The ever-increasing global demand for food and feed, coupled with population growth and climate change, necessitates the development of high-yielding and stress-resilient crops. CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) technology has emerged as a transformative genome-editing tool due to its efficiency, precision, and ability to generate targeted genetic modifications. This review highlights recent advances in CRISPR genome editing, including base editing, prime editing, and novel Cas variants, together with emerging approaches such as epigenome editing, mitochondrial genome editing, and hypercompact Cas systems, and their applications in improving crop productivity, stress tolerance, and nutritional quality. In food and feed crops, these applications also include crop-specific priorities such as grain and nutritional quality in food crops and digestibility, forage quality, and mineral bioavailability in feed and forage crops. Particular emphasis is placed on the complex intellectual property (IP) landscape surrounding CRISPR technologies, including patents on editing systems, delivery platforms, and proprietary vectors. These intellectual property barriers can restrict research access, technology transfer, and commercialization, particularly in developing countries. The review further discusses patent limitations, licensing frameworks, and strategies to enhance equitable global access through collaborative approaches such as non-exclusive licensing, humanitarian licensing, indigenous technology development, and international partnerships. Overall, the review underscores the need to balance innovation, intellectual property protection, and global accessibility to ensure that CRISPR-based technologies contribute effectively to worldwide food and feed security and sustainable agriculture.

The global burden of viral diseases in both humans and agricultural animals is exacerbated by dysregulated inflammatory cell death and cytokine storms. Pyroptosis, executed by gasdermin family proteins, is a key driver of this pathology, yet its functional conservation and therapeutic targeting in avian species remain critically unexplored. Here, we establish the first germline-edited heterozygous DFNA5-mutant chicken model using primordial germ cell mediated genome editing. We demonstrate that genetic disruption of DFNA5 markedly attenuates Raptinal-induced pyroptosis and reduces IL-1β and IL-18 secretion. Using an optimized adenoviral dual-sgRNA delivery system, we successfully generated chimeric chickens with stable germline transmission of a frameshift deletion in DFNA5. Notably, DFNA5 deficiency is associated with reduced expression of the viral gp85 gene following avian leukosis virus subgroup J (ALV-J) infection. Mechanistically, transcriptome analysis reveals that downregulated genes in ALV-J-infected DFNA5-mutant cells are enriched in apoptosis and NOD-like receptor signaling pathways, while upregulated genes are associated with cell adhesion, extracellular matrix organization, and homeostatic pathways, suggesting a possible shift from pyroptotic inflammation toward cellular integrity and repair. This work provides genetic evidence that a possible association between DFNA5-mediated pyroptotic responses and establishes a genetic strategy for poultry disease resistance research and for understanding host-virus evolution and viral-induced inflammatory pathologies across vertebrates.

Sustainable improvement of rice yield requires coordinated modification of multiple agronomic traits, yet the agronomic outcomes of multiplex genome editing remain difficult to predict. Here, we applied CRISPR/Cas12a-mediated multiplex editing in the indica rice cultivar Kasalath to target six yield-related genes, Gn1a, TAD1, ROC5, GS3, GW2, and LARGE2, using a single Agrobacterium-delivered construct. Sanger sequencing of 28 T0 plants detected edits in Gn1a (3.57%), ROC5 (7.14%), GS3 (35.71%), and GW2 (10.71%), whereas TAD1 and LARGE2 showed no detectable T0 edits. Single, double, and triple edits were recovered, and homozygous lines were established for four single mutants, two double mutants, and two triple mutants. Molecular analysis showed that the recovered GW2 alleles were intronic and did not alter the GW2 coding sequence. Phenotypic evaluation across 13 agronomic traits showed that allele type and locus combination, rather than simple additive effects, determined agronomic outcome. The gs3-13d frameshift allele caused strong pleiotropic effects, including increased tillering and panicle number but reduced grain length and yield. The roc5-7d gw2-8d gs3-3i triple mutant maintained wild-type-level yield performance with altered leaf architecture, whereas gw2-8d gs3-3i large2-10d showed increased grain width, grain thickness, and 1000-grain weight but severe reductions in plant height, tillering, seed setting, yield per panicle, and yield per plant. These results demonstrate the feasibility of CRISPR/Cas12a multiplex editing in Kasalath and highlight the importance of allele design, locus combination, and source-sink balance in multiplex genome editing targeting yield-related traits.

