatments for a wide range of blinding eye diseases. The eye is an attractive target organ for genetic engineering approaches, in part due to its relatively immune-privileged status, its accessibility, and the ease of monitoring of efficacy and safety. Consequently, the eye has been at the forefront of genetic engineering advances in recent years. The development of Clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), base editors, prime editors, and transposases have enabled efficient and specific gene modification. Ocular gene therapy continues to progress, with recent advances in delivery systems using viral / non-viral vectors and novel promoters and enhancers. New strategies to achieve neuroprotection and neuroregeneration are evolving, including direct in-vivo cell reprogramming and optogenetic approaches. In this review, we discuss recent advances in ocular genetic engineering, examine their current therapeutic roles, and explore their potential use in future strategies to reduce the growing burden of vision loss and blindness.

Sub-Saharan Africa's agricultural sector faces a multifaceted challenge due to climate change consisting of high temperatures, changing precipitation trends, alongside intensified pest and disease outbreaks. Conventional plant breeding methods have historically contributed to yield gains in Africa, and the intensifying demand for food security outpaces these improvements due to a confluence of factors, including rising urbanization, improved living standards, and population growth. To address escalating food demands amidst urbanization, rising living standards, and population growth, a paradigm shift toward more sustainable and innovative crop improvement strategies is imperative. Genome editing technologies offer a promising avenue for achieving sustained yield increases while bolstering resilience against escalating biotic and abiotic stresses associated with climate change. Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein (CRISPR/Cas) is unique due to its ubiquity, efficacy, alongside precision, making it a pivotal tool for Sub-Saharan African crop improvement. This review highlights the challenges and explores the prospect of gene editing to secure the region's future foods.

The burgeoning field of microbiome research has profoundly reshaped our comprehension of human health, particularly highlighting the potential of probiotics and fecal microbiota transplantation (FMT) as therapeutic interventions. While the benefits of traditional probiotics are well-recognized, the efficacy and mechanisms remain ambiguous, and FMT's long-term effects are still being investigated. Recent advancements in high-throughput sequencing have identified gut microbes with significant health benefits, paving the way for next-generation probiotics (NGPs). These NGPs, engineered through synthetic biology and bioinformatics, are designed to address specific disease states with enhanced stability and viability. This review synthesizes current research on NGP stability, challenges in delivery, and their applications in preventing and treating chronic diseases such as diabetes, obesity, and cardiovascular diseases. We explore the physiological characteristics, safety profiles, and mechanisms of action of various NGP strains while also addressing the challenges and opportunities presented by their integration into clinical practice. The potential of NGPs to revolutionize microbiome-based therapies and improve clinical outcomes is immense, underscoring the need for further research to optimize their efficacy and ensure their safety.

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

The advent of CRISPR-Cas has revolutionized precise gene editing. While pioneering CRISPR nucleases like Cas9 and Cas12 generate targeted DNA double-strand breaks (DSB) for knockout or homology-directed repair, next generation CRISPR technologies enable gene editing without DNA DSB. Base editors directly convert bases, prime editors make diverse alterations, and dead Cas-regulator fusions allow nuanced control of gene expression, avoiding potentially risks like translocations. Meanwhile, the discovery of diminutive Cas12 orthologs and Obligate Mobile Element-Guided Activity (OMEGA) nucleases has overcome cargo limitations of adeno-associated viral vectors, expanding prospects for in vivo therapeutic delivery. Here, we review the ever-evolving landscape of cutting-edge gene editing tools, focusing on miniature Cas12 orthologs and OMEGA effectors amenable to single AAV packaging. We also summarize CRISPR therapies delivered using AAV vectors, discuss challenges such as efficiency and specificity, and look to the future of this transformative field of in vivo gene editing enabled by AAV vectors delivery.

