gene editing

LDLR Variant Classification Through Activity-Normalized Prime Editing Screening.

BACKGROUND Inherited variants in the LDL (low-density lipoprotein) receptor (LDLR) gene are the most common cause of familial hypercholesterolemia, significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt lipid-lowering therapy and cascade testing of at-risk relatives; however, most LDLR variants observed in the population have uncertain or absent clinical classifications, leaving many patients without actionable information. METHODS We developed the first activity-normalized prime editing screening pipeline to measure the impact of 5184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Each prime editing guide RNA is paired with a genotypic outcome reporter to correct for variable editing efficiency, overcoming a key limitation of previous pooled genome editing screens. A statistical framework further improves variant effect estimates by jointly analyzing all missense variants at each amino acid position. RESULTS We show that prime editing of the reporter construct correlates with endogenous variant installation frequency, validating the activity normalization approach. The resulting scores capture a continuous spectrum of functional effects, robustly separate pathogenic versus benign ClinVar variants, and show concordance with LDL-C levels in UK Biobank participants. We calibrate functional evidence strengths to the ACMG/AMP variant interpretation framework, enabling integration into a clinical variant classification workflow. By combining functional, computational, population, and contextual evidence, 322 of 434 LDLR variants currently classified as variants of uncertain significance, conflicting, or absent from ClinVar appear to meet evidence thresholds for reclassification and can be prioritized for expert review, substantially expanding the pool of actionable variant classifications. The screen also reveals a cluster of gain-of-function variants in LDLR class A repeat 5, at least some of which enhance LDL-C uptake through increased apolipoprotein B interaction, with implications for therapeutic genome editing. Last, prime editing uniquely detects splice-altering coding variants missed by cDNA-based screens and pathogenicity predictors, revealing an advantage of endogenous variant installation. CONCLUSIONS Altogether, activity-normalized prime editing provides a scalable framework for LDLR variant classification that substantially expands the proportion of variants with evidence for genetic diagnosis and reveals novel biology with therapeutic relevance.

Editorial: Phenylpropanoid metabolism in plants: functional diversity, stress resilience, and biotechnological applications

