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At least 19 recordsLinked to original sources
PubMed2025

Turbo-charging crop improvement: harnessing multiplex editing for polygenic trait engineering and beyond.

Multiplex CRISPR editing has emerged as a transformative platform for plant genome engineering, enabling the simultaneous targeting of multiple genes, regulatory elements, or chromosomal regions. This approach is effective for dissecting gene family functions, addressing genetic redundancy, engineering polygenic traits, and accelerating trait stacking and de novo domestication. Its applications now extend beyond standard gene knockouts to include epigenetic and transcriptional regulation, chromosomal engineering, and transgene-free editing. These capabilities are advancing crop improvement not only in annual species but also in more complex systems such as polyploids, undomesticated wild relatives, and species with long generation times. At the same time, multiplex editing presents technical challenges, including complex construct design and the need for robust, scalable mutation detection. We discuss current toolkits and recent innovations in vector architecture, such as promoter and scaffold engineering, that streamline workflows and enhance editing efficiency. High-throughput sequencing technologies, including long-read platforms, are improving the resolution of complex editing outcomes such as structural rearrangements-often missed by standard genotyping-when targeting repetitive or tandemly spaced loci. To fully realize the potential of multiplex genome engineering, there is growing demand for user-friendly, synthetic biology-compatible, and scalable computational workflows for gRNA design, construct assembly, and mutation analysis. Experimentally validated inducible or tissue-specific promoters are also highly desirable for achieving spatiotemporal control. As these tools continue to evolve, multiplex CRISPR editing is poised to become a foundational technology of next-generation crop improvement to address challenges in agriculture, sustainability, and climate resilience.

Gene Editing↗
PubMed2026

An efficient woody plant protoplast platform enables transgene-free multiplex genome editing and rapid trait validation in pear.

Multiplex editing is crucial for analysing complex multiple-gene traits in woody plants, yet its application remains limited because of low transformation efficiency and lengthy regeneration cycles. To overcome these barriers, in this study we establish an efficient protoplast isolation protocol for pear (Pyrus) that employs 1.0% cellulase R10 and 0.4% macerozyme R10 with an 8.5 h digestion. Its broad applicability using different digestion times across seven other economically important woody plants is demonstrated. Coupling a 40% PEG-4000-mediated transfection regimen with DNA-free CRISPR/Cas9 ribonucleoprotein (RNP) delivery enables multiplex genome editing in isolated protoplasts. Using this platform, we simultaneously disrupted the key components of the chloroplast division apparatus ARC3, PARC6, and FtsZ2-1a in Pyrus bretschneideri and found that it consistently reproduced macro-chloroplast abnormalities, confirming effective multigene perturbation within a single cellular context. Notably, failure of chloroplast division activated chloroplast-to-nucleus retrograde signaling, as evidenced by the induction of the nuclear stress-response genes RBOHD and ZAT12, a concomitant surge in reactive oxygen species, and progression to severe cellular deformation. Thus, our study establishes a rapid, cross-genus protoplast-RNP workflow that enables DNA-free multiplex editing and accelerates genotype-to-phenotype analyses in woody perennials. The approach provides a practical foundation for functional genomics and supports advances in non-transgenic precision breeding of tree crops.

Protoplasts↗
PubMed2026

Multiplexed genome editing by CRISPR-Un1Cas12f1 restores dystrophin expression in a mouse model of Duchenne muscular dystrophy.

The compact type V clustered regularly interspaced short palindromic repeats (CRISPR) nuclease Un1Cas12f1 is compatible with adeno-associated virus (AAV)-mediated genome editing, although the protospacer adjacent motif (PAM) requirements and capacity for multiplexed genome editing remain undefined. Here, we show that Un1Cas12f1 exhibits a broad tolerance for non-canonical PAMs, including Y-rich motifs with a preference for TTCR and TCTA PAMs, thereby expanding the genomic targeting range. We further demonstrate that a tandem sgRNA array expressed from a single transcript supports Un1Cas12f1-mediated multiplexed genome editing at up to five distinct genomic loci. Leveraging this multiplexing capability, we achieved targeted excision of the Dmd exon 23 through intramuscular delivery of an all-in-one AAV vector encoding Un1Cas12f1 and a CRISPR array. This treatment restored the disrupted open reading frame and dystrophin expression in a mouse model of Duchenne muscular dystrophy (DMD). Together, these findings establish Un1Cas12f1 as a compact CRISPR system capable of multiplexed genome editing and demonstrate its therapeutic potential for DMD.

