Genome editing (GE) has transformed medicine by allowing precise changes to DNA, offering potential treatments for a range of inherited and acquired disorders. Several technologies support these advances, including zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-based systems, of which the latter has emerged as the most accessible, versatile, and popular. While GE holds great promise, its clinical use requires careful attention to safety, ethics and regulatory standards. Inadvertent on- and off-target DNA alterations and unintended modification of non-target cells pose major technical challenges, while bioethical considerations and the need for harmonized safety standards create regulatory challenges. The Food and Drug Administration (FDA) and European Medicines Agency (EMA), as regulatory agencies for key advanced therapy markets, provide detailed guidance on these aspects, emphasizing rigorous preclinical testing, patient monitoring, ethical consent, and compliance with legal frameworks. This concise review summarizes what is currently published in the scientific literature and recommended by regulatory agencies, providing an overview of the responsible clinical application of GE, with emphasis on patient safety, adherence to regulatory guidance, and ethical practice.

Sugarcane (Saccharum spp.) is an important cash crop that provides about 90% of sugar and 40% of bioethanol in China. Due to its large genome and complicated genetic background, conventional breeding is difficult to achieve efficient genetic improvement of sugarcane. Genome editing is a disruptive technology in life sciences, enabling precise and efficient modification of target genes. From zinc-finger nucleases (ZFNs) to transcription activator-like effector nucleases (TALENs), the CRISPR/Cas system and the derived base editing and prime editing, these technologies have greatly advanced genetic research and upgraded biological breeding. With the decoding of the sugarcane genome, genome editing has provided a new technical means for the genetic improvement of polyploid sugarcane. This article provides a comprehensive review of the trajectory of genome editing in plants, the optimization of the CRISPR/Cas system, the genetic transformation status of sugarcane, the development of sugarcane genomics, and the application of genome editing in sugarcane. It focuses on exploring the application prospects of genome editing in breeding lodging-resistant sugarcane varieties. This review aims to provide valuable references for promoting the use of genome editing in sugarcane breeding.

Cyanobacteria harbor sophisticated molecular defense systems that have evolved over billions of years to protect against viral invasion and foreign genetic elements. These ancient photosynthetic organisms possess a diverse array of restriction-modification (R-M) systems and CRISPR-Cas arrays that present challenges for genetic engineering, but also offer unique opportunities for cancer-targeted biotechnological applications. These systems exist in prokaryotes mainly as defense mechanisms but they are currently used in molecular applications as gene editing tools. Moreover, latest developments in nucleases such as zinc finger nucleases (ZFNs), TALENs (transcription-activator-like effector nucleases) are discussed. A comprehensive genomic analysis of 126 cyanobacterial species found 89% encode multiple R-M systems, averaging 3.2 systems per genome, creating formidable barriers to transformation but also providing molecular machinery that could be harnessed for precise recognition and targeting of cancer cells. This review critically examines the dual nature of these defense systems, their ecological functions, and the emerging strategies to translate their molecular precision into advanced anticancer therapeutics. Hence, the review main objectives are to explore the recent understanding of these mechanisms and to exploit the knowledge gained in opening new avenues for cancer-focused targeted interventions, while acknowledging the significant challenges to translate these systems from laboratory curiosities to practical applications.

Over the past two decades, genome editing has advanced dramatically from Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs) to more refined systems such as CRISPR-Cas9, prime editing, and nanoCas technologies. These innovations have opened new frontiers in cancer treatment. This review aims to critically examine and compare recent advances in these genome editing platforms, with a focus on their molecular mechanisms, delivery challenges, oncological applications, and clinical prospects. We systematically explore how CRISPR-Cas9 enables gene knockouts, high-throughput functional genomic screens, and immune editing, while acknowledging its limitations due to off-target effects and genotoxicity. In contrast, base and prime editors offer precise, double-strand breaks (DSBs) free alternatives, suitable for correcting oncogenic mutations such as TP53, KRAS, and EGFR. Prime editing, although versatile, faces delivery and efficiency challenges. The emergence of nanoCas systems, derived from compact Cas orthologs, provides promising delivery advantages for in vivo applications. We also examine how tumor microenvironment, cell-type specificity, and immune barriers impact editing efficacy and safety. Strategies such as high-fidelity variants, optimized guide RNAs, and stimuli-responsive nanoparticles are discussed to enhance precision and minimize risk. Conclusively, integrating these genome editing tools into oncology requires addressing translational barriers while harnessing their precision and therapeutic potential for next-generation cancer treatments.

