Frontiers in genome editing最新文献

{"title":"Gene therapy for non-obstructive azoospermia: therapeutic avenues and translational pathway to clinical application.","authors":"Man-Xi Jiang, Tao Jing, Huai L Feng, Yan Zhu","doi":"10.3389/fgeed.2026.1921175","DOIUrl":"https://doi.org/10.3389/fgeed.2026.1921175","url":null,"abstract":"<p><p>Non-obstructive azoospermia (NOA) represents the most severe male infertility. Although microsurgical testicular sperm extraction combined with intracytoplasmic sperm injection offers opportunities, approximately 50% of NOA patients still fail to obtain sperm for clinical treatments. Fortunately, the emergence of precision medicine, particularly advances in gene therapy, has opened new therapeutic avenues for NOA in preclinical studies, demonstrating proof-of-concept in animal models. This review examines current progress in NOA gene therapy. Firstly, three principal therapeutic strategies: lipid nanoparticle-mediated mRNA delivery for transient protein replacement, CRISPR-based gene editing for permanent correction, and stem cell-based approaches combined with genetic modification are comparatively analyzed. Subsequently, particular emphasis is placed on recent breakthroughs in testicular mRNA delivery that have successfully restored spermatogenesis in NOA mouse models, resulting in viable offspring. Furthermore, the delivery technologies including adeno-associated viral vectors, and non-viral methods are critically evaluated to assess their respective advantages, limitations, and suitability for different testicular cell types. The review also addresses critical safety considerations, such as off-target effects, germline transmission risks, and epigenetic consequences. Finally, we discuss the translational roadmap toward clinical application, and how the obstacles including delivery optimization, long-term safety validation, and the regulatory pathways to affect the transition from preclinical proof-of-concept to first-in-human trials.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1921175"},"PeriodicalIF":5.9,"publicationDate":"2026-09-30","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13662417/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149198344","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"MambaGLA-NPC: a 3D framework integrating CNN and state space models for robust multi-center nasopharyngeal carcinoma segmentation.","authors":"Wei Jin, Xiaobo Chen, Weifeng Jin, Hai Liao, Li Yu","doi":"10.3389/fgeed.2026.1954329","DOIUrl":"https://doi.org/10.3389/fgeed.2026.1954329","url":null,"abstract":"<p><strong>Background and objective: </strong>Accurate 3D segmentation of nasopharyngeal carcinoma (NPC) from MRI is critical for radiotherapy planning but remains challenging due to poor multi-center generalization, lack of spatial continuity, and high computational costs of global modeling. We aim to address these limitations by proposing a novel 3D framework.</p><p><strong>Methods: </strong>We propose MambaGLA-NPC, a 3D deep learning framework integrating State Space Models (SSMs) and Convolutional Neural Networks (CNNs) in a U-shaped encoder-decoder architecture. A Conv-GLSSM module jointly extracts static local features via convolutions and global context via SSMs with a 3D local scanning strategy. A gated channel-spatial attention (GCS Att) module suppresses irrelevant noise in skip connections, and an NPC-specific adapter enables parameter-efficient fine-tuning across multi-center datasets.</p><p><strong>Results: </strong>On a multi-center dataset of 1,098 NPC patients from three hospitals, our model achieves state-of-the-art performance on the internal test set (Dice: 80.47%, IoU: 67.83%, HD95: 4.09 mm). Under cross-center evaluation, it significantly outperforms prior methods (e.g., Dice of 87.13% and 76.70% on two external centers). The NPC-Adapter boosts cross-center Dice by up to 8.84% compared to no adaptation. Ablation studies confirm the contribution of each module, and the Conv-GLSSM reduces computational cost versus Transformers (21.66 GFLOPs vs. OOM for self-attention at <math> <mrow> <msup><mrow><mn>128</mn></mrow> <mrow><mn>3</mn></mrow> </msup> </mrow> </math> resolution).</p><p><strong>Conclusion: </strong>MambaGLA-NPC is the first Mamba-based 3D NPC segmentation framework that effectively balances local-global feature integration with linear complexity, achieving superior accuracy and generalization across multi-center MRI data while maintaining high efficiency.