Journal Article↗
PubMed2026

Advances in CRISPR multiplex gene editing to map and modify stress-responsive transcription factor networks for crop improvement.

The development of CRISPR multiplex genome-editing (MGE) tools is rapidly transforming plant functional genomics and accelerating crop improvements. By simultaneously targeting two or more DNA loci, it allows scientists to precisely edit multiple genes at the single-nucleotide level, within the target genome. Simultaneous manipulation of multiple targets has revolutionized the functional elucidation studies, particularly the dissection of complex genetic pathways. Due to its superior precision and feasibility, CRISPR-MGE is widely accepted and has largely replaced alternative editing tools such as TALENs and ZFNs. Several CRISPR-MGE strategies, including the use of individual expression cassettes, tRNA-processing enzymes, Csy4 or ribozymes, have been successfully deployed in plants. Recent advancements, such as Cpf1, transgene-free methods, or ultra-multiplexing approaches, have further refined the technology into a powerful, efficient, and robust toolkit. MGE enables complex genome editing, including multiple-gene knockouts, base alterations, transcriptional regulation, metabolic engineering, or their combinations. Consequently, it is ideal for elucidating the function of transcription factors that are key molecular players in regulating diverse plant responses, especially in stress pathways. Several stress-responsive TFs have been functionally characterized via CRISPR-MGE, and more advanced tools are being employed. This review evaluates multiplexing tools, their diverse applications, and the current progress toward developing advanced MGE tools. Ultimately, we provide evidence to encourage the use of advanced MGE tools for functional characterization studies of stress-responsive TFs, thereby highlighting their potential to accelerate crop improvement.

Gene Editing↗
PubMed2026

A dual-dimensional CRISPR toolkit enables one-step high-efficiency multiplex genome editing in Komagataella phaffii.

Against the backdrop of green biomanufacturing, engineering methanol-utilizing Komagataella phaffii (K. phaffii) represents an effective strategy to expand the one carbon (C1) product profile and speed up the industrialization of C1-based bioeconomy. To address the technical challenges of low efficiency and cumbersome experimental procedures for multiplex gene editing and precise large-fragment integration during the reconstruction of complex metabolic pathways in K. phaffii, this study established a CRISPR toolkit - Efficient Multi-Gene Editing System 3.0 (EMGES 3.0) - which enabled one-step large-fragment integration coupled with multiplex gene knockout. EMGES 3.0 was constructed through the synergistic optimization of a repair-engineered chassis and an episomal CRISPR vector. For chassis engineering, five DNA repair modules: Δlig4 (DNA Ligase IV, non-homologous end joining end ligation), ppMRE11(The endogenous MRE11 gene from Pichia pastoris) overexpression (The Meiotic Recombination 11, DNA double-strand break end resection), Δrad9 (Radiation-Sensitive 9, DNA damage checkpoint regulation), Δmph1 (Mutator Phenotype Helicase 1, improvement of homologous recombinant strand extension), and PapRecT-PaSSB co-expression (stabilization of recombination intermediates) were integrated to generate the highly recombinogenic strain Y09. For vector engineering, cenARS was replaced by panARS and the endogenous promoter PGAP was employed to drive the double hammerhead ribozyme-single guide RNA-hepatitis delta virus ribozyme (double HH-sgRNA-HDV: dHgH)-mediated sgRNA expression, yielding the optimized vector Nov_pGAP_panARS_pLAT1_Cas9. These two features on K. phaffii together enhanced the EMGES 3.0 to a higher standard of transformation rate and editing efficiency. According to our results, EMGES 3.0 achieved dual-functional gene knockout efficiencies between 76.6% and 100%. For insertion of medium-long fragments (>4.5 kb), the efficiency achieved 93.3%. In addition, the one-step integration of ultra-long fragments (>16 kb) achieved 14.8%, which was reported for the first time. Furthermore, the efficiency of simultaneous long-fragment integration at three neutral loci reached 38.4% (>15 kb). We applied the system for one-step production of free fatty acids (FFAs, yield: 5.82 ∼ 7.30 mg/L/OD600) and resveratrol (yield: 1.14 ∼ 1.28 mg/L) using methanol as the sole carbon source. EMGES 3.0 provides a robust technical foundation for complex compounds biosynthesis and high-yield industrial strains, while also advancing K. phaffii as an industrial synthetic biology chassis for efficient C1 utilization.

