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At least 55 records · Page 3Linked to original sources
PubMed2025

Rat somatic genome editing enables ER+ breast cancer modeling.

Genetically engineered mouse models have advanced cancer research but often fail to capture key features of certain human tumors. Rats, with distinct physiology and tumor biology, offer a powerful alternative, yet their use has been constrained by technical barriers to genome editing. Here, we report efficient somatic genome editing in rats, enabling both Indel and substitution mutations. We then apply this approach to model estrogen receptor (ER)-positive breast cancer, which accounts for ~70% of human cases but remains poorly represented in mice. The resulting rat tumors reproduce hallmarks of human ER+ breast cancer, including ductal histology, hormone responsiveness, and immune-microenvironmental features. By contrast, identical genetic alterations in mice failed to yield ER+ tumors, underscoring critical species differences in tumorigenesis. Together, this work establishes a versatile platform for rapid generation of clinically relevant rat tumor models, opening new avenues to dissect tumor biology, therapeutic response, and immune interactions in previously inaccessible cancer subtypes.

Journal Article↗
PubMed2026

An efficient endogenous type I-E CRISPR-Cas genome-editing platform for producing transglutaminase in Streptomyces mobaraensis.

Streptomyces mobaraensis is an industrially important actinomycete capable of producing transglutaminase (TGase), a valuable crosslinking enzyme that is widely used in the food, pharmaceutical, and textile industries. However, its genetic manipulation remains challenging owing to the lack of efficient genome-editing tools. Here, we characterized an endogenous type I-E CRISPR-Cas system in S. mobaraensis IPIO2 through bioinformatics analysis and plasmid interference assays, identifying the protospacer adjacent motif as 5'-AAC-3'. We engineered an artificial editing plasmid, pCRISPR, by inserting a mini-CRISPR array (repeat-spacer-repeat) and homologous recombination repair templates into the replicative plasmid pJTU1278. This system exhibited high editing efficiencies, achieving 70% for single-gene deletions and 75-80% for large DNA fragment deletions ranging from 10 to 40 kb. Based on this system, deletion of four genes consistently downregulated during TGase production, identified through comparative proteomics, enhanced TGase production by 8.5-18.5%. Furthermore, deleting the pseudouridimycin and piericidin A1 biosynthetic gene clusters using this system significantly improved the safety profile of TGase production, resulting in a 17% increase in TGase yield. This study established a robust and efficient endogenous CRISPR-Cas-based genome-editing platform in S. mobaraensis, providing a powerful tool for strain engineering and industrial optimization of TGase production.

Comparative proteomics↗
PubMed2024

Bone-marrow-homing lipid nanoparticles for genome editing in diseased and malignant haematopoietic stem cells.

Therapeutic genome editing of haematopoietic stem cells (HSCs) would provide long-lasting treatments for multiple diseases. However, the in vivo delivery of genetic medicines to HSCs remains challenging, especially in diseased and malignant settings. Here we report on a series of bone-marrow-homing lipid nanoparticles that deliver mRNA to a broad group of at least 14 unique cell types in the bone marrow, including healthy and diseased HSCs, leukaemic stem cells, B cells, T cells, macrophages and leukaemia cells. CRISPR/Cas and base editing is achieved in a mouse model expressing human sickle cell disease phenotypes for potential foetal haemoglobin reactivation and conversion from sickle to non-sickle alleles. Bone-marrow-homing lipid nanoparticles were also able to achieve Cre-recombinase-mediated genetic deletion in bone-marrow-engrafted leukaemic stem cells and leukaemia cells. We show evidence that diverse cell types in the bone marrow niche can be edited using bone-marrow-homing lipid nanoparticles.

Animals↗
PubMed2024

AAV-mediated genome editing is influenced by the formation of R-loops.

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, may promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

Dependovirus↗
PubMed2024

AAV-mediated genome editing is influenced by the formation of R-loops.