that govern them (Hanusch et al., 2026;Ninkuu et al., 2022;Tohge et al., 2018). Recent studies compiled in this special issue collectively demonstrate that plant chemodiversity is driven by intricate coordination between regulatory networks, metabolic pathways, and environmental signals. These studies, ranging from molecular evolution of biosynthetic enzymes to the ecological consequences of volatile compounds, collectively demonstrate how plants optimize their chemical architecture, highlighting novel perspectives on harnessing metabolic diversity for sustainable agriculture and biotechnological benefits.One fundamental question in plant metabolism is the expansion and diversification of gene families to produce chemical complexity. This question was addressed by Kaur et al through molecular characterization of the 4-Coumarate CoA ligase (4CL) gene family in orchids. 4-Coumarate CoA ligase is a key enzyme in the phenylpropanoid pathway involved in metabolic flux distribution by catalyzing the conversion of p-coumaric acid into p-coumaroyl-CoA. This key branching metabolite simultaneously feeds both the lignin and flavonoid pathways, producing a diverse array of metabolites (Ninkuu et al., 2023).Kaur et al in-silico analysis highlighted the structural diversity, evolutionary conservation, and potential functional specialization of 4CL members in orchids (Figure 1b). A comprehensive understanding of the 4CL enzyme can help elucidate the evolution of novel phenylpropanoid metabolites by plants.Although gene-family diversification expands the metabolic capacity for chemical diversification, this is dynamically regulated by transcriptional, epigenetic, and environmental factors. Hence, plant metabolism is driven by interconnected networks of biosynthetic genes, signaling pathways, transcription factors, and other regulatory mechanisms. Zheng et al. demonstrated this complexity in a transcriptome analysis of leaves from the upper and mid portions of Xanthoceras sorbifolia, revealing distinctions in flavonoid production between leaf tissues and emphasizing that flavonoid accumulation is driven by coordinated pathway components rather than by biosynthetic genes alone. The study also noted that higher flavonoid accumulation was associated with enhanced antioxidant capacity and stress tolerance, with FLS, 4CL, and FG3 regulating this process. These findings demonstrate that chemodiversity is not solely driven by expansion of biosynthetic capacity, but also by context-dependent modulation of metabolic flux. In related findings, Zhang et al. demonstrated that geographical origin can influence acteoside metabolism in Rehmannia glutinosa by reprogramming its transcriptome and metabolomic architecture in a coordinated manner (Figure 1d). This study showed that environmental cues influence the molecular signature underpinning medicinal plant quality. Specifically, Henan-grown roots showed coordinated upregulation of PAL, C4H, 4CL, TyDC, and UGT, supporting enhanced flux through the phenylpropanoid and tyrosinederived branches of acteoside biosynthesis. Understanding the environmental factors that influence medicinal plants' metabolism is essential for optimizing cultivation practices, standardizing these crops, and preserving the authenticity of products derived from them.Plants' internal regulations can also impact their ecological functions. For instance, the volatile profile in specific cultivars of Passiflora edulis (passion fruit) influences the feeding preference of thrips. According to Li et al., plant metabolites can mediate ecological signaling cascades, enhancing plant-organism interactions by using volatile compounds as communication signals for pollinators, herbivores, and microorganisms. Leaf volatile organic compounds (VOCs) in P. edulis influence thrips preference and resistance. A total of 87 differential VOCs, metabolically associated with the phenylpropanoid and αlinolenic acid pathways, were identified, including benzaldehyde and (Z)-3-hexenol as key compounds.Whereas benzaldehyde attracted 58% of thrips, (Z)-3-hexenol repelled 22% of them. This mechanism offers an interesting opportunity for future translational studies to develop environmentally friendly pest management products and provides clues for breeding targets to address pest resistance. These studies collectively showed that plant chemodiversity is generated through an intricate network of regulators, including transcription factors, gene families, environmental conditions, epigenetic modifications, and ecological interactions, rather than through isolated pathways. The intricate interactions among these factors equip plants to respond to external and internal stimuli.Future research should move beyond descriptive metabolite profiling toward predictive models that connect genotype, regulatory state, environment, and metabolic phenotype. Integrating multi-omics with machine learning, genome editing, and synthetic biology could enable researchers to predict and experimentally redirect metabolic flux under defined environmental conditions (Figure 1e). Predicting how environmental conditions, genetic variation, and regulatory mechanisms fine-tune metabolite production provides an opportunity to improve crop breeding, medicinal plant cultivation, and the sustainable production of natural products from plants (Feng et al., 2024).The contributions in this special issue reiterate that metabolites are dynamic outputs of environmental control, evolutionary history, and ecological relationships, rather than mere chemical molecules accumulated in plant tissues. Therefore, harnessing the potential of plant chemodiversity requires reframing an integrated approach beyond cataloging metabolites towards elucidating the biological principles governing their emergence, modulation, and functions. Moreover, the recent impact of climate change and growing demand for sustainable resource production have increased attention on deciphering plants' chemical language as alternative strategies to develop resilient crops, novel bioactive compounds, and build a sustainable future.

Cultivating the Next Generation: Early Career Researchers at the Forefront of Plant Science