CRISPR-Cas Systems↗
PubMed2025

Installation of Dominant-Negative Mutations in FAS and TGFβR2 via Base Editing in Primary T Cells.

Adoptive cell transfer (ACT) of engineered T cells is effective against B-cell malignancies but has faltered against solid tumors due to the immunosuppressive tumor microenvironment (TME). FASL and TGFβ are key mediators of T cell dysfunction in the TME and overexpressing dominant negative (dn) forms of their receptors in T cells increases anti-tumor efficacy in solid tumor models. However, an approach which directly targets the endogenous genes would be more amenable to multiplex editing and reduce competition with WT alleles. Here, we employ base editing (BE) in primary human T cells to install naturally occurring dominant negative FAS and TGFβR2 mutations. In vitro survival and proliferation assays demonstrate that BE T cells are resistant to pro-apoptotic and anti-proliferative effects of FAS and TGFβ signaling. CAR-T cells with BE-installed dn TGFβR2 or dn FAS exhibit improvements in cytotoxicity, while dn TGFβR2 CAR T demonstrate increased persistence and reduced expression of phenotypic markers of exhaustion compared to controls. Moreover, BE-engineered dn CAR T outperform lentiviral-engineered cDNA over expression counterparts in several functional assays. Considering the efficiency of BE and its amenability for multiplex editing, our novel approach lends itself to engineering strategies necessary to overcome T cell dysfunction in solid tumors.

CAR T↗
PubMed2026

Multiplex FAD2/FATB Editing Generates Ultra-High-Oleic, Low-Saturate Soybean With Increased Seed Fatty Acid Content.

Oleic acid (18:1), saturated fatty acid (SFA), and polyunsaturated fatty acid (PUFA) levels are important traits for storage stability and edibility. In this study, we aimed to develop high-oleic soybean (Glycine max) by simultaneously targeting fatty acid desaturase 2 (FAD2) and fatty acyl-ACP thioesterase B (FATB) gene families using CRISPR/Cas9. Considering the paleopolyploid genome of soybean, multiple sgRNAs were designed to target GmFAD2-1, GmFAD2-2, and GmFATB genes expressed during seed development. Compared with targeting GmFAD2-1 alone (~83%), additional editing of GmFAD2-2B, GmFAD2-2C, and GmFATB1a increased the 18:1 fatty acid content to over 90%. Editing of GmFATB significantly reduced the SFA content by more than 40% compared with wild-type (WT). Reduced absolute SFA content was also observed in GmFAD2-only edited lines with decreased GmFATB expression. Notably, all evaluated high-oleic genotypes in the Williams82-background showed higher mean total fatty acid (TFA) content than the WT, both per unit seed mass and per seed. Correlation analysis of 805 individual seed profiles from Williams82-background lines further characterized the relationship between 18:1 fatty acid composition and TFA content per unit seed mass. Gene expression analysis revealed no consistent increase in triacylglycerol (TAG) synthesis genes, whereas the expression of sugar-dependent 1 (SDP1) was reduced. In contrast, genes involved in phosphatidic acid (PA) metabolism, including diacylglycerol kinase (DGK) and phospholipase D (PLD), were partially upregulated. These transcriptional changes support a hypothesis that altered lipid turnover may contribute to the higher TFA phenotype. Collectively, this study defines effective multiplex target combinations for ultra-high oleic, low-saturated soybean.

FAD2↗
PubMed2026

Turnip Mosaic Virus-Based gRNA Delivery System for Plant Genome Editing.