Recombinant adeno-associated viral vectors (rAAV) hold an intrinsic ability to stimulate homologous recombination (AAV-HR) and are the most used in clinical settings for in vivo gene therapy. However, rAAVs also integrate throughout the genome. Here, we describe DNA-RNA immunoprecipitation sequencing (DRIP-seq) in murine HEPA1-6 hepatoma cells and whole murine liver to establish the similarities and differences in genomic R-loop formation in a transformed cell line and intact tissue. We show enhanced AAV-HR in mice upon genetic and pharmacological upregulation of R-loops. Selecting the highly expressed Albumin gene as a model locus for genome editing in both in vitro and in vivo experiments showed that the R-loop prone, 3' end of Albumin was efficiently edited by AAV-HR, whereas the upstream R-loop-deficient region did not result in detectable vector integration. In addition, we found a positive correlation between previously reported off-target rAAV integration sites and R-loop enriched genomic regions. Thus, we conclude that high levels of R-loops, present in highly transcribed genes, promote rAAV vector genome integration. These findings may shed light on potential mechanisms for improving the safety and efficacy of genome editing by modulating R-loops and may enhance our ability to predict regions most susceptible to off-target insertional mutagenesis by rAAV vectors.

Preprint↗
PubMed2026

In vivo genome editing of central nervous system SIV reservoirs in ART-suppressed rhesus macaques.

Latent human immunodeficiency virus type 1 (HIV-1) reservoirs in the central nervous system (CNS) may sustain viral persistence and neuroinflammation contributing to HIV-associated neurocognitive disorders (HAND) despite suppressive ART. AAV9-delivered CRISPR has successfully edited SIV proviral DNA in peripheral tissues with acceptable safety profiles, but the extent of in vivo genome editing in the brain remains unclear. Using SIV-infected rhesus macaques, we mapped intact proviral DNA across CNS regions and tested systemic AAV9-CRISPR-Cas9 targeting conserved sites within Ψ packaging signal and Gag region. Ten adult rhesus macaques were infected with genetically barcoded SIVmac239, suppressed with ART, then randomized to receive intravenous AAV9-SaCas9 with dual gRNAs (Ψ + Gag) or a Cas9-only control. At necropsy after viral rebound, SIV genomes were detected in multiple brain regions as well as lymphoid tissues, confirming the CNS as a persistent reservoir during ART. Barcode analysis revealed region-specific patterns consistent with compartmentalized CNS persistence. In CRISPR-treated animals, proviral editing was measurable across anatomically distinct CNS sites. These findings demonstrate that intact and potentially replication-competent virus persists in the primate brain under ART and that systemic AAV9-CRISPR can reach and edit proviral DNA in this sanctuary, supporting genome editing as a strategy toward durable remission of CNS reservoirs.

ART↗
PubMed2025

New retron systems from environmental bacteria identify triggers of anti-phage defense and expand tools for genome editing.

Retrons are bacterial immune systems that protect a bacterial population against phages by killing infected hosts. Retrons typically comprise a reverse transcriptase (RT), a template noncoding RNA that is partially reverse transcribed into RT-DNA, and a toxic effector. The reverse transcriptase (RT), noncoding RNA, and RT-DNA complex sequester the toxic effector until triggered by phage infection, at which point the toxin is released to induce cell death. Due to their ability to produce single-stranded DNA in vivo, retrons have also been engineered to produce donor templates for genome editing in both prokaryotes and eukaryotes. However, the current repertoire of experimentally characterized retrons is limited, with most retrons sourced from clinical and laboratory strains of bacteria. To better understand retron biology and natural diversity, and to expand the current toolbox of retron-based genome editors, we developed a pipeline to isolate retrons and their bacterial hosts from a variety of environmental samples. Here, we identify seven new retron systems, each isolated from a different host bacterium. We characterize DNA production by these retrons and test their ability to defend against a panel of Escherichia coli phages. We find that two of these retrons are disrupted by other elements, in one case a group II intron and in another a separate defense system, yet both retrons still produce RT-DNA. For two other retrons, we further unravel their mechanism of defense by identifying the phage genes responsible for triggering abortive infection. Finally, we engineer these retrons for genome editing in E. coli, demonstrating their potential use in a biotechnological application.