Scientific exploration is a relay race across generations. From Darwin's observations on plant movement and adaptation, to Mendel's elucidation of fundamental genetic principles using peas, to the modern molecular dissection of plant stress response mechanisms, every advance has depended not only on the strategic vision of established scientists but on their mentorship of young scholars and graduate students in the laboratory as well. It is this intergenerational transmission that enables each generation of researchers to revisit problems and questions with new tools, building on prior knowledge and continually pushing the boundaries of inquiry. Within this chain of succession, one group is critically important yet often overlooked. This group is Early Career Researchers, or ECRs. As defined for this Special Issue, ECRs are investigators who have obtained their PhD within the last 7 years and are under 40 years of age. They are the backbone of daily laboratory operations, deeply involved in experimental design, data acquisition, and technical problem solving. They are adept at emerging technologies and possess a practical understanding of the strengths and limitations of modern research tools. Although ECRs perform the majority of the research execution in most teams, their opportunities to publish as corresponding authors are often limited, and their independent academic identity is frequently intertwined with the guidance of senior mentors. This situation constrains their own career development to some extent and, at the same time, hinders the field from fully benefiting from diverse perspectives and fresh ideas, precisely the kind of vitality that sustains disciplinary progress. Recognizing the importance of identifying and supporting early career talent, several plant science journals under Wiley, including JIPB, Nordic Journal of Botany, Food and Energy Security, Plant Environment Interactions, and our own Plant, Cell and Environment, jointly launched this Special Issue on Early Career Researchers in Focus, Breakthroughs in Plant Science in August 2024. The response was overwhelming, reflecting the remarkable creativity of researchers at this career stage. Over the past 2 years, PCE received over 100 submissions. Following rigorous peer review, 28 papers were ultimately accepted for publication. This Special Issue serves both as a showcase of ECRs' scientific contributions and as a concrete effort to build an academic platform for them. The breadth of research covered in this issue is truly impressive, reflecting the multifaceted nature of modern plant science, ranging from macroecology to molecular mechanisms. Thematically, the papers encompass everything from global-scale ecological surveys to local physiological adaptation mechanisms. Methodologically, they extend from traditional physiological measurements to cutting-edge deep learning applications and CRISPR gene editing technologies. The issue comprises 23 original research articles, 2 brief communications and 3 review articles, covering topics such as volatile-mediated plant defense (Li et al. 2026), the molecular regulation of anthocyanin biosynthesis in apple under low nitrogen (Meng et al. 2026), and transgene-free genome editing in trees using CRISPR ribonucleoproteins (Ramakrishnan et al. 2025). Several contributions integrate or synthesize multi-omics evidence to reveal novel molecular mechanisms, including the role of lysine acetylation in plant immunity (Villette et al. 2025) and lipidome reprogramming that enhances photosynthetic performance (Deepa et al. 2026). These works are not merely incremental advances; they also propose new conceptual frameworks, for example by revealing a universal genome size–cell size relationship across land plants (Mir-Rosselló et al. 2025) and demonstrating the decoupling of water and nitrogen translocation from subsoil to the canopy in forest trees (Mrak et al. 2025). By bringing these outstanding contributions together in a single collection, this Special Issue serves both to highlight the remarkable contributions of ECRs and to inspire future discoveries. We are confident that these emerging leaders will continue to shape the trajectory of plant science, and we hope this issue serves as an entry point for readers to appreciate their exceptional research. Data sharing not applicable to this article as no datasets were generated or analyzed during the current study.

Resistance to modulation of in vivo gene expression by genomic editing

Enhancer editing is widely proposed as a strategy to modulate gene dosage therapeutically. EKLF/KLF1, a master erythroid transcription factor, is an attractive target because haploinsufficiency is benign and associated with hereditary persistence of fetal hemoglobin (HPFH). Here we test whether enhancer disruption can recapitulate this state by targeting two KLF1 regulatory elements, EHS1 and INT1. EHS1 editing reduces KLF1 expression and increases γ-globin, and combined INT1/EHS1 targeting produces additional increases in some samples, although responses are variable. In xenotransplantation models, edited cells engraft and retain indels but fail to sustain consistent γ-globin induction. Epigenetic profiling across the KLF1 genetic locus reveals a remarkable resistance to loss of hypersensitive sites. These results establish a key principle: the transcriptional consequences of enhancer editing are strongly constrained by local genomic architecture. Attempts to mimic KLF1 haploinsufficiency by non-coding indels are blunted by dense regulatory architecture, enhancer redundancy, and long-range interaction. Thus, it may be difficult to develop KLF1 as a target for gene therapy for hemoglobinopathies until tools for recapitulating natural mutations via heterozygous edits are developed. Our findings underscore the observation that disruption of non-coding elements may not always recapitulate the biology of haploinsufficiency from heterozygous loss-of-function mutations. Structural and epigenetic properties of the human KLF1 locus constrain the in vivo effects of efficient editing at its non-coding regulatory elements, providing a cautionary tale for the design of functional enhancer modifications.

