Climate change, rising global food demand, and shrinking resources require transformative innovations in crop breeding. This review outlines recent advances in new breeding technologies (NBTs), including molecular markers, genome-wide association studies (GWAS), genomic selection (GS), next-generation sequencing (NGS), and gene editing (GE) tools such as the clustered regularly interspaced short palindromic repeat (CRISPR/Cas), base editing, and prime editing. These methods enable the accurate improvement of traits, thereby accelerating the development of crops resistant to both abiotic and biotic stresses. The integration of multi-omics platforms, including genomics, transcriptomics, proteomics, metabolomics, and phenomics, provides a comprehensive framework for deciphering and manipulating complex trait architectures. Artificial intelligence (AI) and machine learning (ML) enhance precision breeding by providing data-driven insights and enabling the forecasting of traits. Emphasis is also placed on combining gene editing with other strategies, such as speed breeding, to accelerate the development of traits. This review underscores the importance of an integrated systems biology approach that combines multi-omics, gene editing, AI, and speed breeding to accelerate the development of climate-resilient, high-yielding, and nutritionally enhanced crops. The integration of these innovative technologies holds great promise for addressing global food security, environmental sustainability, and agricultural resilience in the face of climate change. A strategic framework for the future of plant breeding is outlined, emphasizing the importance of interdisciplinary collaboration in building a sustainable agricultural future.

Over the last couple of decades, tremendous progress has been made in legume genomics. Genomics information generated for legume crops is being explored through molecular breeding and transgenic approaches. However, the gap between knowledge generation and its utilization is increasing. In this regard, recent developments in genome editing techniques provide an excellent opportunity to utilize the available knowledge for the improvement of legume crops. This review highlights recent developments with Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated protein 9 (CRISPR/Cas9)-based genome-editing approaches, including Cas variants/orthologs and Protospacer adjacent motif-less (PAMless) Genome Editing, multiplex genome editing, base editing, prime editing, transcriptional regulation, methylome editing, and DNA-free editing methods. Furthermore, the applications of non-homologous end joining (NHEJ) and homology-directed repair (HDR)- based editing, are addressed which enable targeted and precise genomic modifications. Moreover, virus-mediated genome editing, in planta transformation, and mobile guide RNAs are increasingly being leveraged to enhance the efficiency and heritability of genome editing. Additionally, the role of artificial intelligence in guide RNA design, off-target prediction, and the development of novel Cas variants is also discussed, which can speed up the legume improvement. This article highlights the successful examples of efforts utilizing CRISPR/Cas9 for the development of legume crops with biotic and abiotic stress tolerance, desirable plant architecture, improved nutrient uptake, and enhanced yield and quality. The biggest limitation in the genome editing of legume crops is their recalcitrance to both transformation and tissue culture. This article discusses how this particular limitation can be addressed in the context of genome editing of legume crops. Finally, the possibilities of integrating these recently developed tools with translational breeding have also been discussed, which will facilitate the legume production for sustainable agriculture and food security.

Efficient and accurate prime editing system in plants.

Gene editing technologies have revolutionized therapeutic development, offering potentially curative and preventative strategies for cardiovascular disease (CVD), which remains a leading global cause of morbidity and mortality. This review provides an introduction to the state-of-the-art gene editing tools—including ZFNs, TALENs, CRISPR/Cas9 systems, base editors, and prime editors—and evaluates their application in lipid metabolic pathways central to CVD pathogenesis. Emphasis is placed on targets such as PCSK9, ANGPTL3, CETP, APOC3, ASGR1, LPA, and IDOL, supported by findings from human genetics, preclinical models, and recent first-in-human trials. Emerging delivery vehicles (AAVs, LNPs, lentivirus, virus-like particles) and their translational implications are discussed. The review highlights ongoing clinical trials employing liver-targeted in vivo editing modalities (LivGETx-CVD) and provides insights into challenges in delivery, off-target effects, genotoxicity, and immunogenicity. Collectively, this review captures the rapid progress of LivGETx-CVD from conceptual innovation to clinical application, and positions gene editing as a transformative, single-dose strategy with the potential to redefine prevention and long-term management of dyslipidemia and atherosclerotic cardiovascular disease.