CRISPR, Clustered Regularly Interspaced Short Palindromic Repeat, as a powerful genome engineering system, has been widely accepted and employed in gene editing of a vast range of cell types. Compared to zinc finger nucleases (ZFNs) or transcriptionactivator-like effector nucleases (TALENs), CRISPR shows a less complicated process and higher efficiency. With the development of different CRISPR systems, it can be used not only to knock out a gene but also to make precise modifications, activate or repress target genes with epigenetic modifications, and even for genome wide screening. Here we will describe the procedure of generating a stable cell line with a knock-in mutation created by CRISPR. Specifically, this protocol demonstrated how to apply CRISPR to create the point mutation of R249 to S249 on TP53 exon 7 in human embryonic stem cells (hESC) H9 line, which includes three major steps: (1) design CRISPR system targeting TP53 genomic region, (2) deliver the system to H9 hESC and clone selection, and (3) examination and selection of positive clones.

Cereals are crucial sources of food for human and animal populations worldwide. Their grain and fodder primarily serve as sources of energy and nutrition. Cereal production is hampered because of the prevalent abiotic stress worldwide. Abiotic stresses such as drought, salinity, extreme temperatures, and heavy metal toxicity significantly reduce global cereal crop production. Previously, traditional breeding and transgenic technology have been promising and potent approaches used to mitigate unfavourable abiotic stresses, enhancing crop production to some extent. The recent advent of more potent genome-editing technologies, particularly Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR), has revolutionized the pace of crop improvement programs. Genome-editing technology using engineered nucleases offers significant opportunities for crop improvement. Genome editing tools include Meganucleases, Zinc Finger Nucleases (ZFN), Transcription activator-like effector nucleases (TALENs), and CRISPR/CRISPR-associated protein (Cas). Among all genome-editing tools, CRISPR/Cas9 has been widely used to improve crop cultivars due to its specificity, simplicity, robustness, and flexibility. Recent progress in genome-editing technology have improved various plant traits in cereals. Among these traits, cereal genotypes have shown substantial advances in the last decade, particularly in enhanced tolerance to abiotic stress, enabled by genome-editing tools. This review summarizes the recently developed cereal cultivars for abiotic stress tolerance that employ different genome-editing technologies, including the most recent additions, prime editing and base editing. These improved cereal cultivars perform better and maintain higher yields under adverse abiotic stresses.

Genome editing has revolutionized many processes, including crop improvement and production of next-generation hybrid seeds from genic male sterile lines and maintainers. Different genome editing techniques include zinc-finger nucleases, transcription activator-like effector nucleases, and clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein (Cas), and came into existence after the success of engineered meganucleases. Insertion of DSBs at specific sites and induction of the repair process to bring about desirable amendments in gene functions are the prime tasks of genome editing approaches. CRISPR/Cas system, developed recently, relies on CRISPR-RNAs for recognizing the targeted sequence and is far more effective than the previously developed genome editing approaches. The refinement of abiotic and biotic stress tolerance, enhancement of quality traits, and improvement of yield represent the changes that have been achieved through various genome editing practices. This update review presents an overview of genome editing in plants and the currently available genome-editing tools to evaluate their effectiveness, along with their positive and negative aspects.

Innovative strategies for augmenting Omega-3-Fatty acid production in microalgae: Sustainable approaches for vegan food applications.

Mitochondria, as crucial organelles within eukaryotic cells, have their proteins and RNAs encoded by both the nuclear genome and the mitochondrial genome. They play vital roles in energy regulation, cellular metabolism, signal transduction, and various other physiological activities. Additionally, mitochondria interact with multiple organelles to collectively maintain cellular homeostasis. Mitochondria can also be transferred between cells and tissues through mechanisms such as migrasomes. Mitochondrial DNA (mtDNA) mutations often cause severe inherited rare diseases, characterized by tissue specificity, heterogeneity, multiple mutation sites, and challenges in achieving a complete cure. Gene editing of mtDNA holds promise for fundamentally curing such diseases. Traditional gene-editing nucleases, such as zinc-finger nucleases (ZFNs) and transcription activator-like effector nuclease (TALENs), as well as novel gene editors like DddA-derived cytosine base editors (DdCBEs), have been demonstrated to correct certain mtDNA mutations. However, CRISPR-based technologies-despite their superior programmability and efficiency-are currently limited due to the technical bottleneck of inefficient sgRNA delivery into mitochondria. This article systematically reviews the structure and function of mitochondria, related diseases, and the current state of mtDNA gene-editing therapies. Furthermore, it explores future directions for optimizing therapeutic tools to overcome the challenge of sgRNA delivery, thereby addressing the treatment barriers posed by pathogenic mtDNA mutations in inherited rare diseases.