Cullin 3 ubiquitin ligase regulates redox defense and metabolic programs in proximal tubule cells

Abstract Background Chronic kidney disease (CKD) is characterized by proximal tubule (PT) stress, oxidative injury, and metabolic dysfunction. Human kidney single-nucleus RNA-sequencing (snRNA-seq) identified enrichment of ubiquitin-dependent protein catabolic processes in injured PT cells, suggesting activation of the ubiquitin-proteasome system during tubular stress. Because Cullin 3 (CUL3), scaffold of ubiquitin ligases, regulates oxidative stress signaling through the KEAP1-NRF2 axis, we investigated its role in PT injury and stress adaptation. Methods Human CKD snRNA-seq data and kidney immunostaining were used to define PT cell states, CUL3-associated pathways, and CUL3 localization. Injury-associated CUL3 regulation was examined in wildtype mice after ischemia-reperfusion injury (IRI). Inducible PT-specific knockout mice (Slc34a1-CreER; Cul3flox/flox) were analyzed at baseline and after injury by histology, immunostaining, proteomics, and injury assessment. In addition, proteomic analysis of a whole-tubule epithelial knockout model (Pax8-rtTA/LC1; Cul3flox/flox) was performed. In immortalized human PT cells, CUL3 was suppressed or activated using CRISPR interference and CRISPR activation, followed by bulk RNA sequencing. Results CUL3 transcript and protein expression was enriched in stressed PT states in human CKD. In mice, CUL3 protein abundance increased after injury, supporting injury-associated induction in vivo. PT-specific CUL3 deletion increased antioxidant NQO1 expression without causing overt baseline injury. Proteomic analysis of isolated CUL3-deficient PT cells revealed induction of antioxidant, detoxification, proteostasis, and lipid metabolic programs, together with suppression of mitochondrial oxidative metabolism. Similar changes were observed in whole-tubule Cul3 knockout model. In gene-edited human PT cells, CUL3 suppression recapitulated stress-associated and metabolic remodeling programs, whereas CUL3 activation induced reciprocal transcriptional changes. Despite induction of antioxidant pathways, PT-specific CUL3 deletion did not alter disease severity after IRI or aristolochic acid nephropathy. Conclusion CUL3 is an injury-induced regulator of PT metabolic and stress-associated states and modulates antioxidant defense and mitochondrial metabolism in PT cells.

Isolation, Cloning and Sequence Characterization of the Salinity-Responsive Transcription Factor Gene OsbHLH024 from Rice (Oryza sativa L.) Cv. Ratnagiri-8

Oryza sativa L. (rice) is a staple food crop whose productivity is increasingly constrained by soil salinity, a stress that disrupts ionic balance and limits growth and yield. Basic helix–loop–helix (bHLH) transcription factors are known regulators of plant abiotic stress responses, and OsbHLH024 has been reported to act as a negative regulator of salinity tolerance in rice, making it a promising candidate gene for functional and genome-editing studies. As a foundational step towards such studies, the present investigation reports the isolation, cloning and sequence-level validation of OsbHLH024 from the salt-tolerant indica cultivar Ratnagiri-8. The gene sequence (locus Os01g0575200) was retrieved from the Rice Annotation Project Database (RAP-DB), and five overlapping primer pairs were manually designed and evaluated in silico to amplify the ~4.35 kb gene as five fragments (P1–P5; 1001, 1001, 1001, 1051 and 701 bp). High-molecular-weight genomic DNA extracted from Ratnagiri-8 leaves by the CTAB method served as a template, and all five fragments were amplified as single, specific bands. The fragments were purified, ligated into the pJET1.2/blunt vector and transformed into Escherichia coli DH5α; colony PCR and plasmid-based PCR identified positive recombinant clones for all five fragments, which were subsequently confirmed by Sanger sequencing. Alignment of the assembled consensus sequence against the RAP-DB reference using BLAST and Clustal Omega showed 99.10% sequence identity, confirming successful isolation and cloning of OsbHLH024 from Ratnagiri-8. This sequence-validated clone constitutes a genomic resource for future comparative and functional characterisation of OsbHLH024 in salinity tolerance.

