1.Introduction Crop breeding is transitioning to engineering by synthetic biol-ogy.Conventional breeding,constrained by limited genetic varia-tion and lengthy development cycles,cannot meet the challenges of micronutrient malnutrition and yield reductions from climate change with sufficient speed or precision[1].

A growing global population and the increasing prevalence of diet-related health issues such as “hidden hunger”, obesity, hypertension, and diabetes necessitate a fundamental rethinking of crop design and breeding. Synthetic metabolic engineering offers a method to modify and redesign metabolic pathways to increase the nutritional value of crops. We summarize recent advances in the biofortification of key nutrients including provitamin A, vitamin C, vitamin B9, iron, zinc, anthocyanins, flavonoids, and unsaturated fatty acids. We discuss the potential of multi-gene stacking, gene editing, enzyme engineering, and artificial intelligence in synthetic metabolic engineering. We propose future research directions and potential solutions centered on leveraging AI-driven systems biology, precision gene editing, enzyme engineering, agrobacterium-mediated genotype-independent transformation, and modular metabolic engineering strategies to develop next-generation nutritionally enhanced super crops and transform global food systems.

The growth rate of global grain production can no longer meet the demands arising from population expansion. Meanwhile, climate change, cultivated land degradation and environmental stress are further exacerbating the vulnerability of the food production system. Ensuring food security is fundamental to maintain the stability and sustainable development of human society. Crop breeding is the pivotal technical means to achieve this goal. Developing new crop varieties with high yield, superior quality, and multiple resistances have become the core pathway to safeguard agricultural sustainable development and global food supply. However, traditional breeding techniques suffer from long cycles, low accuracy and limited efficiency, which cannot satisfy the development requirements of modern seed industry. The rapid iteration of artificial intelligence (AI) technology has injected new intelligent momentum into the innovation of crop breeding, driving the transformation of breeding technology from traditional experience-based breeding to precise, intelligent and efficient breeding. In this review, we summarized the development of crop breeding, and focused on the innovative breakthroughs of genomic selection, precision genome editing, protein design and high-throughput phenotyping driven by AI. We also further elaborated the intelligent driving effects exerted by these technologies on key breeding links involving germplasm mining, gene function analysis, directional trait improvement, intelligent phenotypic assessment and intelligent factory breeding. Finally, we discussed the challenges and future developmental prospects of AI deployment in crop breeding, aiming to provide a reference for the innovation and industrial application of intelligent breeding technologies.

CRISPR-based prime editors (PEs) install precise edits into genomic DNA without generating double-strand breaks. Their editing efficiency is highly dependent on reverse transcriptases (RTs), but efficient RT candidates remain limited. Here, we identified 19 novel active RTs by screening 558 candidates. Among them, RERV-RT, derived from Rattus norvegicus, exhibited the highest activity. Through structure-guided engineering and deep mutational scanning, we developed an optimized variant, enRERV-RT, which outperforms conventional M-MLV-RT-based PE systems by 1.20-fold in mammalian and plant cells, and by 1.88-fold at hard-to-edit loci, while enabling precise multiplex editing of functionally relevant genes. Additionally, we developed a high-throughput platform, TRAP-seq-PE, to systematically evaluate prime editor performance. Across diverse mutation types, we found that PE systems based on enRERV-RT exhibited higher editing efficiencies than those based on M-MLV-RT. Collectively, our work establishes a versatile, high-efficiency PE system, thereby facilitating advances in clinical gene therapy and precise crop breeding.

Recent advances in clustered regularly interspaced short palindromic repeats (CRISPR) technology enable precise genetic modifications and produce genetically modified organism -free crops that match consumer preferences. By 2035, we will be able to consume CRISPR-edited crops, addressing food security issues and boosting economies for individual countries. This review highlights the progress of genetically modified crops and the regulatory challenges involved in bringing CRISPR-edited crops to market based on product- and process-based approaches across different regions. We also examine public preferences regarding these technologies and the current status of CRISPR-edited crops in terms of market availability. Furthermore, we stress the importance of establishing clear safety standards, effective patent management, and guidance on regulatory pathways for crop approval, as well as exploring future directions for integrating these technologies with artificial intelligence.