Plant virus-based gRNA delivery systems offer a rapid alternative to stable transformation for CRISPR-mediated genome editing, but potyvirus-based platforms in Cas9-expressing plants are still underexplored. Here, we developed a turnip mosaic virus (TuMV)-based system for gRNA delivery in Cas9-expressing Nicotiana benthamiana and tested whether Csy4-mediated gRNA processing could improve editing efficiency. A TuMV construct carrying a gRNA targeting PHYTOENE DESATURASE (NbPDS) induced detectable editing in both infiltrated and systemic tissues, although editing frequencies were low. Incorporation of the bacterial endoribonuclease Csy4 increased editing efficiencies in the two NbPDS genes, raising editing in infiltrated leaves to 7.1%-13.8% for NbPDSa and 7.6%-23.0% for NbPDSb, whereas lower but reproducible editing was detectable in systemic leaves. The TuMV-Csy4 platform also supported editing of a second endogenous target, MAGNESIUM CHELATASE SUBUNIT H (NbChlH), and enabled multiplex editing of NbPDS and NbChlH regardless of guide order. Editing efficiencies were consistently higher in infiltrated leaves than in systemic leaves, and no visible photobleaching or chlorosis was observed in systemic tissues despite confirmed molecular editing. To assess the potential for heritable editing, a tRNAIle mobility element was fused to the NbPDS gRNA. Although this construct increased somatic editing, no albino progeny were recovered after screening approximately 20,000 seedlings, demonstrating that heritable editing was not achieved under these conditions or did not result in mutations in all copies of the two NbPDS genes. Together, these results establish TuMV as a platform for Cas9-based gRNA delivery and show that Csy4-mediated processing improves editing efficiency, supports multiplex targeting, and demonstrates the feasibility of potyvirus-based genome editing systems in plants.

genome editing platform↗
PubMed2026

Discovery and Engineering of a Rat Endogenous Retrovirus Reverse Transcriptase for Efficient Prime Editing.

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.

Animals↗
PubMed2026

CasY7: An optimized Cas12i system for enhanced genome editing in monocot crops.

The CRISPR-Cas12 family nucleases, particularly the Cas12i subtypes, are considered promising alternatives to Cas9 for genome editing in plants. We previously developed a new Cas12i variant, CasY7, which has been successfully applied in clinical trials; its performance in plants remains to be investigated. Initial testing in stable transgenic maize and rice showed that the codon-optimized CasY7 (pCasY7e1) achieved average editing efficiencies of 58.7% and 62.3% across five target sites, respectively, outperforming the typical Cpf1 (pCpf1) control that targets the same sites. To further enhance activity, we fused T5 exonuclease to CasY7 (pCasY7e2), which shifted mutation profiles toward larger deletions, and subsequently integrated an MS2 aptamer into the crRNA scaffold (pCasY7e3). The optimized pCasY7e3 system increased editing efficiencies to 87.7% in maize and 82.9% in rice-approximately 2.7-fold higher than pCpf1. We further demonstrated multiplexed editing in maize, generating biallelic dwarf mutants, and validated functionality in hexaploid wheat with editing efficiencies up to 58.8%. Overall, our comprehensive validation across 942 transgenic plants confirmed robust editing in maize, rice, and wheat, establishing CasY7 as a high-efficiency addition to the CRISPR toolkit.

Zea mays↗
PubMed2026

Beyond the salt barrier: CRISPR-mediated DNA reprogramming to uncouple yield from tolerance in Rice: A review.