Transgenic plants and genome editing technologies are revolutionizing agriculture through sustainable approaches to food security, pest management, and adaptation to climate change; but their widespread use is hampered by regulatory systems that are fragmented, ethics considerations, and an ongoing lack of trust from the general public. In contrast to other literature that evaluates regulation processes and public acceptance separately, our review paper introduces a new, holistic approach that includes both technical risk assessment from a scientific perspective, and Codex Alimentarius and OECD standards, and the socio-legal and judicial environment of how the national policy decisions are actually made. The paper provides a comparative, historical analysis of the key difference between product- and process-based risk governance in the USA, the EU, and India. Through the use of case studies with global significance like MON810 maize, Bt Brinjal, and the April 2024 Philippine Court of Appeals' order for cease-and-desist of Golden Rice, we discuss the increasing tension between administrative scientific approvals and precautionary judicial orders. We further explore the emerging exemptions to regulation of Site-Directed Nuclease (SDN-1 and SDN-2) genome edited crops which led to India's revolutionary 2025 commercialization of climate-resilient rice crops. Our review ends with a forward-looking approach to biotechnology regulation policy, making an appeal to shift from static historical dichotomies towards flexible risk-proportionate and internationally coordinated regulatory systems. Finally, we show that global success of agricultural biotechnology is not just about safety verification, but rather about establishment of transparent and communicable institutions that can transform scientific risk assessments into legitimate risk management decisions.

Brassica juncea is a common cruciferous crop, which can be used not only for oil extraction but also as condiments and medicinal materials. It is regarded by both traditional medicine and modern nutrition science as a food with combined dietary and health promoting value. Leaf trichomes are hair-like structures differentiated from epidermal cells and constitute an important barrier against biotic and abiotic stresses, playing a crucial role in enhancing plant resistance and thus possessing significant scientific relevance. In this study, the phenotype and gene editing site of BjA06.GL1 and BjB02.GL1 knockout mustard T1 generation plants were identified. Then, RNA sequencing was performed to compare the leaf transcriptome profiles between gene-edited lines and wild-type plants, with the aim of elucidating the molecular regulatory mechanisms by which BjGL1 controls leaf trichome development and associated biological processes in mustard. The sequencing data showed that, on average, 90.64% of the reads uniquely aligned to the Brassica juncea (Xuecai) reference genome. A total of 4,604 differentially expressed genes were identified in this study. Compared with the gene knockout mutant, 1,831 genes were significantly upregulated and 2,773 genes were downregulated in mustard leaves with trichomes. The differentially expressed genes were mainly enriched in pathways related to cytochrome P450 (CYP), transporters, environmental adaptation, and plant-pathogen interactions. These pathways are closely associated with secondary metabolite biosynthesis, transmembrane transport, and responses to abiotic stress and pathogen defense. qRT-PCR validation confirmed consistent expression trends of trichome regulatory genes screened from transcriptome data. This study provides an important theoretical basis for elucidating molecular mechanisms potentially contributing to trichome formation in mustard.

Global climate change and shrinking freshwater supplies threaten agricultural sustainability. A major translational bottleneck exists: drought-tolerant genotypes developed in labs often fail in open-field conditions due to phenotypic mismatch. Traditional single-gene approaches and static lab tests overlook genotype-by-environment (G × E) interactions, hormonal tradeoffs (involving Abscisic Acid, Jasmonic Acid, Brassinosteroids, and Melatonin), and artificial effects of pots. This review presents an integrated quantitative selection framework to bridge lab and field results. In this review, we propose a conceptual three-stage Predictive Translational Workflow designed to bridge lab and field outcomes by integrating multi-omics envirotyping with advanced mixed models like (G × E)-BLUP and MegaLMM. These methods group complex transcriptomic and metabolomic data into functional networks, filtering out lab artifacts and pinpointing core regulatory nodes stable in real climates. Finally, we show how this pipeline enables precise CRISPR/Cas9 editing of native promoters, reducing agronomic yield penalties in normal conditions and offering a streamlined, non-GMO path to field-resilient crops.