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.

Enhancing Metabolic Engineering in Medicinal Plants Through Prime Editing.

Abstract Introduction and aims Dystrophic epidermolysis bullosa (DEB) is an inherited blistering skin disease with over 800 causative mutations identified in the COL7A1 gene. eePASSIGE is a breakthrough DNA editing technology that pairs prime editing (PE) with serine integrase technology, allowing for targeted integration of large DNA constructs. We aim (i) to use eePASSIGE to integrate a full-length cDNA of COL7A1 into the AAVS1 safe harbour locus, and (ii) to develop lipid-based nanoparticles (LNPs) capable of delivering eePASSIGE machinery to human skin cells. Together, these two strategies form the basis for a proposed permanent ‘one-size-fits-all’ DEB cure. Methods We have used lipofection of HEK293T, N/TERT, and human fibroblast cells to provide proof of concept. For PE and eePASSIGE, lipofectamine was used to deliver plasmid DNA encoding editing constructs and pegRNAs. For PE, Sanger sequencing of target site amplicons was used to identify successful edits. For eePASSIGE, puromycin selection was used to isolate successfully edited cells. For LNP studies, fluorescent reporter mRNA was delivered to N/TERTs and fibroblasts; successful transfection was quantified with flow cytometry. Results In N/TERT cells, we achieved up to 17% PE efficiency in transfected cells, although only 1% of cells were successfully transfected. eePASSIGE was used to successfully integrate a puromycin resistance gene into HEK293T cells at the AAVS1 safe harbour locus. Lipid nanoparticles were used to successfully transfect human fibroblasts and keratinocytes in submerged culture with reporter constructs with up to 80% efficiency and with lower toxicity than lipofectamine. Conclusions Our experiments thus far have laid the groundwork for future study of eePASSIGE to integrate large coding constructs into disease-relevant cells. Our next steps are (i) to improve transfection of eePASSIGE machinery in cells of interest, and (ii) to optimize LNP formulations for use in three-dimensional culture and in vivo mouse skin.

Organoid-based platforms in livestock: Current advances and future prospects.

Emerging trends in the development of efficient CAS nucleases for meticulous gene editing in plants.

CRISPR's impact on cancer: From fundamental models to clinical solutions.

CRISPR/cas genome editing for neurodegenerative diseases: Mechanisms, therapeutic advances, and clinical prospects.

Vanishing White Matter Disease (VWMD) is a devastating, currently incurable neurodevelopmental disorder primarily affecting white matter. The prevailing view attributes VWMD to the activation of the canonical integrated stress response (c-ISR). However, recent studies have identified a novel, distinct pathway called the split ISR (s-ISR), though its activation has so far only been documented in mouse stem cells harboring a single eIF2B mutation, leaving uncertainty about whether it occurs in human cells, whether other mutations can trigger it, and what role it plays in the disease. Here, we used prime editing (PE) to engineer multiple eIF2B pathogenic mutations into HEK293T and induced pluripotent stem cells (iPSCs), generating human models. We demonstrated PE's effectiveness and safety, marking the first successful application of PE for modeling VWMD. We found that all modeled mutations activate the s-ISR, indicating that this response is a common feature across VWMD mutations, and that it can be further amplified by stress-induced c-ISR and effectively suppressed by ISRIB. Mechanistically, we show that s-ISR hinders mutant iPSCs from achieving the high protein synthesis levels necessary for proper differentiation, expecially into astrocytes. This impairment disrupts their maturation process, directly linking s-ISR activation to the white matter abnormalities of VWMD.

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.