Multi-layer Cis-molQTLs Reveal Regulatory Architecture and Enhance Heritability Explanation for Complex Traits in Cattle.

Genome-wide association study (GWAS) analyses have identified numerous loci associated with economic traits in cattle. Many of these loci reside in noncoding regions, and the regulatory mechanisms through which they influence complex traits remain poorly understood. Here, we integrated 657 RNA-seq libraries from 275 Huaxi cattle across three tissues (longissimus dorsi muscle, liver, and subcutaneous backfat) with ∼ 10 million imputed SNP genotypes to systematically map cis-molecular quantitative trait loci (cis-molQTLs) across four transcriptomic regulatory layers: gene expression (eQTLs), splicing (sQTLs), alternative polyadenylation (aQTLs), and RNA editing (edQTLs). These cis-molQTL classes display distinct genomic distributions and functional enrichments, yet operate in a coordinated manner within complex trait regulatory networks and are significantly enriched near GWAS- and QTLdb-reported loci for growth, carcass, and meat quality traits. Using 1788 genotyped and phenotyped Huaxi cattle, a GREML framework showed that these multi-layer cis-molQTL SNPs collectively explain 61.9% of total SNP-based heritability across 19 complex traits. Incorporating cis-molQTL annotations into genomic prediction models, including MultiBLUP, BayesRC, and molGBLUP, improved prediction accuracy for most traits relative to the baseline GBLUP model (mean increase of 0.05), highlighting the value of multi-layer regulatory variation for functionally informed genomic prediction and precision breeding.

Beyond the single gene: Integrating genomic selection and genome editing for the improvement of polygenic traits in crop plants

Polygenic traits such as yield, drought tolerance, water-use efficiency, and nutritional quality are central to crop improvement but remain challenging targets because they are controlled by numerous loci and influenced by complex genotype-by-environment (G×E) interactions. These characteristics limit the effectiveness of single-gene approaches and highlight the need for integrated breeding strategies. This review examines the complementary roles of genomic selection (GS) and genome editing (GE) for the improvement of complex traits in crop plants. The genetic architecture of polygenic traits, the principles underlying genomic prediction, and recent advances in CRISPR-based genome editing are critically evaluated. Building on current knowledge, five integration strategies are discussed: GWAS-guided target prioritization, favorable allele deployment with polygenic background optimization, SpeedGS-assisted breeding, regulatory network editing, and environment-informed genome editing. The review also examines key challenges associated with GS–GE integration, including causal variant identification, epistasis, pleiotropy, genotype-by-environment interactions, transformation efficiency, and regulatory considerations. Finally, emerging developments in machine learning, multi-omics, and high-throughput phenotyping are highlighted as enabling technologies for future breeding programs. Overall, the integration of GS and GE is presented as a promising framework for accelerating genetic gain and improving complex polygenic traits in crops.

Advances in the Molecular Regulatory Mechanisms of Testicular Development and Spermatogenesis in Yaks

Yaks are indigenous livestock species of the Qinghai–Tibet Plateau. Due to long-term exposure to extreme environmental stressors, including high altitude, hypoxia, and low temperatures, its male reproductive system has evolved distinct adaptive strategies. Specifically, testicular weight is markedly lower than that of cattle. Nevertheless, according to reported data from different experiments, although its single ejaculate volume is lower than that of Tibetan cattle, it can still maintain comparable fresh sperm motility. These reproductive phenotypes make the yak an ideal model organism for investigating plateau adaptation and sperm energy metabolism. The present review synthesizes continuous molecular events in yak testes spanning embryonic development through senescence. It focuses on dissecting the core regulatory networks governing spermatogonial stem cell self-renewal, meiosis, and spermiogenesis. It integrates the latest advances in testicular microenvironment dynamics and epigenetic modifications. Additionally, male sterility in cattle-yak serves as a natural mutant model. Essential regulatory modules governing spermatogenesis can be inferred from its spermatogenic-arrest phenotype. Nevertheless, comparative omics analyses between yaks and cattle are confounded by seasonal variation and differing genetic backgrounds. It is therefore critical to distinguish signatures driven by genetic adaptation from those arising from environmental plasticity. Finally, this paper presents research prospects regarding how to utilize single-cell multi-omics and gene-editing technologies to deeply dissect the underlying mechanisms of plateau reproductive adaptation to provide theoretical support for improving yak fecundity and hybrid breeding.

