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Results for “Genomic editing”

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At least 73 records · Page 4Linked to original sources
PubMed2026

Long-term functional synaptic integration of genome-edited retinal organoids in a primate model of macular degeneration.

Retinal organoids represent a promising regenerative strategy for restoring vision in retinal degenerative diseases, but the capacity of host cone bipolar cells in the primate macula to rewire with transplanted photoreceptors has not been established. In this study, we transplanted genome-edited ISL1-/- human retinal organoids lacking ON-bipolar cells into an acute laser-induced macular photoreceptor ablation non-human primate model. Using immunohistochemistry, ultrastructural imaging, and focal macular electroretinography, we demonstrate that host rod and cone bipolar cells actively extend dendrites toward grafted photoreceptors and form synaptic contacts, with evidence of functional signal transmission in a subset of transplanted eyes. Longitudinal, per-eye analyses revealed that host ON-bipolar responses improved in two of four eyes with ISL1-/- graft by up to 21.6% and remained stable for up to 2 years post transplantation. Moreover, OFF-pathway connectivity showed potential progressive maturation, with delayed increase in d-wave after 13 months in one of those eyes. These findings provide the first demonstration of long-term anatomical host-graft synaptic integration in the primate macula, establishing that central cone bipolar circuits retain the capacity for durable rewiring with human stem-cell-derived grafts. Our results highlight ISL1-/- retinal organoids as a promising approach for central vision restoration in macular degeneration.

Animals↗
PubMed2025

Efficient genome editing in Pseudomonas syringae pv. actinidiae using the CRISPR/FnCas12a system.

CRISPR-based gene editing has rarely been studied in plant pathogens. In this report, the CRISPR/FnCas12a system was successfully established for gene editing in Pseudomonas syringae pv. actinidiae (Psa), which causes bacterial canker (BC) of kiwifruit. The system was constructed in a Psa-suitable vector pBBR1-MCS2 to edit hopH1 or/and hopZ5, which encode effectors only present in Psa biovar 3 (Psa3) responsible for BC pandemic in kiwifruit worldwide. Two different CRISPR RNAs (crRNA) were designed to edit either hopH1 or/and hopZ5, and two different sets of PCR primers were used to screen deletions of the target genes and the presence of the vector in Psa. The deletion in Psa was impacted by the position of the DNAs targeted by the crRNAs. The vector-eliminated mutant could receive the editing system iteratively. Interestingly, the double mutant ΔhopZ5ΔhopH1 showed significantly stronger virulence than the wild-type Psa on Actinidia eriantha cv. White (resistant to BC), but weak virulence on A. chinensis cv. Hongyang (highly susceptible to BC), respectively, suggesting that hopH1 or/and hopZ5 potentially matches an unknown resistance gene in White. In summary, we have established the CRISPR/FnCas12a genome-editing system to probe gene function in the pathogen and to explore effector-target interactions in kiwifruit-Psa-pathosyetem.

Bacterial canker↗
PubMed2024

The modern human aryl hydrocarbon receptor is more active when ancestralized by genome editing.

The aryl hydrocarbon receptor (AHR) is a transcription factor that has many functions in mammals. Its best known function is that it binds aromatic hydrocarbons and induces the expression of cytochrome P450 genes, which encode enzymes that metabolize aromatic hydrocarbons and other substrates. All present-day humans carry an amino acid substitution at position 381 in the AHR that occurred after the divergence of modern humans from Neandertals and Denisovans. Previous studies that have expressed the ancestral and modern versions of AHR from expression vectors have yielded conflicting results with regard to their activities. Here, we use genome editing to modify the endogenous AHR gene so that it encodes to the ancestral, Neandertal-like AHR protein in human cells. In the absence of exogenous ligands, the expression of AHR target genes is higher in cells expressing the ancestral AHR than in cells expressing the modern AHR, and similar to the expression in chimpanzee cells. Furthermore, the modern human AHR needs higher doses of three ligands than the ancestral AHR to induce the expression of target genes. Thus, the ability of AHR to induce the expression of many of its target genes is reduced in modern humans.

Receptors, Aryl Hydrocarbon↗
PubMed2025

From convention to innovation: the role of genetic modification and genome editing in Australian wheat breeding.