As global temperatures rise and arid climates intensify, the reserves of Earth's resources and the future development of humankind are under unprecedented pressure. Traditional methods of food production are increasingly inadequate in meeting the demands of human life while remaining environmentally sustainable and resource-efficient. Consequently, the sustainable supply of lipids is expected to become a pivotal area for future food development. Lignocellulose biomass (LB), as the most abundant and cost-effective renewable resource, has garnered significant attention from researchers worldwide. Thus, bioprocessing based on LB is appearing as a sustainable model for mitigating the depletion of energy reserves and reducing carbon footprints. Currently, the transformation of LB primarily focuses on producing biofuels, such as bioethanol, biobutanol, and biodiesel, to address the energy crisis. However, there are limited reports on the production of single cell oil (SCO) from LB. This review, therefore, provides a comprehensive summary of the research progress in lignocellulosic pretreatment. Subsequently, it describes how the capability for lignocellulosic use can be conferred to cells through genetic engineering. Additionally, the current status of saccharification and fermentation of LB is outlined. The article also highlights the advances in synthetic biology aimed at driving the development of oil-producing microorganism (OPM), including genetic transformation, chassis modification, and metabolic pathway optimization. Finally, the limitations currently faced in SCO production from straw are discussed, and future directions for achieving high SCO yields from various perspectives are proposed. This review aims to provide a valuable reference for the industrial application of green SCO production.

In individuals with Down syndrome (DS), an additional HSA21 chromosome copy leads to the overexpression of a myriad of HSA21 genes, disrupting the transcription of the entire genome. This dysregulation in transcription and post-transcriptional modifications contributes to abnormal phenotypes across nearly all tissues and organs in DS individuals. The array of severe clinical symptoms associated with trisomy 21 poses a considerable challenge in the quest for a cure for DS. Fortunately, a wealth of research suggests that chromosome therapy, hinging on cutting-edge genome editing technologies, can potentially eliminate the extra copy of the human chromosome 21. Genome editing tools have demonstrated their efficacy in restoring trisomy to a normal diploid state in vitro DS cell models. Furthermore, we delve into the noteworthy findings in cellular therapy for DS, with recent studies showcasing the increasing feasibility of strategies involving stem cells and CAR T-cells to address corresponding clinical phenotypes.

Soybean (Glycine max [L.] Merr.) is one of the world's most important sources of oil and vegetable protein. Much of the energy required for germination and early growth of soybean seeds is stored in fatty acids, mainly as triacylglycerols (TAGs), and the main seed storage proteins are β-conglycinin (7S) and glycinin (11S). Recent research advances have deepened our understanding of the biosynthetic pathways and transcriptional regulatory networks that control fatty acid and protein synthesis in organelles such as the plastid, ribosome and endoplasmic reticulum. Here, we review the composition and biosynthetic pathways of soybean oils and proteins, summarizing the key enzymes and transcription factors that have recently been shown to regulate oil and protein synthesis/metabolism. We then discuss the newest genomic strategies for manipulating these genes to increase the food value of soybeans, highlighting important priorities for future research and genetic improvement of this staple crop.

R2 retrotransposons are model site-specific eukaryotic non-LTR retrotransposons that copy-and-paste into gene loci encoding ribosomal RNAs. Recently we demonstrated that avian A-clade R2 proteins achieve efficient and precise insertion of transgenes into their native safe-harbor loci in human cells. The features of A-clade R2 proteins that support gene insertion are not characterized. Here, we report high resolution cryo-electron microscopy structures of two vertebrate A-clade R2 proteins, avian and testudine, at the initiation of target-primed reverse transcription and one structure after cDNA synthesis and second strand nicking. Using biochemical and cellular assays we discover the basis for high selectivity of template use and unique roles for each of the expanded A-clade zinc-finger domains in nucleic acid recognition. Reverse transcriptase active site architecture is reinforced by an unanticipated insertion motif in vertebrate A-clade R2 proteins. Our work brings first insights to A-clade R2 protein structure during gene insertion and enables further improvement and adaptation of R2-based systems for precise transgene insertion.