Gene Editing↗
PubMed2026

Mechanisms of and mitigating strategies for cellular immune responses to CRISPR-associated nucleases in genome editing therapy.

Immunogenicity of CRISPR-associated nucleases (Cas) is a critical barrier to the development of safe and effective genome editing therapies. These proteins inherently pose a risk of immune recognition due to their prokaryotic origins. A multitude of factors, such as the delivery vehicle, the route of administration, components of the therapeutics, tissue microenvironment, and pre-existing immunity, also contribute to the complexity of the host immune response to Cas proteins. As CRISPR-based therapies advance into clinical settings, it is imperative to elucidate and address the immunogenicity of Cas proteins. Here, using Cas9 as an example, we review the current understanding of Cas protein immunogenicity, the challenges it poses for therapeutic application, and strategies to mitigate cellular immune responses to Cas proteins.

AAV↗
PubMed2025

Strain-Promoted mRNA Transdermal Delivery by Lipoic Lipid Nanoparticles for Therapeutic Skin Genome Editing.

Lipid nanoparticle (LNP)-mRNA formulations have revolutionized the field of nucleic acid therapeutics, yet their broader clinical application is constrained by inflammatory side effects and oxidative stress, particularly in the context of inflammatory diseases. Herein, we report the rational design and synthesis of a lipoic acid-based ionizable lipid library to address these limitations. By leveraging the antioxidant properties and thiol-mediated uptake potential of lipoic acid, we identified LA-A2B2CD3 as an optimal candidate through a structure-activity relationship study and design of experiment (DOE) optimization. LA-A2B2CD3 LNPs exhibited superior reactive oxygen species scavenging, enhanced mRNA translation, and reduced inflammatory cytokine production in vitro and in vivo. Mechanistic studies revealed that the efficient cellular uptake and the transdermal delivery capacity of LA-A2B2CD3 heavily rely on the reducible disulfide ring of lipoic acid. Application of LA-A2B2CD3 LNPs for the localized transdermal delivery of Cas9 mRNA and CD93 sgRNA in a murine model of psoriasis resulted in effective CD93 genome editing and the inhibition of the CD93-p38 MAPK-AKT-SMAD2/3 pathway, leading to significant therapeutic improvement. This work presents a robust, biocompatible LNP platform with minimized immunogenicity and strong potential for genome-editing therapies in inflammatory conditions, offering a transformative approach for the mRNA-based treatment of skin and other inflammation-related disorders.

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

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↗
PubMed2025

Quantifying Protein-Nucleic Acid Interactions for Engineering Useful CRISPR-Cas9 Genome-Editing Variants.

Numerous high-specificity Cas9 variants have been engineered for precision genome editing. These variants typically harbor multiple mutations designed to alter the Cas9-single guide RNA (sgRNA)-DNA complex interactions for reduced off-target cleavage. By dissecting the contributions of individual mutations, we attempt to derive principles for designing high-specificity Cas9 variants. Here, we computationally modeled the specificity harnessing mutations of the widely used Cas9 isolated from Streptococcus pyogenes (SpCas9) and investigated their individual mutational effects. We quantified the mutational effects in terms of energy and contact changes by comparing the wild-type and mutant structures. We found that these mutations disrupt the protein-protein or protein-DNA contacts within the Cas9-sgRNA-DNA complex. We also identified additional impacted amino acid sites via energy changes that constitute the structural microenvironment encompassing the focal mutation, giving insights into how the mutations contribute to the high-specificity phenotype of SpCas9. Our method outlines a strategy to evaluate mutational effects that can facilitate rational design for Cas9 optimization.

Gene Editing↗
PubMed2025

Ultra-high field strength electroporation enables efficient DNA transformation and genome editing in nontuberculous mycobacteria.