Recessive dystrophic epidermolysis bullosa (RDEB) is one of the main types of epidermolysis bullosa, a rare, severe, genetic disease characterized by systemic chronic inflammation, blisters, erosions and extensive chronic wounds on the skin and a tendency to squamous cell carcinoma development. The disease is caused by pathogenic variant of COL7A1 gene encoding the synthesis of type VII collagen (C7), that is the main component of the anchorage fibrils, which maintain the adhesion between the epidermis and the underlying dermis. The disease is debilitating. Treatment methods mainly include supportive measures so the development of treatment methods that directly affect the cause of the disease is very relevant. The article is devoted to the possibility of using one of the most promising treatment strategies of RDEB — gene editing in patients with RDEB by means of specific nucleases, namely TALEN (Transcription activator-like effector nucleases) and CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/Crispr-Associated protein) that allows to almost irreversibly restore the C7 synthesis. The method is at preclinical stage. The results of preclinical studies, comparative analysis of used methods, as well as approaches that may help to implement this method of RDEB gene therapy in clinical practice in the nearest future are presented.

In recent years, using modern technologies, researchers have harnessed the potential of yeast species for various industrial uses, such as the bioproduction of biopharmaceuticals, food additives, industrial biocatalysts, and biofuels. To improve the efficiency and potential of yeast species for industrial uses, genetic modification is carried out. Various genome engineering techniques, including Cre-loxP, homing endonucleases, zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9 (CRISPR/Cas9), have been employed by different research groups for the genetic manipulation of yeast species. Among different genome engineering techniques, CRISPR/Cas9 has become popular because of its precise editing at targeted loci with increased efficiency. The ease of use, effectiveness, and adaptability of CRISPR/Cas9 make multiplexing possible for simultaneously targeting multiple genes, which was earlier very challenging through traditional methods. Moreover, the ability to perform marker-free editing is the significant advantage offered by CRISPR/Cas9. This review focuses on the applications of the CRISPR/Cas9 system in both conventional and non-conventional yeast species. Further, we discussed the advancements of CRISPR/Cas9, including the regulation of gene transcription-activation/repression and other genome engineering aspects. Additionally, innovations in CRISPR/Cas9, such as cloning-free CRISPR/Cas9 assembly, CRISPR-targeted in vivo editing (ACtive), CRISPR/Cas9-induced gene conversion, and selective ploidy ablation (CRI-SPA) are also discussed for enhancing the potential applications of CRISPR/Cas9 in diverse yeast species.

Genome editing tools have great potential for medicinaluse. Amongthem, zinc finger nucleases (ZFNs) are smaller in size than transcriptionalactivator-like effector nucleases and CRISPR-Cas9. Therefore, ZFNsare easily packed into a viral vector with limited cargo space, includingadeno-associated viral vectors. Furthermore, because ZFN patents expiredin 2020, high patent royalties are not required for application. Althoughfunctional 6-finger ZFNs can be easily prepared by modular assembly,it has been extremely difficult to produce functional 7-finger ZFNs,which are expected to have higher target specificity than 6-fingerZFNs in some cases. Herein we describe the construction of 7-fingerZFNs and the improvement in genome editing efficiency, which is generallylower in 7-finger ZFNs than in 6-finger ZFNs. Modular assembly of7-finger ZFNs was achieved using a specific mutation, and the originalgenome editing efficiency was increased by up to 19%. Furthermore,7-finger ZFNs showed reduced off-target effects, exhibiting highertarget specificity than the corresponding 6-finger ZFNs. Our studyprovides critical insights for safer and more specific genome editing.