Remarkable progress has been made in the field of genome engineering after the discovery of CRISPR/Cas9 in 2012 by Jennifer Doudna and Emmanuelle Charpentier. Compared to any other gene-editing tools, CRISPR/Cas9 attracted the attention of the scientific community because of its simplicity, specificity, and multiplex editing possibilities for which the inventors were awarded the Nobel prize for chemistry in 2020. CRISPR/Cas9 allows targeted alteration of the genomic sequence, gene regulation, and epigenetic modifications using an RNA-guided site-specific endonuclease. Though the impact of CRISPR/Cas9 was undisputed, some of its limitations led to key modifications including the use of miniature-Cas proteins, Cas9 Retron precise Parallel Editing via homologY (CRISPEY), Cas-Clover, or development of alternative methods including retron-recombineering, Obligate Mobile Element Guided Activity(OMEGA), Fanzor, and Argonaute proteins. As cancer is caused by genetic and epigenetic alterations, gene-editing was found to be highly useful for knocking out oncogenes, editing mutations to regain the normal functioning of tumor suppressor genes, knock-out immune checkpoint blockade in CAR-T cells, producing 'off-the-shelf' CAR-T cells, identify novel tumorigenic genes and functional analysis of multiple pathways in cancer, etc. Advancements in nanoparticle-based delivery of guide-RNA and Cas9 complex to the human body further enhanced the potential of CRISPR/Cas9 for clinical translation. Several studies are reported for developing novel delivery methods to enhance the tumor-specific application of CRISPR/Cas9 for anticancer therapy. In this review, we discuss new developments in novel gene editing techniques and recent progress in nanoparticle-based CRISPR/Cas9 delivery specific to cancer applications.

Cancer is a multifaceted disease influenced by both intrinsic cellular traits and extrinsic factors, with the tumor microenvironment (TME) being crucial for cancer progression. To satisfy their high proliferation and aggressiveness, cancer cells always plunder large amounts of nutrients and release various signals to their surroundings, forming a dynamic TME with special metabolic, immune, microbial and physical characteristics. Due to the neglect of interactions between tumor cells and the TME, traditional cancer therapies often struggle with challenges such as drug resistance, low efficacy, and recurrence. Importantly, the development of gene editing technologies, particularly the CRISPR-Cas system, offers promising new strategies for cancer treatment. Combined with nanomaterial strategies, CRISPR-Cas technology exhibits precision, affordability, and user-friendliness with reduced side effects, which holds great promise for profoundly altering the TME at the genetic level, potentially leading to lasting anticancer outcomes. This review will delve into how CRISPR-Cas can be leveraged to manipulate the TME, examining its potential as a transformative anticancer therapy.

The objective of present review is to provide a scientific overview of sugarcane as a potential feedstock for biofuel and use of genome editing approach for improvement of industrial and agronomical traits in sugarcane. Sugarcane, a perennial tropical grass with a high biomass index, is a promising feedstock for bioethanol production, and its bagasse, rich in lignocellulosic material, serves as an ideal feedstock for producing second-generation bioethanol. To improve the conversion of sugarcane biomass into biofuels, developing varieties with improved biomass degradability and high biomass and sucrose content is essential. The complex genome architecture and earlier lack of sequence data hindered biotechnological advancements in sugarcane, but recent genome sequence updates offer new opportunities for sugarcane improvement. The first genetically modified sugarcane was developed in 1992 by Bower and Birch using microprojectile bombardment of embryogenic callus. Since then, transgenic techniques have rapidly evolved, leading to the advancement of genome editing technologies. Application of genome editing tools particularly CRISPR/Cas system has been successfully used in sugarcane for editing. Recently, multiple alleles of the magnesium chelatase and acetolactate synthase genes in sugarcane have been successfully edited through multiplexing. Additionally, CRISPR-edited sugarcane varieties with modified cell wall components and increased sucrose content for enhanced bioethanol production have been developed. At the end, the future of CRISPR-edited crops will depend on how well regulatory frameworks adapt to the rapidly evolving technology.