Sheep represent an important large-animal model for biomedical research and biopharmaceutical production. Although the piggyBac transposon system offers efficient and stable genomic integration, the optimal gene delivery strategy for ovine embryos remains unclear. This study evaluated piggyBac-mediated transgenesis using pronuclear injection (PNI) in both in vivo- and in vitro-derived embryos and assessed cytoplasmic injection (CTI) as an alternative approach. In vivo-derived embryos were obtained from superovulated Kıvırcık ewes approximately 40 h after gonadotropin-releasing hormone administration, whereas in vitro-derived embryos were produced from slaughterhouse-derived oocytes. All embryos were injected with the hyperactive piggyBac transposase-based pmhyGENIE-3 construct (10 ng/µL). In vivo-derived embryos were transferred immediately after injection, whereas in vitro-derived embryos were cultured for 3 days and screened for EGFP expression prior to transfer. Among 65 in vitro-derived embryos injected by PNI, no transgenic offspring was obtained. In contrast, PNI of 19 in vivo-derived embryos resulted in one transgenic lamb (5.3%). CTI of 12 in vivo-derived embryos similarly produced one transgenic lamb (8.3%). Whole-genome sequencing of the healthy founder male produced by CTI identified a single detectable genomic integration locus on chromosome 10 within a non-coding RNA locus (LOC121820439). Germline transmission was confirmed by in vitro fertilization using sperm from the founder male, with EGFP expression detected in 10.1% (10/99) resulting embryos. These findings provide proof-of-concept evidence for piggyBac-mediated transgenesis in sheep and support the feasibility of cytoplasmic injection as an alternative gene-delivery approach under the conditions tested.

The study aimed to construct and evaluate an independently generated transgenic mouse model applied to the short-term carcinogenicity assessment. Mutated human HRAS fragment containing an intron point-mutation was inserted into C57BL/6JGpt mice via bacterial artificial chromosome transgenic technology, eventually generating BALB/c;B6J-Tg(hHRAS)16/Gpt mice, abbreviated as HRAS mice. The inserted human HRAS fragment in HRAS mice was characterized, revealing five tandem copies at chromosome 19. Baseline profiles, including biochemical, hematological, immunophenotypic, survival, and carcinogenic data of HRAS mice, were collected. To evaluate the tumor susceptibility in HRAS mice, we applied N-Nitroso-N-methylurea (MNU) to HRAS mice in a short-term carcinogenicity assessment conducted according to Good Laboratory Practice. The genetic characteristics of HRAS mice include five tandem arrays of mutated human HRAS fragments located in genomic coordinate 7,755,606 on chromosome 19 and the duplication of a 9-kilobase genome sequence (genomic coordinate 7,755,606-7,746,509) located on chromosome 19. HRAS mice showed a relatively lower incidence and range of spontaneous tumor formation during long-term observation compared to CByB6F1-Tg(HRAS)2Jic (Tg.rasH2) transgenic mice. The short-term carcinogenicity assessment showed a strong tumor response to MNU, with high incidences of lymphoma (≥ 90%) and stomach squamous cell papilloma (≥ 90%) in both male and female HRAS mice. The HRAS mice showed susceptibility to MNU and exhibited baseline characteristics distinct from those of Tg.rasH2 mice. The co-expression of HRAS and MKI67 at the cellular localization level was found in neoplasms of HRAS mice. These findings preliminarily evaluated the feasibility of HRAS mice applied to the short-term carcinogenicity assessment.