Efficient DNA delivery is essential for genetic manipulation of mycobacteria and for dissecting their physiology, pathogenesis, and drug resistance. Although electroporation enables transformation efficiencies exceeding 10⁵ CFU per µg DNA in Mycobacterium smegmatis and Mycobacterium tuberculosis, it remains highly inefficient in many nontuberculous mycobacteria (NTM), including Mycobacterium abscessus. Here, we discovered that NTM such as M. abscessus exhibit exceptional tolerance to ultra-high electric field strengths and that hypertonic preconditioning partially protects cells from electroporation-induced damage. Using ultra-high electric field strength (3 kV/mm) electroporation, we achieved dramatic improvements in plasmid transformation efficiency-up to 106-fold in M. abscessus, 83-fold in Mycobacterium marinum, and 37-fold in Mycobacterium kansasii-compared to standard conditions (1.25  kV/mm). Transformation efficiency was further influenced by the choice of selectable marker. Ultra-high field strength electroporation also markedly enhanced allelic exchange in M. abscessus expressing Che9c RecET recombinases, increasing the recovery of gene deletion mutants by over 1,000-fold relative to conventional electroporation. In parallel, oligonucleotide-mediated recombineering for targeted point mutations produced nearly 10,000-fold more mutants under ultra-high field conditions. Together, these findings establish ultra-high field electroporation as a robust, broadly applicable platform for genetic engineering of NTMs. This method substantially enhances transformation efficiency and enables construction of advanced genetic tools-including expression libraries and CRISPRi knockdown libraries-in species that have historically resisted genetic manipulation.IMPORTANCEInfections caused by nontuberculous mycobacteria (NTM), including Mycobacterium abscessus, are increasing globally, yet genetic manipulation of these pathogens remains technically challenging due to inefficient DNA delivery and low gene editing success. The ultra-high electric field strength electroporation strategy described here overcomes these barriers, enabling dramatic improvements in both transformation and genome editing efficiency. This advance paves the way for high-throughput functional genomics in NTMs, including the construction of genome-wide knockout, CRISPRi knockdown, and expression libraries. Broad adoption of this approach will accelerate discovery of genetic determinants of virulence and drug resistance, facilitating the development of antimicrobials and vaccines.

Electroporation↗
PubMed2026

An effective method for isolation and regeneration of Solanum tuberosum mesophyll protoplasts for transgene-free genome editing.

An effective system for isolating and regenerating protoplasts is crucial for research in genome engineering. This study focused on refining a protocol for the isolation and regeneration of mesophyll protoplasts from the leaves of Solanum tuberosum cv. Kufri Jyoti. Key factors influencing protoplast yield and viability, such as dark pretreatment, pre-plasmolysis, enzyme concentrations, and osmoticum levels, were thoroughly assessed and optimized. The highest protoplast yield and viability were achieved with an enzyme mixture of 1.0% cellulase R-10 and 0.5% macerozyme R-10 after 16 h of incubation. Furthermore, culturing on a Murashige and Skoog-based medium (MSPI) without ammonium nitrate, enriched with an osmoticum concentration of 0.4 M and a carefully adjusted auxin-to-cytokinin ratio, successfully facilitated protoplast division, microcalli proliferation, and minicalli formation. Callus proliferation and shoot induction were accomplished on MS13K medium supplemented with naphthaleneacetic acid (NAA) and zeatin riboside. Root initiation and elongation were promoted on MS basal medium supplemented with indole-3-butyric acid (IBA) at 1 mg/L. The regenerated plantlets were subsequently acclimatized and hardened under controlled greenhouse conditions. This robust protoplast-to-plant protocol serves as a crucial resource for the introduction of ribonucleoprotein complexes into plant cells, facilitating accurate, transgene-free genome editing.

Callus induction↗
PubMed2025

Targeting the "bitterness gene" by genome editing abolishes synthesis of bitter flavanones in citrus; prospects for new varieties and extended climates for cultivation.