Cardiomyopathy is a group of disease characterized by structural and functional damage to the myocardium. The etiologies of cardiomyopathies are diverse, spanning from genetic mutations impacting fundamental myocardial functions to systemic disorders that result in widespread cardiac damage. Many specific gene mutations cause primary cardiomyopathy. Environmental factors and metabolic disorders may also lead to the occurrence of cardiomyopathy. This review provides an in-depth analysis of the current understanding of the pathogenesis of various cardiomyopathies, highlighting the molecular and cellular mechanisms that contribute to their development and progression. The current therapeutic interventions for cardiomyopathies range from pharmacological interventions to mechanical support and heart transplantation. Gene therapy and cell therapy, propelled by ongoing advancements in overarching strategies and methodologies, has also emerged as a pivotal clinical intervention for a variety of diseases. The increasing number of causal gene of cardiomyopathies have been identified in recent studies. Therefore, gene therapy targeting causal genes holds promise in offering therapeutic advantages to individuals diagnosed with cardiomyopathies. Acting as a more precise approach to gene therapy, they are gradually emerging as a substitute for traditional gene therapy. This article reviews pathogenesis and therapeutic interventions for different cardiomyopathies.

This review article aims to summarize broadly recent developments in the treatment of HPV-associated cancers, including cervical cancer and head and neck squamous cell carcinoma. Relatively new treatments targeting the key HPV E6 and E7 oncoproteins, including gene editing with TALENs and CRISPR/Cas9, are discussed. Given the increased immunogenicity of HPV-related diseases, other therapies such as PRR agonists, adoptive cell transfer, and tumor vaccines are reaching the clinical trial phase. Due to the mechanism, immunogenicity, and reversibility of HPV carcinogenesis, HPV-related cancers present unique targets for current and future therapies.

Chinese cabbage, Brassica rapa L. subsp. pekinensis is a crucial and extensively consumed vegetable in the world, especially Eastern Asia. The market demand for this leafy vegetable increases year by year, resulting in multiple challenges for agricultural researchers worldwide. Multi-omic approaches and the integration of functional genomics helps us understand the relationships between Chinese cabbage genomes and phenotypes under specific physiological and environmental conditions. However, challenges exist in integrating multi-omics for the functional analysis of genes and for developing potential traits for Chinese cabbage improvement. However, the panomics platform allows for the integration of complex omics, enhancing our understanding of molecular regulator networks in Chinese cabbage agricultural traits. In addition, the agronomic features of Chinese cabbage are significantly impacted by the environment. The expression of these agricultural features is tightly regulated by a combination of signals from both the internal regulatory network and the external growth environment. To comprehend the molecular process of these characteristics, it is necessary to have a prior understanding of molecular breeding for the objective of enhancing quality. While the use of various approaches in Chinese cabbage is still in its early stages, recent research has shown that it has the potential to uncover new regulators both rapidly and effectively, leading to updated regulatory networks. In addition, the utilization of the efficient transformation technique in conjunction with gene editing using CRISPR/Cas9 will result in a reduction in time requirements and facilitate a more precise understanding of the role of the regulators. Numerous studies about Chinese cabbage have been conducted in the past two decades, but a comprehensive review about its genome still limited. This review provides a concise summary of the latest discoveries in genomic research related to Brassica and explores the potential future developments for this species.

While the advent of CAR-T therapies has heralded a new era of efficacious therapies in relapsed/refractory Multiple Myeloma, access continues to be a major limiting factor due to prolonged manufacturing times of autologous products and apheresis and/or manufacturing failures. Allogeneic adoptive cellular therapy products (CAR-T, CAR-NK), currently investigational, are "off-the-shelf" products that may address availability and manufacturing bottlenecks. Novel rapid manufacturing platforms that decrease adoptive cell therapy product development time by weeks are currently being tested in clinical trials and may additionally help bridge the demand-supply chasm. This review provides a comprehensive overview of allogeneic adoptive cellular therapies and rapid manufacturing platforms in development.