Prime editing provides precise base changes, minute insertions (Small insertions ≤3 bp showed efficiencies of 2–8%) or deletions, and more defined substitutions without cutting both DNA strands or finding a donor. This is clearly better for safety and control. Plants have quickly taken on, but not in identical way. Changing editor backbones, reshaping pegRNAs, and evaluating out different delivery methods have often made things more efficient, but these improvements don't always work for all species or tissues. Simple design choices like PBS length, RTT layout, adding a 3′ structural tail, or employing paired pegRNAs can have greater implications on results than the editor itself. Editing efficiencies in rice protoplasts ranged from 0.26% to 2.2% for different targets. Rice showed that it was possible, as subsequent initiatives certain of which turned out far more successful than others—propelled into wheat, several dicots, and even some trees. While improvements in editor engineering, more advanced promoters, and computational design all got better, functionality still varies from locus to locus and genotype to genotype. In the real world, the transformation techniques and the local target context often define the outcome. This review summarizes collectively the greatest developments about plant prime editing, focusing on how it can be deployed for specific crops, how procedures can be strengthened, and design guidelines. The degree to which prime editing has been utilized in breeding and functional genomics will depend on further study on pegRNA stabilization, backbone variations, and various methods of delivering it.

Micronutrient deficiencies, particularly in low and middle-income countries, pose significant health challenges due to reliance on staple crops with low nutritional value. Biofortification, the process of enhancing micronutrient content in crops, offers a strategic solution to improve public health. This review examines how genome editing technologies, including CRISPR/Cas9, base editors, and prime editors, can accelerate biofortification efforts by directly modifying genetic pathways that influence micronutrient content. The review highlights various biofortification approaches and their integration with sustainable agriculture practices. It discusses the technical, agronomic, regulatory, and social challenges of developing biofortified crops. It presents case studies where genome-edited crops, such as high-iron rice and zinc-enriched wheat, have been successfully developed. The paper also evaluates the effectiveness of these crops in improving nutritional outcomes and their potential for scaling. By aligning genome editing with sustainable farming practices, biofortification can enhance food security while maintaining environmental resilience. This integrated approach holds promise for tackling micronutrient.

Prime editing (PE) enables the precise installation of intended base substitutions, small deletions or small insertions into the genome of living cells. While the use of Cas9 nickase can avoid DNA double-strand breaks (DSB), undesired insertions and deletions (indels) often accompany the correct edits, particularly when PE activity increased. Here we show that the anti-CRISPR (Acr) protein AcrIIA5 can significantly enhance PE activity by up to 8.2-fold while markedly reducing byproduct indels. Further investigation reveals that AcrIIA5 can promote PE across various approaches (PE2, PE3, PE4, PE5, and PE6), edit types (substitutions, insertions and deletions), and endogenous loci. Mechanistically, AcrIIA5 appears to inhibit the re-nicking activity of PE complex rather than enhancing the core editing machinery itself, suggesting a distinct mode of interaction with Cas9. Overall, we demonstrate that a known "inhibitor" Acr protein can unexpectedly acting as an "enhancer" of CRISPR/Cas-based genome editing, providing an effective strategy to optimize PE specificity and activity.

The CRISPR-Cas system originates from the adaptive immune mechanism of microorganisms and has now evolved into a revolutionary gene-editing tool, driving rapid development in the field of molecular biology. This system is systematically classified into two major categories: Class 1 (multi-protein complexes) and Class 2 (single-protein effectors), covering six main types, including DNA-targeting Cas9, Cas12 and RNA-targeting Cas13. These types possess diverse editing and regulatory functions. In recent years, this technology has not only achieved precise gene knockout and repair, but also derived new tools such as base editing and prime editing, which have significantly improved editing accuracy and expanded application scope. In addition, CRISPR technology has gone beyond gene editing itself and been applied in transcriptional regulation (CRISPRa/i) and molecular diagnostics (e.g., high-sensitivity nucleic acid detection). However, this technology still faces challenges in terms of delivery efficiency, safety and specificity. In particular, the limitations of viral vectors in packaging capacity and immunogenicity have promoted research on non-viral vectors (such as lipid nanoparticles, LNP) and physical delivery methods. This article reviews three aspects of the CRISPR system: classification and functions, evolution and optimization of editing tools, and delivery technologies, and looks forward to its broad prospects in the fields of genetic disease treatment, infectious disease detection and cell programming.