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1954329"},"PeriodicalIF":5.9,"publicationDate":"2026-09-29","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13658446/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149186542","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Combined RNA- and DNA-targeting CRISPR ribonucleoproteins enhance suppression of HBV transcripts.","authors":"Puja Banik, Addison C Hill, Madison B Schank, Yi Zhang, Juan Zhao, Jaeden S Pyburn, Holly Orfield, Yuezu Wu, Mohammad J Hajipour, Mohamed El Gazzar, Shunbin Ning, Jonathan P Moorman, Zhi Q Yao, Ling Wang","doi":"10.3389/fgeed.2026.1824895","DOIUrl":"10.3389/fgeed.2026.1824895","url":null,"abstract":"<p><strong>Introduction: </strong>Chronic hepatitis B virus (HBV) infection remains a major global health problem and there is no curative treatment currently available. RNA-targeting CRISPR systems offer a potential alternative to DNA-editing nucleases by enabling direct degradation of viral transcripts without introducing genomic breaks. In this report, we evaluated the antiviral activity of synthetic guide RNA (gRNA)/Cas13d ribonucleoproteins (RNPs) targeting HBV RNA and a combination use with DNA-targeting RNPs.</p><p><strong>Methods: </strong>We designed 10 gRNAs targeting conserved regions across the HBV transcriptome followed by screening for best candidate gRNA for Cas13d. To enhance antiviral efficacy, we evaluated multiplex RNA targeting using combined gRNA6+gRNA7/Cas13d RNPs, and combined RNA-targeting gRNA7/Cas13d with previously validated HBV DNA-targeting gRNA9/Cas9 and gRNA4/Cas12a RNPs for HBV treatment.</p><p><strong>Results: </strong>Amongst these 10 gRNAs, gRNA7/Cas13d exhibited selective knockdown of its specific target RNA transcript without inducing a broad suppression of total HBV DNA, mRNAs, and proteins. The antiviral effect of gRNA7/Cas13d RNP was transient, diminishing by Day 3 post the treatment. The combined gRNA6+gRNA7/Cas13d treatment also did not further induce global HBV suppression compared with single gRNAs, although gRNA7/Cas13d still showed a robust target-specific RNA cleavage during the combined treatment in HBV cellular models. While gRNA9/Cas9 and gRNA4/Cas12a exhibited the dominant antiviral effects, either alone or in combination, leading to substantial reductions in HBV replication and expression, the addition of gRNA7/Cas13d further enhanced the suppression of the gRNA7-specific HBV RNA transcript, achieving up to an 80% reduction in the target RNA level.</p><p><strong>Discussion: </strong>Collectively, these findings demonstrate that gRNA7/Cas13d can mediate target-specific HBV RNA cleavage but has limited overall antiviral activity when used alone as a single-agent; however, the combination of DNA- and RNA-targeting CRISPR-RNPs may provide a complementary approach for enhancing target-specific HBV RNA suppression.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1824895"},"PeriodicalIF":5.9,"publicationDate":"2026-09-23","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13642003/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149129342","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Engineering TnpB as a compact RNA-guided genome editor from molecular constraints to design principles.","authors":"Bin Ma, Yimiao Li, Dongsheng Zhang, Yu Wu, Jie Gao, He Li, Shuang Jia","doi":"10.3389/fgeed.2026.1945303","DOIUrl":"10.3389/fgeed.2026.1945303","url":null,"abstract":"<p><p>Recent discoveries of non-CRISPR RNA-guided nucleases have expanded the landscape of programmable genome editing beyond canonical Cas systems. Among these, TnpB proteins derived from IS200/IS605-family transposable elements are ultra-compact RNA-guided DNA endonucleases with potential for delivery-constrained genome engineering. Here, we review the structural and mechanistic basis of TnpB function, including protein architecture, transposon-associated motif (TAM) recognition, ωRNA/reRNA scaffold organization, RNA-DNA heteroduplex formation, and RuvC-mediated DNA cleavage. We highlight that TnpB activity emerges from coordinated interactions between the nuclease and its RNA scaffold, which jointly influence ribonucleoprotein assembly, target recognition, catalytic activation, and specificity. We further discuss engineering strategies involving ortholog selection, protein and TAM-recognition engineering, RNA scaffold optimization, delivery, and specificity control, and relate these approaches to current applications across biological systems. Although TnpB remains less mature than established CRISPR-Cas platforms, its compact architecture and growing engineering toolkit support its development as a complementary genome editing platform. Future progress will benefit from establishing predictive models that integrate target accessibility, RNA-protein compatibility, activity, and fidelity.