Exo-Cas12i2 v1, a fusion of the 5' exonucleases T5E and PapE, facilitates editing of TA-rich regions and mediates deletions of large genomic fragments. Exo-Cas12i2 v1-driven MITE manipulation enables precise regulation of genes involved in gibberellin-mediated cell elongation and root ethylene responses to generate favorable agronomic traits.

Betalain, an economically valuable water-soluble natural plant pigment, is prized for its strong antioxidant activity, making it popular as a dietary supplement and a visual marker for plant transformation. However, market demand significantly outstrips current production capacity. This study reports the development of an efficient push-and-pull multigene strategy based on polycistronic expression and metabolic flux regulation to enhance betalain biosynthesis in transgenic maize (Zea mays L.) endosperm. We engineered a novel enhanced RUBY (eRUBY) system derived from the original polycistronic RUBY construct (CYP76AD1P2ADODA1P2ADOPA5GT unit, abbreviated CDG) by introducing arogenate dehydrogenase (ADHa) to increase the L-tyrosine substrate supply. All the genes were driven by the endosperm-specific promoter. Fusion of ADHa into a single polycistronic eRUBY construct (CDGA) produced significantly higher betanin (6.88 mg g-1 dry weight) and isobetanin (1.81 mg g-1 dry weight) levels than in CDG + A, which stacked the ADHa cassette independently with CDG. The high betalain accumulation in CDGA lines (which also exhibited higher transgene copy number) resulted in a 2.85-7.58-fold improvement in endosperm antioxidant capacity compared to WT (versus 2.48-2.80-fold in CDG + A). Importantly, transgenic plants maintained a normal phenotype. Transcriptome and metabolome analyses further indicated that metabolism of phenylalanine, alanine, aspartate, and glutamate contributes to betalain production. Hybridization with sweet corn successfully created a high-sugar eRUBY maize variety. Collectively, these results demonstrate the successful development of a novel maize germplasm with significantly enhanced nutritional value through high betalain accumulation. (c) 2025 Crop Science Society of China and Institute of Crop Science, CAAS. Production and hosting by Elsevier B.V. on behalf of KeAi Communications Co., Ltd. This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/).

Genome editing using CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated protein) or other systems has become a cornerstone of numerous biological and applied research fields. However, detecting the resulting mutations by analyzing sequencing data remains time consuming and inefficient. In response to this issue, we designed SuperDecode, an integrated software toolkit for analyzing editing outcomes using a range of sequencing strategies. SuperDecode comprises three modules, DSDecodeMS, HiDecode, and LaDecode, each designed to automatically decode mutations from Sanger, high-throughput short-read, and long-read sequencing data, respectively, from targeted PCR amplicons. By leveraging specific strategies for constructing sequencing libraries of pooled multiple amplicons, HiDecode and LaDecode facilitate large-scale identification of mutations induced by single or multiplex target-site editing in a cost-effective manner. We demonstrate the efficacy of SuperDecode by analyzing mutations produced using different genome editing tools (CRISPR/Cas, base editing, and prime editing) in different materials (diploid and tetraploid rice and protoplasts), underscoring its versatility in decoding genome editing outcomes across different applications. Furthermore, this toolkit can be used to analyze other genetic variations, as exemplified by its ability to estimate the C-to-U editing rate of the cellular RNA of a mitochondrial gene. SuperDecode offers both a standalone software package and a web-based version, ensuring its easy access and broad compatibility across diverse computer systems. Thus, SuperDecode provides a comprehensive platform for analyzing a wide array of mutations, advancing the utility of genome editing for scientific research and genetic engineering.