This study reports the first isolation and characterization of the buffalo EF1α1 promoter, demonstrating its strong gene expression activity both in vitro across diverse cultured cell types and in vivo across multiple mouse organs. Although viral promoters, such as cytomegalovirus (CMV) and simian virus (SV40), are widely used for their strong expression in various cell lines in mammalian expression systems and in animal tissues, they are prone to methylation-induced transcriptional silencing and subsequent loss of exogenous gene expression. The most effective alternative to viral promoters is the synthetic hybrid CAG promoter (cytomegalovirus major immediate-early enhancer combined with the chicken beta-actin promoter) or mammalian cellular promoter such as human elongation factor 1 alpha (hEF1α), which drives strong gene expression but lacks consistency and is limited in their in vivo expression potential due to their vulnerability to epigenetic silencing. To overcome these challenges, the bbEF1α1 promoter was cloned and evaluated both in vitro and in vivo. It consistently drives higher levels of exogenous gene expression than CMV in diverse cell lines. Importantly, transgene expression was achieved in various organs of transgenic mice and in muscle tissue following in vivo electroporation. These findings establish the bbEF1α1 promoter as a powerful ubiquitous driver of gene expression, offering high stability with broad applications in gene therapy, biopharmaceutical production, and functional genomics.

Noccaea caerulescens is a metal hyperaccumulator plant species with the capacity to accumulate high concentrations of zinc (Zn), cadmium (Cd) and nickel (Ni). Several candidate genes have so far been associated with accumulation and tolerance of these metals, but gene function analysis has been cumbersome in the absence of an efficient stable plant transformation method for N. caerulescens. A previously identified mutation in the FLOWERING LOCUS C gene, conferring early flowering to the calamine accession St. Felix de Pallières, has been introgressed in the genetic background of five different genotypes, originating from the calamine populations Clough Wood (CLW) and Le Blémard (BLE), the ultramafic population Cira (CIR), and the non-metallicolous populations St. Baudille (SBD) and Werschmatt (WER). Agrobacterium tumefaciens mediated floral dipping transformation was employed in five introgression lines in these backgrounds and in Arabidopsis thaliana as control. Three introgression lines, of genetic backgrounds CLW, BLE and SBD, were successfully transformed, achieving an average transformation efficiency ≥ 0.29% for each background. A reproducible, stable transformation method based on floral dipping is presented here as an improvement on previously reported methods. The early flowering N. caerulescens flc mutation can be readily introgressed into different genetic backgrounds to facilitate genetic transformation studies, which is expected to provide a highly desired extra genetic tool to study gene function analysis in the metal hyperaccumulator N. caerulescens.

Cyclophilins are ubiquitous proteins that are essential for cis-trans isomerisation of peptide bonds preceding the proline residue. Apart from catalysing the cis-trans isomerization, these proteins are also implicated in diverse cellular functions. We investigated the role of a cyclophilin gene PoxCYP62-2, isolated from a halotolerant strain of Penicillium oxalicum, in abiotic stress tolerance of plants. Ectopic expression of PoxCYP62-2 gene in transgenic Arabidopsis thaliana plants conferred tolerance to cold stress. The transgenic plants exhibited higher rosette fresh and dry mass, lower MDA content, and increase in chlorophyll and proline content. Metabolite profiling revealed cold-induced increase in several metabolites that are known to enhance cold tolerance of plants in transgenic plants. Confocal microscopy suggested that PoxCYP62-2 protein was localized to nucleus in agroinfiltrated leaves of Nicotiana benthamiana. These findings suggests that PoxCYP62-2 gene is a potential candidate for enhancing cold stress-tolerance of crop plants.

This review evaluates CRISPR/Cas applications in agriculture from a global perspective with explicit reference to Türkiye. Using a literature gap-matrix approach organised around four analytical dimensions-environmental, economic, social and policy, and scientific and technological-we synthesize the primary evidence on water and input use, productivity, disease resistance, and product quality. The literature concentrates on water and fertilizer use, productivity, and off-target accuracy, whereas soil health, biodiversity, consumer acceptance, ethical considerations and regulatory frameworks remain systematically under-represented. Global deployment of CRISPR is already delivering measurable advantages in food security, shelf life and nutritional value, while in Türkiye the research base is at an early stage but has clear potential in wheat, barley, tomato and olive. Translating CRISPR into Turkish agricultural sustainability requires (i) a domestic biosafety framework aligned with the emerging European New Genomic Techniques approach, (ii) sustained investment in multi-location primary field trials, and (iii) inclusive deployment mechanisms-particularly through producer cooperatives-that allow smallholder farmers to benefit from edited varieties.