Bitterness in citrus fruit is conferred by flavanone-neohesperidosides, whose accumulation is catalyzed by a single enzyme flavanone-7-O-glucosides-1,2-rhamnosyltransferase (1,2RhaT), expressed in both leaves and fruit. To eliminate citrus bitterness, we used CRISPR/Cas9 genome editing to inactivate the 1,2RhaT gene in grapefruit (Citrus paradisi) and "Carrizo" citrange (Citrus sinensis × Citrus trifoliata). Edited lines displayed frameshift mutations that introduced premature stop codons, effectively abolishing the synthesis of the bitter neohesperidosides naringin, neohesperidin, and poncirin. Metabolomic analyses in leaves from 1,2RhaT-mutant lines confirmed the absence of bitter flavanone-neohesperidosides and a compensatory increase in the tasteless flavanone-rutinosides hesperidin, didymin, and narirutin. Since 1,2RhaT is encoded by a single gene, our findings in leaves are expected to be identical for fruit and thus demonstrate a strategy for developing non-bitter citrus cultivars while retaining health-benefitting flavonoid levels. Furthermore, cold-hardy citrus species that are currently unacceptably bitter due to high flavanone-neohesperidoside levels may become useful sources for introduction of cold-hardiness following inactivation of the 1,2RhaT gene. This approach thus paves the way for expanding grapefruit markets and breeding cold-hardy, palatable citrus varieties that are better suited to a wider range of climates.

Flavanones↗
PubMed2026

Molecular Mobility of N-Acetylgalactosamine-Modified Cyclodextrins on a Polyrotaxane for Highly Efficient Liver Targeting of Antibody Chimeras and Genome-Editing Ribonucleoproteins.

Triantennary N-acetylgalactosamine (triGalNAc), which interacts strongly with the trimeric structure of asialoglycoprotein receptors (ASGPRs), is a validated platform for liver targeting. However, the intricate design and synthesis of its linkers impose high production costs and significant technical challenges. In this study, we report an alternative strategy for targeting ASGPR using monovalent GalNAc (monoGalNAc) conjugated to the cyclic molecules of polyrotaxane, which can rotate and translocate along the axial polymer chain. The intracellular uptake efficacy of monoGalNAc-modified polyrotaxane is comparable to that of triGalNAc-modified polyrotaxane and significantly higher than that of triGalNAc- or monoGalNAc-modified immobile control polymers. These results suggest that the inherent mobility of polyrotaxanes allows monoGalNAc moieties to cluster in a trivalent-like manner, thereby enhancing multivalent interactions with multiple ASGPR oligomers. The successful application of monoGalNAc-modified polyrotaxane to lysosome-targeting antibody chimeras and genome-editing nanoparticles demonstrates that this facile technology is a highly promising alternative to conventional triGalNAc.

Rotaxanes↗
PubMed2025

Mutational scanning of TnpB reveals latent activity for genome editing.

TnpB is a diverse family of RNA-guided endonucleases associated with prokaryotic transposons. Due to their small size and putative evolutionary relationship to CRISPR-Cas12, TnpB enzymes hold significant potential for genome editing. However, most TnpBs lack robust gene editing activity, and unbiased profiling of mutational effects on editing activity has not been explored. Here, we mapped comprehensive sequence-function landscapes of a TnpB ribonucleoprotein and discovered many activating mutations in both the protein and RNA. One- and two-position RNA mutants outperform existing variants, highlighting the utility of systematic RNA scaffold mutagenesis. Leveraging the protein's mutational landscape, we identified enhanced TnpB variants from a combinatorial library of activating mutations. These variants enhanced editing in human cells, N. benthamiana, pepper, and rice, with up to a fifty-fold increase compared to wild-type TnpB. These findings highlight previously unknown elements critical for regulating TnpB endonuclease activity and reveal surprising latent activity accessible through mutation.

Journal Article↗