Base editing enables precise nucleotide substitutions within a relatively broad editing window (5-6 nucleotides). However, considerable bystander editing significantly compromise its accuracy. Point mutagenesis, a powerful approach for gradient-tuning protein function, facilitates the generation of diverse plant phenotypes to meet the demands of complex environments and consumer preferences. Here, a series of plant base editors is engineered by fusing three optimized TadA8e variants, TadA9, TadA-LM, and TadA-dual, with a PAM-flexible SpRY nickase (SpRYn, with 5'-NNN PAM recognition). These editors enable A-to-G, C-to-T, and dual-base (simultaneous A-to-G and C-to-T) conversions within a highly condensed active window (1-3 nucleotides). Performance evaluations reveal that the TadDBE (TadA Dual-Base Editor) achieves the most robust outcomes, delivering dual-base editing efficiencies ranging from 2.3% to 61.4%, while maintaining minimal off-target activity. Utilizing TadDBE, targeted point mutagenesis is performed on OsBadh2, a gene encoding betaine aldehyde dehydrogenase that plays a critical role in the biosynthesis of 2-acetyl-1-pyrroline (2-AP), a key aromatic compound. This approach yields rice lines exhibiting gradient-tuned aromatic profiles and optimized levels of 2-AP and γ-aminobutyric acid (GABA). These evolved TadA-derived editors provide a precise, PAM-flexible platform for base editing and represent a versatile strategy for generating genome-edited plants with gradient-tuned agronomic traits.

The canonical CRISPR/Cas editors are constrained by the requirement for specific PAMs, which substantially limits their editable target range. Rice (particularly indica rice) is sensitive to low temperature, which impacts the yield and restricts the geographic distribution of rice. In this study, we developed PAM-flexible multiplex genome editing tools based on SpG (recognising NGN-PAMs) and SpRY (recognising NNN-PAMs) variants. We then tested the feasibility of using a sweet potato leaf curl virus (SPLCV) replicon-based expression vector and single-stranded DNA-binding domain (DBD) to improve the editing efficiency of these PAM-flexible editors. Furthermore, we used SpG-mediated multiplex genome editing to achieve comprehensive improvement in cold tolerance in indica rice by editing WRKY transcription factors OsWRKY53 and OsWRKY63, to generate high cold-resistant indica rice lines. We concluded that these PAM-flexible multiplex genome editors are powerful tools for multi-gene editing for crop genetic improvement.

Ginsenosides, the primary bioactive components of Panax ginseng, exhibit diverse pharmacological properties, ranging from anticancer to neuroprotective effects. However, traditional production by ginseng cultivation faces limitations due to extended growth cycles, insufficient yields, intricate extraction processes, and significant environmental dependencies. Synthetic biology and synthetic metabolic engineering offer promising alternatives for sustainable manufacturing of essential bioactive compounds, including ginsenosides. First, this review describes the ginsenoside biosynthesis pathways, emphasizing crucial enzymes (e.g., HMG-CoA reductase, squalene epoxidase, dammarenediol-II synthase, amyrin synthase, and various UDP-glycosyltransferases) and their regulatory networks. Understanding these fundamental pathways enables rational engineering of production systems. Second, it examines current synthetic biology approaches, encompassing plant cell, tissue, and hairy root cultures, engineered microbial hosts including Saccharomyces cerevisiae and Escherichia coli, and cell-free enzymatic synthesis. Third, it evaluates the medicinal significance, market prospects, and industrial feasibility of these biomanufactured compounds. Finally, it analyzes the sustainability of production models and explores the emerging potential of engineered plant chassis. These advanced methodologies directly address traditional agricultural constraints and establish a robust framework for future ginsenoside synthesis.

eRUBY is a powerful visual reporter combining a feedback-insensitive TyrA arogenate dehydrogenase (ADHα) gene with original RUBY structure, and substantially promotes betalain biosynthesis in rice endosperm.

Plant science has entered a transformative era as genome editing enables precise DNA modifications to address global challenges such as climate adaptation and food security. These modifications are primarily driven by the integration of three modular components—DNA-targeting modules, effector modules, and control modules—that can be selectively activated or suppressed. The field has evolved from protein-based systems (e.g., zinc finger nucleases and transcription activator-like effector nucleases) to RNA-guided systems (e.g., CRISPR-Cas) that can control both genetic and epigenetic states. Modular pairing of DNA-targeting and effector domains, with or without inducible control, enables precise transcriptional regulation and chromatin remodeling. The present review examines these three modules and highlights strategies for their optimization. It also outlines innovative tools, such as optogenetic and receptor-integrated systems, that enable spatiotemporal control over genome editor expression. These modular approaches bypass traditional limitations and allow scientists to create plants with desirable traits, decipher complex gene networks, and promote sustainable agriculture.