Genetic and biotechnological advances in eggplant improvement for food security

Eggplant, a key strategic horticultural crop within the Solanaceae family, serves as a crucial source of micronutrients, bioactive phytochemicals, and functional foods significantly contributing to food security and sustainable agriculture. Its wide agro-climatic adaptability, economic importance, and versatile culinary applications have driven escalating demand amidst population growth, urbanization, and climate change-induced stresses. However, the genetic improvement of eggplant is hindered by the complex inheritance patterns of critical agronomic traits, including fruit yield, quality attributes, stress tolerance, and morphological diversity. These traits are often governed by polygenic genomic architectures, low heritability, epistasis, and genotype-by-environment interactions, limiting the efficacy of traditional breeding methods. Advancements in high-throughput genetic and genomic studies have paved the way for transformative interventions results configured whole-genome and assemblies, genome-wide associations, and molecular markers enable the dissection of genetic determinants underlying key agronomical traits. Furthermore, functional gene annotation via transcriptomics, and miRNAs cohorts offers unparalleled insights into phenotypic plasticity and molecular adaptation mechanisms underlying these traits. Moreover, the integration of these discoveries with next-generation gene editing technologies, particularly CRISPR-Cas9 systems, has revolutionized targeted trait introgression, enabling rapid development of elite, climate-resilient eggplant genotypes. In this review, we provide a comprehensive overview of recent breakthroughs in eggplant genetics and genomics, highlighting cutting-edge genetic and genomic resources catalyzing trait discovery and assisted breeding. Moreover, we discuss how the synergy between omics-driven insights and genome-editing platforms is reshaping breeding paradigms. Altogether, these integrative strategies not only accelerate trait enhancement but also fortify food security frameworks by fostering genetic diversity, adaptive capacity, and nutritional fortification in eggplant production systems.

Efficient Microinjection and CRISPR/Cas9 Mediated Genome Editing in Urechis unicinctus

Applications and Future Perspectives of CRISPR/Cas9 Gene Editing Technologies in CAR-T Cell Therapy

A recent major breakthrough in cancer immunotherapy is the Chimeric antigen receptor-T cell (CAR-T cell) therapy, which has shown significant clinical efficacy in haematological malignancy treatment. Nonetheless, its application in a more general way is limited by a number of challenges, such as T-cell exhaustion, off-target associated toxicities, and the difficulty of personalised manufacturing. Recently, new opportunities have come into solving these problems with the introduction of clustered regularly interspaced short tandem repeats (CRISPR)-Cas9 genome editing, which has made it possible to perform precise and combinatorial genetic editing in CAR-T cells. The important applications of CRISPR in CAR-T cell engineering, which include the disruption of inhibitory immune checkpoints to enhance antitumour activity, the generation of universal allogeneic CAR-T cells by deletion of T-cell receptor and human leukocyte antigen (HLA) genes, and the modulation of cytokine signalling pathways to reduce toxicity are discussed in this review. Moreover, novel approaches, including targeted CAR integration and multiplex gene editing, are discussed as having the potential to enhance the therapeutic efficacy and scalability. In spite of these improvements, there are issues of off-target effect, delivery efficacy, genomic instability, and unaddressed issues of long-term safety. CAR-T cell therapies are likely to be improved further in future through advancements in genome editing technology, delivery methods, and synthetic biology. In general, CRISPR/Cas9-based engineering is a promising way of developing the next generation of precision cancer immunotherapy.