Rice (Oryza sativa L.) feeds half of humanity, yet its cultivation is increasingly threatened by soil salinization, which now affects 1.4 billion hectares globally. Decades of breeding and engineering have focused on Na+ exclusion, principally through the Saltol QTL and the xylem-unloading transporter OsHKT1;5, yet this strategy has reached a physiological ceiling. Excluder genotypes survive salinity but fail to fill grain, because the ATP-intensive cost of continuous ion extrusion starves reproductive sinks, while ABA-mediated stomatal closure imposes chronic carbon limitation. The resulting "survival-yield gap" exposes a fundamental flaw in single-trait approaches to a polygenic stress. In this review, we argue that durable, yield-stable salt tolerance requires a coordinated systems-level intervention spanning five mechanistic tiers: (i) CRISPR/Cas9-mediated removal of negative regulatory brakes (OsRR22, RST1, PC1) that suppress plant's latent stress-adaptive capacity; (ii) reinforcement of actin-myosin cytoskeletal transport to sustain SOS1, NHX1, and HKT1;5 delivery under ionic stress; (iii) importation of halophyte design principles from Oryza coarctata, including salt gland architecture and superior Na+ compartmentalization; (iv) recalibration of the ROS-photosynthesis axis via the DHHC09-STRK1-CatC molecular switch and stomatal density engineering; and (v) pyramiding these modules into a "Salt-Shield Rice" genotype through multiplex editing, marker-assisted introgression, speed breeding, and genomic selection. We propose a phased ten-year roadmap that integrates synthetic biology circuit design with conventional breeding to deliver field-ready, multi-module varieties with greater than 70% yield stability at 8-10 dS m-1. This remains an aspirational design target rather than a demonstrated outcome, as three of the five tiers-halophyte-derived structural traits, cytoskeletal reinforcement, and full multi-module pyramiding-remain unvalidated in rice.

CRISPR/Cas9↗
PubMed2026

The TANG cluster comprising ten nitrate transporter genes controls fruit sweetness and size in tomato.

Sucrose is a major transport form of photoassimilated carbon in tomato, Arabidopsis, and many other plant species, and plays a critical regulatory role in plant growth, development, and fruit quality. Plant vacuoles function as storage organelles, accumulating substantial quantities of metabolically inactive nitrates as a nitrogen reserve and soluble sugars as a carbon reserve. Consequently, the balance between nitrate and sucrose accumulation determines plant growth dynamics and fruit taste. In this study, we identified a gene cluster designated TANG (Total soluble solidsAccumulation viaNitrate transporterGene cluster), comprising ten nitrate transporter genes that are significantly associated with sucrose accumulation in tomato. This gene cluster mediates the transport of nitrate between the cytoplasm and vacuole, thereby influencing its storage. Functional disruption of TANG8, a member of the gene cluster, results in either enhanced sugar accumulation or increased fruit size. Selective disruption of multiple TANG cluster members yields fruits with elevated sweetness and increased fruit size in S. pimpinellifolium. The interaction between the TANG members and a tonoplast localized Sucrose Transporter 4 provides insight into the competitive accumulation of nitrate and sugar. The multiplex editing of a gene cluster provides a successful example of engineering crops with high quality and yield.

Gene cluster↗
PubMed2025

Enzymatic depletion of transposable elements in sequencing libraries and its application for genotyping multiplexed CRISPR-edited plants.

Whole-genome sequencing has become a common strategy to genotype individual plants of interest. Although a limited number of genomic regions usually need to be surveyed with this strategy, excess sequencing information is almost always generated at an appreciable financial cost. Repetitive sequences (e.g., transposons), which can account for more than 80% of the genome of some plants, are often not required in these genotyping projects. Therefore, strategies that enrich DNA coding for the protein-coding genes prior to sequencing can lower the cost to obtain sufficient sequence information. Here, we present the development and application of methylation-sensitive reduced representation sequencing (MsRR-Seq), which relies on the cytosine methylation-sensitive restriction enzyme MspJI to deplete constitutive heterochromatic DNA before library construction. By applying MsRR-Seq to citrus and maize, we show that protein-coding genes can be enriched in sequencing datasets. We then describe the application of MsRR-Seq to facilitate the identification of complex mutants from populations of citrus plants resulting from multiplex CRISPR/Cas9 editing of four genes. Overall, this work demonstrates an easy and low-cost method to enrich non-repetitive DNA in high-throughput sequencing libraries, an approach that is especially useful for large plant genomes with an excessively high proportion of methylated repetitive sequences.

DNA Transposable Elements↗
PubMed2025

Efficient and multiplexed somatic genome editing with Cas12a mice.