As an efficient gene editing tool, the CRISPR/Cas9 system has been widely employed to investigate and regulate the biosynthetic pathways of active ingredients in medicinal plants. CRISPR technology holds significant potential for enhancing both the yield and quality of active ingredients in medicinal plants. By precisely regulating the expression of key enzymes and transcription factors, CRISPR technology not only deepens our understanding of secondary metabolic pathways in medicinal plants but also opens new avenues for drug development and the modernization of traditional Chinese medicine. This article introduces the principles of CRISPR technology and its efficacy in gene editing, followed by a detailed discussion of its applications in the secondary metabolism of medicinal plants. This includes an examination of the composition of active ingredients and the implementation of CRISPR strategies within metabolic pathways, as well as the influence of Cas9 protein variants and advanced CRISPR systems in the field. In addition, this article examines the long-term impact of CRISPR technology on the progress of medicinal plant research and development. It also raises existing issues in research, including off-target effects, complexity of genome structure, low transformation efficiency, and insufficient understanding of metabolic pathways. At the same time, this article puts forward some insights in order to provide new ideas for the subsequent application of CRISPR in medicinal plants. In summary, CRISPR technology presents broad application prospects in the study of secondary metabolism in medicinal plants and is poised to facilitate further advancements in biomedicine and agricultural science. As technological advancements continue and challenges are progressively addressed, CRISPR technology is expected to play an increasingly vital role in the research of active ingredients in medicinal plants.

CRISPR/Cas-based genome-editing technology serves as a powerful and versatile tool for genome modification. It has been broadly utilized in crop breeding to enhance traits such as yield, various quality attributes, and biotic and abiotic stress tolerance. Because of public safety concerns over genetically modified organisms (GMOs), many countries have established stringent regulatory policies for genetically modified plants, dramatically limiting the application of related products. However, genome editing in stably transformed plants can result in transgene-free progeny through self-pollination or hybridization or yield DNA-free gene-edited plants via transient transformation. These edited plants materially differ from GMOs and are referred to as genome-edited organisms (GEOs). GEOs have the potential to alleviate regulatory burdens and aid in commercialization. Various methods have been developed to expedite the creation of transgene-free or DNA-free GEOs. This review summarizes the various strategies for creating these types of GEOs based on the CRISPR/Cas systems. It also covers the advantages and drawbacks of these strategies. Additionally, we examine off-target effects and mitigation strategies for plant genome editing and outline regulatory policies for gene-edited crops in selected countries and regions. We hope this review offers valuable references for the advancement of transgene-free and DNA-free GEOs.

Research into plant gene function is crucial for developing strategies to increase crop yields. The recent introduction of large language models (LLMs) offers a means to aggregate large amounts of data into a queryable format, but the output can contain inaccurate or false claims known as hallucinations. To minimize such hallucinations and produce high‐quality knowledge‐based outputs, the abstracts of over 60 000 plant research articles are compiled into a Chroma database for retrieval‐augmented generation (RAG). Then linguistic data are used from 13 993 Arabidopsis ( Arabidopsis thaliana ) phenotypes and 23 323 gene functions to fine‐tune the LLM Llama3‐8B, producing PlantGPT, a virtual expert in Arabidopsis phenotype–gene research. By evaluating answers to test questions, it is demonstrated that PlantGPT outperforms general LLMs in answering specialized questions. The findings provide a blueprint for functional genomics research in food crops and demonstrate the potential for developing LLMs for plant research modalities. To provide broader access and facilitate adoption, the online tool http://www.plantgpt.icu is developed, which will allow researchers to use PlantGPT in their scientific investigations.