Genetic alterations and other undesirable changes in human cells contribute to various health issues, such as genetic disorders, cancer, and degenerative illnesses. The rise of gene editing technologies has opened up new possibilities to not only grasp the mechanisms of these conditions but also to prevent or rectify them at their genetic origin. This research paper examines the development of gene editing technologies, starting with initial methods like zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs) and progressing to contemporary techniques such as CRISPR-Cas9, base editing, and prime editing.The article employs a literature review approach, integrating scholarly articles, books, and reports to examine how these technologies operate, their comparative advantages and drawbacks, and their possible uses in human health. The results emphasize the revolutionary effectiveness of CRISPR-Cas9, the accuracy of base and prime editing for point mutations, and the essential contribution of TALENs and ZFNs in establishing modern techniques. Summarizing research, ethical supervision, and technological improvements had suggested that these tools could revolutionize precision medicine and greatly lessen the impact of genetic disorders.

Human Immunodeficiency Virus (HIV) infection remains a major global health issue, including in Indonesia. Gene therapy (GT) has emerged as a promising therapeutic approach for various diseases, including HIV. However, its application also raises significant ethical challenges, particularly within the Indonesian context. This article aims to explore the ethical considerations, potential, and challenges of implementing GT for patients with HIV in Indonesia. A comprehensive narrative review was conducted by examining currentscientific literature and ethical frameworks related to GT and HIV management, with a focus on clinical feasibility, safety, and social implications within the Indonesian context. Gene therapy technologies such as zinc-finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and ClusteredRegularly Interspaced Short Palindromic Repeats (CRISPR) have shown promising potential in suppressing HIV infection. However, concerns remain regarding onand off-target effects that may cause genomic instability and oncogenesis. Ethical challenges include the high cost of therapy, limited public understanding of GT,and the absence of specific regulations governing its application in HIV treatment. Indonesia’s diverse sociocultural landscape further complicates equitable access and acceptance of this advanced technology. The implementation of GT for HIV in Indonesia requires careful ethical consideration, transparent communication,and robust policy development. Establishing national guidelines and conducting further research are essential to ensure that the adoption of GT is safe, equitable, and ethically responsible within the Indonesian healthcare system.

Microalgae are a broad class of photosynthetic, eukaryotic microorganisms that transform carbon dioxide and solar energy into high-value products (HVPs), which have significant commercial value. They are viewed as promising platforms for HVP production. With the global population estimated to reach approximately 9.22 billion by 2075, microalgae are recognised for their resilient and remarkably effective biofactories. However, despite their industrial relevance and environmental advantages over land plants, microalgae-based HVP production requires further optimisation to become commercially viable. Hence, genome editing tools such as clustered regularly interspaced short palindromic repeats/Cas protein 9 (CRISPR-Cas9) are a potential strategy to generate microalgae strains that promote the production of HVP more efficiently to meet industrial demand compared to other genome editing techniques such as zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). This review paper examines the potential and effectiveness of CRISPR-Cas9 in microalgae for enhancing the production of HVP, particularly PUFA, carotenoids, mycosporine-like amino acids, and vitamins. The literature search used online databases to consider the inclusion and exclusion criteria. In conclusion, due to its effectiveness, CRISPR-Cas9 is recognised as the most widely used genome editing technique for enhancing microalgae HVP production.

Some nucleases may be programmed to break just certain portions of DNA; examples of such enzymes include zinc-finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). Insertions and deletions are used by cellular machinery to repair damaged DNA. By specifically targeting long terminal repeats (LTRs), zinc-finger nucleases (ZFNs) efficiently and accurately remove HIV-1 proviral DNA from inactive human T cells, offering a new and different way to eradicate HIV-1 infections. This paper examines the potential, evaluates the current situation, and draws attention to the challenges surrounding the use of TALENs and ZFNs as therapeutic tools for the treatment of HIV infection, to mitigate the adverse off-target effects that result from their extended expression. There is less off-target editing and higher success in targeting HIV escape mutations using TALENs and ZFNs than with CRISPR/Cas-9. The use of ZFNs and TALEN has resulted in changes to many host genes. These include the entrance receptors CCR5 and CXCR4, as well as the proviral integration protein LEDGF/p75. One of the viral targets is the big terminal repeats of proviral DNA. The advancement of gene therapy from the laboratory to the clinic is hindered by the need to reduce immunogenicity, cytotoxicity, and off-target editing while simultaneously enhancing cleavage efficiency and dispersion. However, TALENs technology and breakthroughs in ZFNs are making cleavage more efficient and selective. The strategy for treating HIV might be drastically changed, and maybe even eradicated, by the creation of synthetic nucleases like ZFNs and TALENs. This review explores the current developments about ZFNs and TALENs for the treatment of HIV.