With the development of new technologies in recent years, researchers have made significant progress in crop breeding. Modern breeding differs from traditional breeding because of great changes in technical means and breeding concepts. Whereas traditional breeding initially focused on high yields, modern breeding focuses on breeding orientations based on different crops' audiences or by-products. The process of modern breeding starts from the creation of material populations, which can be constructed by natural mutagenesis, chemical mutagenesis, physical mutagenesis transfer DNA (T-DNA), Tos17 (endogenous retrotransposon), etc. Then, gene function can be mined through QTL mapping, Bulked-segregant analysis (BSA), Genome-wide association studies (GWASs), RNA interference (RNAi), and gene editing. Then, at the transcriptional, post-transcriptional, and translational levels, the functions of genes are described in terms of post-translational aspects. This article mainly discusses the application of the above modern scientific and technological methods of breeding and the advantages and limitations of crop breeding and diversity. In particular, the development of gene editing technology has contributed to modern breeding research.

Tandem repeat proteins (TRPs) are widely distributed and bind to a wide variety of ligands. DNA-binding TRPs such as zinc finger (ZNF) and transcription activator-like effector (TALE) play important roles in biology and biotechnology. In this study, we first conducted an extensive analysis of TRPs in public databases, and found that the enormous diversity of TRPs is largely unexplored. We then focused our efforts on identifying novel TRPs possessing DNA-binding capabilities. We established a protein language model for DNA-binding protein prediction (PLM-DBPPred), and predicted a large number of DNA-binding TRPs. A subset was then selected for experimental screening, leading to the identification of 11 novel DNA-binding TRPs, with six showing sequence specificity. Notably, members of the STAR (Short TALE-like Repeat proteins) family can be programmed to target specific 9 bp DNA sequences with high affinity. Leveraging this property, we generated artificial transcription factors using reprogrammed STAR proteins and achieved targeted activation of endogenous gene sets. Furthermore, the members of novel families such as MOON (Marine Organism-Originated DNA binding protein) and pTERF (prokaryotic mTERF-like protein) exhibit unique features and distinct DNA-binding characteristics, revealing interesting biological clues. Our study expands the diversity of DNA-binding TRPs, and demonstrates that a systematic approach greatly enhances the discovery of new biological insights and tools.

Different neurological diseases including, Parkinson's, Alzheimer's, and Huntington's diseases extant momentous global disease burdens, affecting millions of lives for imposing a heavy disease burden on the healthcare systems. Despite various treatment strategies aimed at alleviating symptoms, treatments remain elusive and ineffective due to the disease's complexity. However, recent advancements in gene therapy via the CRISPR-Cas system offer ground-breaking and targeted treatment options. Based on a bacterial immune mechanism, the CRISPR-Cas system enables precise genome editing, allowing for the alteration of different genetic mutations and the possible cure of genetic diseases. In the context of neurological disorders, the CRISPR-Cas system shows a promising avenue by allowing researchers to conduct genome-editing which is implicated in neurodegenerative disease therapeutics. This book chapter provides an updated overview of the application of the CRISPR-Cas system for addressing target-specific therapeutic approaches for neurodegenerative disorders. Furthermore, we discuss the principles of the CRISPR-Cas mechanism, its role in modeling neurological disorders, identifying molecular targets, and developing gene-based therapies. Additionally, the chapter explores the recent clinical trials and CRISPR-Cas-mediated treatments for neurological conditions. By leveraging the accuracy and versatility of the CRISPR-Cas system, scientists can more effectively handle the genetic underpinnings of neurodegenerative diseases. Furthermore, the chapter extends the critical viewpoints on ethical considerations and technical limitations related to the clinical deployment of this revolutionizing technique.

Terpenoids are widely used as medicines, flavors, and biofuels. However, the use of these natural products is largely restricted by their low abundance in native plants. Fortunately, heterologous biosynthesis of terpenoids in microorganisms offers an alternative and sustainable approach for efficient production. Various genome-editing technologies have been developed for microbial strain construction. Clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR associated protein 9 (Cas9) is the most commonly used system owing to its outstanding efficiency and convenience in genome editing. In this review, the basic principles of CRISPR-Cas9 systems are briefly introduced and their applications in engineering bacteria for the production of plant-derived terpenoids are summarized. The aim of this review is to provide an overview of the current developments of CRISPR-Cas9-based genome-editing technologies in bacterial engineering, concluding with perspectives on the challenges and opportunities of these technologies.