Somatic genome editing in mouse models has increased our understanding of the in vivo effects of genetic alterations. However, existing models have a limited ability to create multiple targeted edits, hindering our understanding of complex genetic interactions. Here we generate transgenic mice with Cre-regulated and constitutive expression of enhanced Acidaminococcus sp. Cas12a (enAsCas12a), which robustly generates compound genotypes, including diverse cancers driven by inactivation of trios of tumour suppressor genes or an oncogenic translocation. We integrate these modular CRISPR RNA (crRNA) arrays with clonal barcoding to quantify the size and number of tumours with each array, as well as the impact of varying the guide number and position within a four-guide array. Finally, we generate tumours with inactivation of all combinations of nine tumour suppressor genes and find that the fitness of triple-knockout genotypes is largely explainable by one- and two-gene effects. These Cas12a alleles will enable further rapid creation of disease models and high-throughput investigation of coincident genomic alterations in vivo.

Animals↗
PubMed2026

Combinatorial base editing couples disease correction with lineage amplification in hematopoietic stem and progenitor cells.

First-generation genome editing therapies have largely focused on correcting or compensating for pathogenic variants. However, as these approaches enter the clinic, emerging biological constraints limit maximal therapeutic impact. Because globin genes are activated late during erythroid differentiation, genome-corrected hematopoietic stem and progenitor cells (HSPCs) gain little selective advantage in the bone marrow. Here, we establish a strategy that links therapeutic genome edits to an erythroid fitness-enhancing allele to amplify the output of clinically relevant cells. We develop a multiplex base editing strategy that couples fetal hemoglobin (HbF) reactivation with erythroid lineage expansion. Introduction of a naturally occurring erythropoietin receptor truncation (tEPOR) associated with benign erythrocytosis increased erythroid cell production without impairing viability or differentiation. Combinatorial editing of tEPOR together with the BCL11A erythroid enhancer and HBG1/2 promoters in healthy donor, sickle cell disease, and β-thalassemia HSPCs synergistically increased erythroid proliferation and HbF expression beyond single base-edited or Casgevy-treated controls. Multiplex base-edited HSPCs retained long-term lineage repopulation and engraftment capacity in vivo, establishing a modular strategy that pairs disease correction with lineage amplification to improve therapeutic potency.

Journal Article↗
PubMed2025

The FIBRILLIN multigene family in tomato, their roles in plastoglobuli structure and metabolism.

Plastoglobuli (PG) are plant lipoprotein compartments, present in plastid organelles. They are involved in the formation and/or storage of lipophilic metabolites. FIBRILLINs (FBNs) are one of the main PG-associated proteins and are particularly abundant in carotenoid-enriched chromoplasts found in ripe fruits and flowers. To address the contribution of different FBNs, independently and in combination, to isoprenoid formation and sequestration, a multiplex gene editing approach was undertaken in tomato. This approach generated a suite of single and high-order fbn mutants that were shown to lack transcripts and respective protein products. The major PG-related FBNs in tomato chosen for this study are SlFBN1, SlFBN2a, SlFBN4 and SlFBN7a. When knocked out independently, functional redundancy was revealed. However, paralog-specific roles were detected regulating specific isoprenoids (e.g. plastochromanol 8) or plastidial esterification capability. In addition, high-order fbn mutants displayed altered isoprenoid chromoplast sequestration patterns, notably with a significant reduction in carotenes (phytoene and phytofluene) in the PG fraction. Proteomic analysis confirmed the absence of PG-core associated proteins, including NAD(P)H-ubiquinone oxidoreductase C1, tocopherol cyclase (VTE1) and phytol esterase (PES1/PYP). Perturbations to the ultrastructure of the plastid were revealed, with aberrant PG formation and morphology predominating in high-order mutants. Global lipidome profiles also highlighted broader changes directly affecting storage and plastid membrane lipids, for example, tri- and diacylglycerides and galactolipid species. Collectively, these results support both structural and metabolic roles of SlFBNs in PGs. The findings expose fundamental aspects of metabolic compartmentalisation in plant cells and the importance of lipoprotein particles for plastid integrity and functionality.

Solanum lycopersicum↗