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1945303"},"PeriodicalIF":5.9,"publicationDate":"2026-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13639567/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149119340","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Genome edited sheep generated by zygote electroporation of prime editor ribonucleoproteins.","authors":"Zhixuan Yu, Junjin Li, Menghao Liu, Qi Qi, Zengyi Duan, Ziyu Yang, Boyuan Guan, Jiawang Chen, Jian Hou","doi":"10.3389/fgeed.2026.1931608","DOIUrl":"10.3389/fgeed.2026.1931608","url":null,"abstract":"<p><p>Prime editing (PE) enables precise genome modifications without double-strand breaks, yet its application in large mammals has been hindered by low efficiency and the technical bottlenecks of zygote microinjection. This study aimed to establish a scalable PE platform in sheep via the direct electroporation of a PE3 RNP formulation comprising PE2 protein, an epegRNA, and a nicking sgRNA into zygotes. We first validated functional PE2 protein and screened highly efficient enhanced pegRNAs (epegRNAs) targeting two high-value traits, including short-tail (<i>TBXT</i>) and prolificacy (<i>FecB</i>), in primary sheep fibroblasts, achieving editing efficiencies of up to 22.78%. Systematic optimization of zygote electroporation determined that 60 V combined with a PE2: pegRNA mass ratio of 3.5:1 maximized delivery efficiency, yielding gene editing efficiencies of 19.8% and 20.1% for <i>TBXT</i> and <i>FecB</i>, respectively, with no statistically significant differences detected in cleavage or blastocyst rates between electroporated and untreated zygotes. Following dual-target co-delivery, intended edits were detected at both the <i>TBXT</i> and <i>FecB</i> loci in separate preimplantation embryo subsets, with locus-specific editing rates comparable to those observed under single-target conditions. Following the transfer of 54 electroporated blastocysts, 14 live lambs were born. Targeted deep sequencing detected intended edits in five lambs (35.71%), including three at the <i>FecB</i> locus and two at the <i>TBXT</i> locus. Notably, a <i>TBXT</i>-edited lamb exhibiting a distinct short-tail phenotype (15 cm vs. 24.5 cm in wild-type) carried the highest allele frequency, reaching up to 50.97%, providing direct <i>in vivo</i> functional validation. Targeted analysis of predicted off-target sites detected no credible off-target editing, supporting the specificity of this transient RNP delivery strategy. Collectively, this RNP-electroporation framework provides a feasible and precise pathway for scalable prime editing in livestock, bypassing the limitations of conventional injection-based methods.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1931608"},"PeriodicalIF":5.9,"publicationDate":"2026-09-22","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13638620/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149119363","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR/Cas9-mediated disruption of <i>OsPLDβ1</i> enhances salt stress tolerance in rice through modulation of antioxidant defense.","authors":"Sangeetha Karippadakam, V Mohan M Achary, Dalia Vishnudasan, Hemangini Parmar, Alexander Balamurugan, Mallesham Bulle, Ganesan Prakash, Malireddy K Reddy","doi":"10.3389/fgeed.2026.1904040","DOIUrl":"https://doi.org/10.3389/fgeed.2026.1904040","url":null,"abstract":"<p><p>Soil salinity is a critical abiotic stress that severely constrains rice productivity and threatens global food security. Phospholipase D (PLD) enzymes play pivotal roles in plant stress signaling; however, the specific function of <i>OsPLDβ1</i> in rice salt stress responses remains unclear. We here investigated the role of <i>OsPLDβ1</i> using knockout mutants to elucidate its regulatory function under saline conditions. <i>Ospldβ1</i>-mutant lines exhibited substantially improved salt tolerance in contrast to wild-type plants. Improved tolerance was associated with elevated reactive oxygen species (ROS) scavenging capacity, indicating improved oxidative stress management. Furthermore, the mutant lines displayed increased expression of ion transport and homeostasis genes and maintained photosynthetic efficiency and agronomic performance. Collectively, these physiological and molecular responses demonstrate that <i>OsPLDβ1</i> negatively regulates salt stress tolerance in rice. This research highlights the role of <i>OsPLDβ1</i> in salt tolerance in rice and lays the groundwork for developing salinity-resistant crop varieties through genetic manipulation.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1904040"},"PeriodicalIF":5.9,"publicationDate":"2026-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13634860/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149119384","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Precision engineering of upstream open reading frames for translational regulation and crop improvement.","authors":"Linjie Xu, Jiaojiao Ren, Jinjie Zhu","doi":"10.3389/fgeed.2026.1818972","DOIUrl":"https://doi.org/10.3389/fgeed.2026.1818972","url":null,"abstract":"<p><p>Upstream open reading frames (uORFs) are pervasive cis-regulatory elements in the 5'untranslated regions (UTRs) of eukaryotic mRNAs that modulate translation of downstream main open reading frames (mORFs) by shaping ribosome scanning, initiation, and reinitiation. By operating downstream of transcription, uORFs enable fine-scale control of protein abundance, offering a distinctive regulatory layer for precise tuning of gene expression in