Wheat is the most cultivated crop worldwide, and Australia consistently ranks among the top wheat-exporting countries. Although modern technology has expanded the speed and accuracy of conventional breeding, progress is constrained by limited genetic diversity and linkage drag, with new wheat varieties often taking 8-12 years to reach the market. Biotech methods involving the transformation of foreign DNA into genomes [genetic modification (GM)], or editing of native DNA [genome editing (GEd)], provide novel opportunities to efficiently improve traits alongside conventional breeding. In 2020, the world's first GM drought-tolerant bread wheat (HB4) hit the market in Argentina. The USA recently approved HB4 wheat for commercial cultivation, and human consumption of HB4 wheat has been approved by nine countries, including Australia. Currently, 25 countries, Australia included, have deregulated GEd crops in some form, and many other countries have indicated that they will follow suit. As of March 2025, no GM or GEd wheat is commercially grown in Australia. The rate at which private industry integrates GM and GEd into wheat breeding programmes will depend on several factors, including the regulatory consistency governing GM and GEd crops within Australia and among international trading partners, the return on investments relative to deregulation costs including licensing, the level of acceptance amongst growers and consumers, and technical considerations including wheat's amenability to tissue culture. This review contextualizes GM and GEd applications in wheat, often drawing on examples from crop species where biotechnology has been more widely employed, and considers the key stakeholders that will shape the future of GM and GEd wheat in Australia.

GMO↗
PubMed2026

Efficient genome editing in tomato using Cas12i3 variants.

Systematic comparison of Cas12i gene-editing tools in tomato identifies Cas-SF01 as the most efficient variant, achieving editing efficiencies comparable to conventional Cas9 at some genomic sites and providing a compact, highly specific, and versatile tool for studying gene function, engineering fruit metabolism, and developing improved tomato varieties through precision breeding.

Journal Article↗
PubMed2026

A streamlined protocol for small-scale protoplast generation and CRISPR/Cpf1-mediated genome editing in Fusarium oxysporum.

Fusarium oxysporum is a significant threat to agriculture and One Health, requiring advanced molecular tools for functional genomic analyses and biological control agent development. Existing gene-editing methods are hampered by costly protoplast preparation protocols and by CRISPR-Cas9 limitations, such as restricted protospacer adjacent motif (PAM) sequences and complex guide RNA requirements. We engineered an efficient CRISPR/Cpf1 system that overcomes these issues through three main innovations: small-scale protoplast generation using filter column-based methods that greatly reduce enzyme consumption while simplifying workflows, a CRISPR/Cpf1 system with shorter guide RNA design and staggered DNA cleavage to promote homologous recombination, and minimal homology arm strategies that significantly decrease cloning complexity. Extensive validation confirms successful gene targeting with molecular verification and functional analysis via standardized pathogenicity assays. This integrated platform offers affordable, accessible tools for systematic F. oxysporum research, enhancing fundamental understanding of plant-pathogen interactions and supporting high-throughput screening vital for agricultural biotechnology and biological agent development.

CRISPR/Cpf1↗
PubMed2025

Agptools: a utility suite for editing genome assemblies.

SUMMARY: The AGP format is a tab-separated table format describing how components of a genome assembly fit together. A standard submission format for genome assemblies is a fasta file giving the sequence of contigs along with an AGP file showing how these components are assembled into larger pieces like scaffolds or chromosomes. For this reason, many scaffolding software pipelines output assemblies in this format. However, although many programs for assembling and scaffolding genomes read and write this format, there is currently no published software for making edits to AGP files when performing assembly curation. We present agptools, a suite of command-line programs that can perform common operations on AGP files, such as breaking and joining sequences, inverting pieces of assembly components, assembling contigs into larger sequences based on an AGP file, and transforming between coordinate systems of different assembly layouts. Additionally, agptools includes an API that writers of other software packages can use to read, write, and manipulate AGP files within their own programs. AVAILABILITY AND IMPLEMENTATION: Source code and binaries freely available for download at https://github.com/WarrenLab/agptools, implemented in Python and supported on all operating systems.

Software↗
PubMed2025

Genome editing in the green alga Chlamydomonas: past, present practice and future prospects.