Delivery of genetic information to the interior of target cells in vivo has been a major challenge facing gene therapies. This barrier is now being overcome, owing in part to dramatic advances made by lipid-based systems that have led to lipid nanoparticles (LNPs) that enable delivery of nucleic acid-based vaccines and therapeutics. Examples include the clinically approved COVID-19 LNP mRNA vaccines and Onpattro (patisiran), an LNP small interfering RNA therapeutic to treat transthyretin-induced amyloidosis (hATTR). In addition, a host of promising LNP-enabled vaccines and gene therapies are in clinical development. Here, we trace this success to two streams of research conducted over the past 60 years: the discovery of the transfection properties of lipoplexes composed of positively charged cationic lipids complexed with nucleic acid cargos and the development of lipid nanoparticles using ionizable cationic lipids. The fundamental insights gained from these two streams of research offer potential delivery solutions for most forms of gene therapies.

This conference celebrates the 40th anniversary of AETE. Over the past 40 years, AETE has served as a forum for scientists, practitioners, and students working in assisted animal reproduction in livestock species. AETE conferences have reflected developments in the field, from basic to applied science, as well as regulatory changes in assisted animal reproduction practices. Europe has led the way in these developments for many years, progressing from artificial insemination, embryo transfer, and cryopreservation to semen sexing, in vitro production of embryos, cloning by nuclear transfer, genomic selection, and the rescue of highly endangered species. These significant contributions were made possible by the support of funding agencies, both at the national and European levels, promoting cooperation between scientists and practitioners. Assisted reproduction, and animal breeding more generally, face opposition from various groups, including animal rights activists, vegetarians, proponents of organic farming, environmentalists, certain political parties, and increasing regulatory burdens. These challenges seriously affect funding for scientific research, the work of practitioners, and the breeding industry as a whole. It is crucial to invest time and resources in communication to remind the public, politicians, and regulators of the achievements in this field and the contributions made to the food supply chain and the care of the rural and natural environment.

Exploiting the inherent compatibility of DNA-based data storage with living cells, various cellular recording approaches have been developed for recording and retrieving biologically relevant signals in otherwise inaccessible locations, such as inside the body. This review provides an overview of the current state of engineered cellular memory systems, highlighting their design principles, advantages, and limitations. We examine various technologies, including CRISPR-Cas systems, recombinases, retrons, and DNA methylation, that enable these recording systems. Additionally, we discuss potential strategies for improving recording accuracy, scalability, and durability to address current limitations in the field. This emerging modality of biological measurement will be key to gaining novel insights into diverse biological processes and fostering the development of various biotechnological applications, from environmental sensing to disease monitoring and beyond.

CRISPR-Cas systems have revolutionised precision medicine by enabling personalised treatments tailored to an individual's genetic profile. Various CRISPR technologies have been developed to target specific disease-causing genes in blood cancers, and some have advanced to clinical trials. Although some studies have explored the in vivo applications of CRISPR-Cas systems, several challenges continue to impede their widespread use. Furthermore, CRISPR-Cas technology has shown promise in improving the response of immunotherapies to blood cancers. The emergence of CAR-T cell therapy has shown considerable success in the targeting and correcting of disease-causing genes in blood cancers. Despite the promising potential of CRISPR-Cas in the treatment of blood cancers, issues related to safety, ethics, and regulatory approval remain significant hurdles. This comprehensive review highlights the transformative potential of CRISPR-Cas technology to revolutionise blood cancer therapy.