Background:Inherited variants in the LDL receptor (LDLR) gene are the most common cause of familial hypercholesterolemia (FH), significantly increasing coronary artery disease risk. Early identification of pathogenic LDLR variants enables prompt intervention with lipid-lowering therapies; however, the majority of LDLR variants observed in the population have uncertain or absent clinical classifications, limiting the potential to improve clinical management.Methods:We developed an innovative, activity-normalized prime editing screening pipeline to measure the impact of 5,184 LDLR coding variants on LDL-cholesterol (LDL-C) uptake. Through pairing a genotypic outcome reporter with every prime editing guide RNA (pegRNA), we adjust phenotypic measurements to account for variable editing efficiency, extending activity normalization to prime editing for the first time at this scale. Further, we use a statistical estimation approach that leverages measurements for all missense variants at a given position to denoise the resulting scores.Results:We show that prime editing-mediated reporter editing correlates with endogenous variant installation frequency, allowing activity normalization to improve imputation of LDLR variant effect. Our optimized prime editing assay identifies a broad, continuous spectrum of variant functional effects. We achieve robust separation of pathogenic vs. benign ClinVar variants and concordance between experimentally derived functional scores and LDL-C levels measured in UK Biobank participants. Further, when calibrating the strength of evidence provided by this functional screening data to align with the ACMG/AMP variant interpretation guidelines, and integrating additional sources of evidence, a majority of currently unclassified rare LDLR variants meet evidence thresholds for reclassification. We use the broad coverage of this screen to gain insight into how apolipoproteins bind to LDLR. In particular, we identify and characterize rare LDLR variants that enhance LDL-C uptake through increased interaction with apolipoprotein B. Finally, we compare prime editing-based functional scores with those derived from recent base editing and cDNA-based LDLR variant screens, showing that these approaches all show robust correlation with clinically observed LDL-C levels and computational scores, while prime editing identifies candidate splice-altering coding variants that are not modeled by cDNA screening.Conclusions:Altogether, our approach demonstrates the power of prime editing to significantly improve understanding of how variants in LDLR impact function and contribute to FH.

Glioblastoma multiforme (GBM) remains one of the most aggressive and treatment-resistant human cancers, characterized by highly infiltrative growth, extensive cellular heterogeneity, and a profoundly immunosuppressive microenvironment. Although CAR-T cell therapy has revolutionized the treatment of hematologic malignancies, its clinical impact in GBM has been limited by antigenic escape, poor tumor infiltration, T-cell exhaustion, and significant toxicity risks. Recent advances in CRISPR-Cas9 genome editing offer unprecedented opportunities to overcome these barriers by enabling precise, multiplex genetic reprogramming of T cells. In this review, we synthesize current progress in CRISPR-enhanced CAR-T engineering for GBM, focusing on strategies to overcome immune checkpoint suppression, optimize metabolic fitness, enhance trafficking across the blood–brain barrier, reduce neuroinflammation-associated toxicities, and generate universal allogeneic CAR-T products. We also compare the genomic target spaces of candidate guide RNAs (crRNA, d10r10, X37) and highlight their predicted off-target profiles relevant to GBM therapeutic design. Preclinical studies demonstrate that CRISPR-edited CAR-T cells targeting EGFRvIII, IL-13Rα2, HER2, and B7-H3 significantly enhance survival in murine GBM models, while emerging clinical trials indicate acceptable safety and early evidence of anti-tumor activity. We further discuss technological innovations—including base editing, prime editing, CRISPRi/a, non-viral delivery platforms, and precision-medicine–guided CAR design—as well as regulatory, ethical, and manufacturing considerations required for clinical translation. Collectively, these advances underscore the transformative potential of CRISPR-engineered CAR-T therapies to reshape GBM treatment and pave the way toward more effective and accessible cellular immunotherapies. Keywords. Glioblastoma multiforme (GBM), CAR-T cell therapy, CRISPR-Cas9 genome editing, T-cell engineering, Immune checkpoint resistance, Tumor microenvironment, Guide RNA design, EGFRvIII, IL-13Rα2, Universal allogeneic CAR-T cells, Preclinical models, Precision immunotherapy

A Rice Endogenous Small RNA-Binding Protein Improves Prime Editing for Precise Sequence Insertion and Replacement.