The green alga Chlamydomonas is an important and versatile model organism for research topics ranging from photosynthesis and metabolism, cilia, and basal bodies to cellular communication and the cellular cycle and is of significant interest for green bioengineering processes. The genome in this unicellular green alga is contained in 17 haploid chromosomes and codes for 16 883 protein coding genes. Functional genomics, as well as biotechnological applications, rely on the ability to remove, add, and change these genes in a controlled and efficient manner. In this review, the history of gene editing in Chlamydomonas is put in the context of the wider developments in genetics to demonstrate how many of the key developments to engineer these algae follow the global trends and the availability of technology. Building on this background, an overview of the state of the art in Chlamydomonas engineering is given, focusing primarily on the practical aspects while giving examples of recent applications. Commonly encountered Chlamydomonas-specific challenges, recent developments, and community resources are presented, and finally, a comprehensive discussion on the emergence and evolution of CRISPR/Cas-based precision gene editing is given. An outline of possible future paths for gene editing based on current global trends in genetic engineering and tools for gene editing is presented.

Gene Editing↗
PubMed2025

Low-temperature embryo incubation suppresses off-target mutagenesis during CRISPR-Cas9 genome editing in medaka (Oryzias latipes) and zebrafish (Danio rerio).

Gene knockout using CRISPR-Cas9 is often employed in research aimed at elucidating gene functions in fish. However, CRISPR-Cas9 sometimes introduces unintended alterations, known as off-target mutations. These mutations can reduce the robustness of data during phenotypic analysis. In this study, we focused on the culture temperature, which is known to significantly influence mutagenesis, and examined whether low-temperature culture after introducing CRISPR-Cas9 into early embryos of medaka and zebrafish suppresses off-target mutations. Continuous incubation of medaka at 16 °C significantly reduced off-target mutation rates compared to those at 28 °C; the drawback is that it decreased the survival rate of medaka embryos. Therefore, low-temperature incubation was limited to early development in both zebrafish and medaka, and then the temperature was increased to 28 °C. Under these conditions, the mutation rates of the three off-target regions in medaka (Off-D, Off-P, and Off-A) significantly decreased, whereas those of the three target regions (DJ-1, p4hb, and avt) were unaffected. Similarly, the mutation rate of the zebrafish target region (ywhaqa) remained high, whereas the off-target (Off-Y1) mutation rate significantly reduced. Furthermore, this method effectively suppressed the germ line transmission of off-target mutations in medaka. This approach is effective to obtain more reliable data from the G0 generation of medaka and zebrafish and may reduce the screening effort required to remove individuals with off-target mutations in the F1 generation.

Animals↗
PubMed2025

Optimized genomic editing of a common Duchenne muscular dystrophy mutation in patient-derived muscle cells and a new humanized mouse model.

Duchenne muscular dystrophy (DMD) is a fatal X-linked, recessive disease caused by mutations in the DMD gene encoding dystrophin, a membrane-associated protein necessary for maintaining muscle structure and function. One of the common DMD mutations is the deletion of exon 52 (Δ52), which introduces a premature stop codon in exon 53, preventing the expression of functional dystrophin protein. Patients with this mutation could benefit from skipping or reframing exon 53 to restore the dystrophin open reading frame. In this study, we investigated the efficacy of single-cut CRISPR gene editing with Staphylococcus pyogenes Cas9 (SpCas9)-LRVQR to restore dystrophin expression in patient-derived induced pluripotent stem cells (iPSCs) and a newly generated humanized DMD mouse model. We compared two injection routes for adeno-associated virus (AAV) serotype 9 to deliver gene-editing components to neonatal mice: intraperitoneal (IP) and facial vein (FV) injection. We observed efficient restoration of dystrophin protein expression across multiple skeletal muscle groups and the heart. The AAV9-mediated CRISPR single-cut approach ameliorated key DMD hallmarks, including histopathological phenotypes, impaired grip strength, and elevated serum creatine kinase levels. Our optimized strategies for dystrophin restoration in humanized DMD mice with exon 52 deletion represent a promising treatment for DMD.

AAV↗
PubMed2026

Endogenous tagging of the TBXT (Brachyury) gene with 2A-EGFP in BJNhem19 human embryonic stem cells using CRISPR-Cas9 genome editing.

Brachyury (TBXT) is a T-box transcription factor essential for directing human embryonic stem cells (hESCs) toward mesodermal lineage specification. Here, we report the generation of a Brachyury/TBXT-2A-EGFP knock-in reporter line in the BJNhem19 hESC line using CRISPR/Cas9-mediated homology-directed repair. Accurate integration of the reporter cassette was confirmed by DNA sequencing and validated by the comparison of EGFP expression to endogenous TBXT gene expression following directed mesodermal differentiation. This reporter line provides a tractable fluorescence-based platform for real-time monitoring of mesodermal commitment, which will help in high-content screening of differentiation protocols.

Journal Article↗
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↗