Fungi contain a wide range of bioactive secondary metabolites (SMs) that have numerous applications in various fields, including agriculture, medicine, human health, and more. It is common for genes responsible for the production of secondary metabolites (SMs) to form biosynthetic gene clusters (BGCs). The identification and analysis of numerous unexplored gene clusters (BGCs) and their corresponding substances (SMs) has been significantly facilitated by the recent advancements in genomic and genetic technologies. Nevertheless, the exploration of secondary metabolites with commercial value is impeded by a variety of challenges. The emergence of modern CRISPR/Cas technologies has brought about a paradigm shift in fungal genetic engineering, significantly streamlining the process of discovering new bioactive compounds. This study begins with an examination of fungal biosynthetic gene clusters (BGCs) and their interconnections with the secondary metabolites (SMs) they generate. Following that, a brief summary of the conventional methods employed in fungal genetic engineering is provided. This study explores various sophisticated CRISPR/Cas-based methodologies and their utilization in examining the synthesis of secondary metabolites (SMs) in fungi. The chapter provides an in-depth analysis of the limitations and obstacles encountered in CRISPR/Cas-based systems when applied to fungal genetic engineering. It also proposes promising avenues for future research to optimize the efficiency of these systems.

Cell and gene therapy are innovative biomedical strategies aimed at addressing diseases at their genetic origins. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) systems have become a groundbreaking tool in cell and gene therapy, offering unprecedented precision and versatility in genome editing. This chapter explores the role of CRISPR in gene editing, tracing its historical development and discussing biomolecular formats such as plasmid, RNA, and protein-based approaches. Next, we discuss CRISPR delivery methods, including viral and non-viral vectors, followed by examining the various engineered CRISPR variants for their potential in gene therapy. Finally, we outline emerging clinical applications, highlighting the advancements in CRISPR for breakthrough medical treatments.

This review presents a comprehensive exploration of gene editing technologies and their potential applications in the treatment of liver fibrosis, a condition often leading to serious complications such as liver cancer. Through an in-depth review of current literature and critical analysis, the study delves into the intricate signaling pathways underlying liver fibrosis development and examines the promising role of gene editing in alleviating this disease burden. Gene editing technologies offer precise, efficient, and reproducible tools for manipulating genetic material, holding significant promise for basic research and clinical practice. The manuscript highlights the challenges and potential risks associated with gene editing technology. By synthesizing existing knowledge and exploring future perspectives, this study aims to provide valuable insights into the potential of precision gene editing to combat liver fibrosis and its associated complications, ultimately contributing to advances in liver fibrosis research and therapy.

The homologous recombination strategy has a long history of editing Saccharomyces cerevisiae target genes. The application of CRISPR/Cas9 strategy to editing target genes in S. cerevisiae has also received a lot of attention in recent years. All findings seem to indicate that editing relevant target genes in S. cerevisiae is an extremely easy event. In this study, we systematically analyzed the advantages and disadvantages of homologous recombination (HR) strategy, CRISPR/Cas9 strategy, and CRISPR/Cas9 combined homology-mediated repair (CRISPR/Case9-HDR) strategy in knocking out BY4742 ade2. Our data showed that when the ade2 was knocked out by HR strategy, a large number of clones appeared to be off-target, and 10 %-80 % of the so-called knockout clones obtained were heteroclones. When the CRISPR/Cas9 strategy was applied, 60% of clones were off-target and the rest were all heteroclones. Interestingly, most of the cells were edited successfully, but at least 60 % of the clones were heteroclones, when the CRISPR/Cas9-HDR strategy was employed. Our results clearly showed that the emergence of heteroclone seems inevitable regardless of the strategies used for editing BY4742 ade2. Given the characteristics of BY4742 defective in ade2 showing red on the YPD plate, we attempted to build an efficient yeast gene editing strategy, in which the CRISPR/Cas9 combines homology-mediated repair template carrying an ade2 expression cassette, BY4742(ade2Δ0) as the start strain. We used this strategy to successfully achieve 100 % knockout efficiency of trp1, indicating that technical challenges of how to easily screen out pure knockout clones without color phenotype have been solved. Our data showed in this study not only establishes an efficient yeast gene knockout strategy with dual auxotrophy coupled red labeling but also provides new ideas and references for the knockout of target genes in the monokaryotic mycelium of macrofungi.