The prodigious human genome is composed of 3 billion nucleotides - a 4-letter DNA alphabet. According to the National Institutes of Health (NIH), just a single typo in our body’s ‘instruction book’ can lead to tragic abnormalities and diseases. However, the ability to quickly fix DNA ‘spelling errors’ or, in more scientific terms, edit any genome precisely to prevent such errors is now possible and being widely used. This tool, known as CRISPR, is proving to be revolutionary: researchers have successfully edited the disease-causing mutation in blood-forming cells taken directly from people with sickle-cell disease, creating malaria-resistant mosquitoes, and correcting gene errors in diseases known to be caused by one or just a few mutations (NIH, 2025). First, it's important to define genome editing: Genome editing is he process of making permanent modifications to DNA sequences at specific locations (Szczesna, 2023). Genomic editing, until the recent discovery of innovative nucleases like CRISPR-associated nucleases (CRISPR-Cas9), meganucleases (MNs), zinc-finger nucleases (ZFNs), and transcription activator-like effector nucleases (TALENs), was initially performed by introducing breaks to DNA via radiation or using cleavage proteins (endonucleases). DNA is repaired either by non-homologous end joining (NHEJ), which directly rejoins broken ends, or by homologous repair (HR), which uses a similar DNA sequence as a template. Repair templates may include selection markers like antibiotic resistance genes or fluorescent tags to identify cells with the desired DNA modification.

Human mesenchymal stem cells (MSCs) are a promising stem cell source; however, their therapeutic efficacy in chronic wound healing remains limited. This study evaluates the therapeutic potential of transforming growth factor (TGF)-β1-modified, three-dimensionally cultured MSCs (A/T-3D) for enhancing wound healing. The TGF-β1 gene was inserted into a safe genomic locus in adipose-derived MSCs (ASCs) using transcription activator-like effector nucleases. Quantitative polymerase chain reaction (qPCR) analysis showed that A/T-3D upregulated key factors related to wound healing, including epidermal growth factor (EGF), TGF-β1, fibroblast growth factor (FGF), and vascular endothelial growth factor (VEGF)-A, compared to unmodified ASCs. In vitro scratch wound assays indicated that co-culture with A/T-3D-conditioned medium significantly accelerated wound closure in fibroblasts. In vivo, skin excision in nude mice treated with A/T-3D injections resulted in rapid wound closure, enhanced cellularity, and increased reepithelialization. High engraftment rates of A/T-3D and elevated expression of angiogenic factors were observed in the wound bed, highlighting their direct contribution to tissue repair. Collectively, these findings suggest that A/T-3D MSCs have significant therapeutic potential for wound healing through the secretion of epithelialization and angiogenic factors, along with enhanced engraftment capacity.

Structural and mechanical properties of engineered silkworm-spider composite silk.

Chronic infection with the hepatitis B virus (HBV) results in over 1 million deaths annually. Although currently licensed treatments, including pegylated interferon-α and nucleoside/nucleotide analogs, can inhibit viral replication, they rarely eradicate covalently closed circular DNA (cccDNA) reservoirs. Moreover, vaccination does not offer therapeutic benefit to already infected individuals or non-responders. Consequently, chronic infection is maintained by the persistence of cccDNA in infected hepatocytes. For this reason, novel therapeutic strategies that permanently inactivate cccDNA are a priority. Obligate heterodimeric transcription activator-like effector nucleases (TALENs) provide the precise gene-editing needed to disable cccDNA. To develop this strategy using a therapeutically relevant approach, TALEN-encoding mRNA targeting viral core and surface genes was synthesized using in vitro transcription with co-transcriptional capping. TALENs reduced hepatitis B surface antigen (HBsAg) by 80% in a liver-derived mammalian cell culture model of infection. In a stringent HBV transgenic murine model, a single dose of hepatotropic lipid nanoparticle-encapsulated TALEN mRNA lowered HBsAg by 63% and reduced viral particle equivalents by more than 99%, without evidence of toxicity. A surveyor assay demonstrated mean in vivo HBV DNA mutation rates of approximately 16% and 15% for Core and Surface TALENs, respectively. This study presents the first evidence of the therapeutic potential of TALEN-encoding mRNA to inactivate HBV replication permanently.