Gene therapy is emerging as a powerful tool to modulate abnormal gene expression, a hallmark of most CNS disorders. The transformative potentials of recently approved gene therapies for the treatment of spinal muscular atrophy (SMA), amyotrophic lateral sclerosis (ALS) and active cerebral adrenoleukodystrophy are encouraging further development of this approach. However, most attempts to translate gene therapy to the clinic have failed to make it to market. There is an urgent need not only to tailor the genes that are targeted to the pathology of interest but to also address delivery challenges and thereby maximize the utility of genetic tools. In this Review, we provide an overview of gene therapy modalities for CNS diseases, emphasizing the interconnectedness of different delivery strategies and routes of administration. Important gaps in understanding that could accelerate the clinical translatability of CNS genetic interventions are addressed, and we present lessons learned from failed clinical trials that may guide the future development of gene therapies for the treatment and management of CNS disorders.

With a common aim of restoring physiological function of defective cells, optogenetics and targeted gene therapies have shown great clinical potential and novelty in the branch of personalized medicine and inherited retinal diseases (IRDs). The basis of optogenetics aims to bypass defective photoreceptors by introducing opsins with light-sensing capabilities. In contrast, targeted gene therapies, such as methods based on CRISPR-Cas9 and RNA interference with noncoding RNAs (i.e., microRNA, small interfering RNA, short hairpin RNA), consists of inducing normal gene or protein expression into affected cells. Having partially leveraged the challenges limiting their prompt introduction into the clinical practice (i.e., engineering, cell or tissue delivery capabilities), it is crucial to deepen the fields of knowledge applied to optogenetics and targeted gene therapy. The aim of this in-depth and novel literature review is to explain the fundamentals and applications of optogenetics and targeted gene therapies, while providing decision-making arguments for ophthalmologists. First, we review the biomolecular principles and engineering steps involved in optogenetics and the targeted gene therapies mentioned above by bringing a focus on the specific vectors and molecules for cell signalization. The importance of vector choice and engineering methods are discussed. Second, we summarize the ongoing clinical trials and most recent discoveries for optogenetics and targeted gene therapies for IRDs. Finally, we then discuss the limits and current challenges of each novel therapy. We aim to provide for the first time scientific-based explanations for clinicians to justify the specificity of each therapy for one disease, which can help improve clinical decision-making tasks.

Traditional therapeutic approaches such as chemotherapy and radiation therapy have burdened cancer patients with onerous physical and psychological challenges. Encouragingly, the landscape of tumor treatment has undergone a comprehensive and remarkable transformation. Emerging as fervently pursued modalities are small molecule targeted agents, antibody-drug conjugates (ADCs), cell-based therapies, and gene therapy. These cutting-edge treatment modalities not only afford personalized and precise tumor targeting, but also provide patients with enhanced therapeutic comfort and the potential to impede disease progression. Nonetheless, it is acknowledged that these therapeutic strategies still harbour untapped potential for further advancement. Gaining a comprehensive understanding of the merits and limitations of these treatment modalities holds the promise of offering novel perspectives for clinical practice and foundational research endeavours. In this review, we discussed the different treatment modalities, including small molecule targeted drugs, peptide drugs, antibody drugs, cell therapy, and gene therapy. It will provide a detailed explanation of each method, addressing their status of development, clinical challenges, and potential solutions. The aim is to assist clinicians and researchers in gaining a deeper understanding of these diverse treatment options, enabling them to carry out effective treatment and advance their research more efficiently.

The continued development of novel genome editors calls for a universal method to analyze their off-target effects. Here we describe a versatile method, called Tracking-seq, for in situ identification of off-target effects that is broadly applicable to common genome-editing tools, including Cas9, base editors and prime editors. Through tracking replication protein A (RPA)-bound single-stranded DNA followed by strand-specific library construction, Tracking-seq requires a low cell input and is suitable for in vitro, ex vivo and in vivo genome editing, providing a sensitive and practical genome-wide approach for off-target detection in various scenarios. We show, using the same guide RNA, that Tracking-seq detects heterogeneity in off-target effects between different editor modalities and between different cell types, underscoring the necessity of direct measurement in the original system.