plants. Recent advances in ribosome profiling, translation initiation-site mapping, peptidomics, computational annotation, and genome editing have expanded the discovery, functional validation, and targeted manipulation of plant uORFs. In this review, we summarize current understanding of uORF-mediated translational regulation, discuss computational and experimental approaches for candidate identification and functional validation, and highlight genome-editing strategies used to modify endogenous uORFs in crops. We further synthesize representative studies in which uORF editing has been used to modulate metabolic traits, plant development, nutritional quality, and responses to biotic and abiotic stresses. Collectively, available evidence suggests that uORF manipulation can support quantitative and context-dependent trait modulation, in some cases preserving basal gene function and reducing growth fitness penalties. We further discuss outstanding challenges, including transcript isoform complexity, context-dependent translational effects, and the need for validation across diverse genetic and environmental backgrounds. Overall, uORF engineering represents a promising strategy for translational fine-tuning of plant gene expression, with potential implications for crop improvement and sustainable agriculture.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1818972"},"PeriodicalIF":5.9,"publicationDate":"2026-09-21","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13635735/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149119357","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"A novel minimally humanized mouse model of aniridia for preclinical evaluation of CRISPR gene editing strategies.","authors":"Seyedeh Zeinab Mirjalili Mohanna, Andrea J Korecki, Pardis Kazemian, Tess C Lengyell, Bethany A Adair, Blair R Leavitt, Elizabeth M Simpson","doi":"10.3389/fgeed.2026.1910209","DOIUrl":"https://doi.org/10.3389/fgeed.2026.1910209","url":null,"abstract":"<p><strong>Introduction: </strong>Aniridia is a rare congenital vision-loss disorder primarily caused by variants in the paired box 6 (<i>PAX6</i>) gene. Current treatments do not maintain vision long term, nor address the underlying disease mechanism. CRISPR-mediated genome editing holds great promise for treating genetic diseases but requires demonstration of efficacy on human DNA to support translational relevance. The aim of this work was to generate and test a minimally humanized mouse model of aniridia, carrying a FLAG-tagged <i>Pax6</i> and the most common recurring pathogenic aniridia-patient variant, introduced for the first time in mice.</p><p><strong>Methods: </strong>Ocular assessments (gross examination, slit lamp imaging, and fluorescent and confocal microscopy) were performed in mice homozygous for humanization and heterozygous for the variant, paralleling patients. Mice were examined immediately post-wean and at adulthood, with the former chosen as a likely time for treatment initiation. As an exploratory proof-of-principle, a lipid nanoparticle-delivered adenine base editor (ABE8e) was tested to demonstrate the utility of the model.</p><p><strong>Results: </strong>Ocular assessments revealed the expected phenotypes for an aniridia mouse model, supporting successful generation and phenotypic validation of the humanized mouse. The ABE8e treatment resulted in a modest correction (4.8% ± 0.4% SEM) of the pathogenic variant in clinically relevant ex vivo cortical neurons.</p><p><strong>Discussion: </strong>Although designed for the development of genome editing therapies, this humanized mouse is also applicable to other therapies requiring interaction with human DNA or RNA. It also enables evaluation of nonsense-suppression drugs since the readthrough proteins retain the FLAG tag. More broadly, this methodology is applicable to other pathogenic variants for aniridia, CRISPRa approaches, and other genetic diseases for which CRISPR-based therapy holds promise.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1910209"},"PeriodicalIF":5.9,"publicationDate":"2026-09-18","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13631079/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149119261","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"CRISPR/Cas9 in soybean research: a measured leap toward genetic refinement.","authors":"Smrutirekha Sahu, Pandian Rajendran, Sanket Arunrao Ghumde, Diksha Prakash, Shikha Dixit, Deepak Pawar, Dharavath Nagaraju, Vinutha T, Sandeep B Adavi, Palaiyur Nanjappan Sivalingam, Ashish Marathe, Pramod Kumar Rai","doi":"10.3389/fgeed.2026.1821910","DOIUrl":"https://doi.org/10.3389/fgeed.2026.1821910","url":null,"abstract":"<p><p>Soybean [<i>Glycine max</i> (Linn.) Merr] is a globally important source of plant-based protein and edible oil, widely cultivated in India as a major oilseed crop. However, conventional breeding approaches for soybean are laborious, constrained by the genomic complexity of soybean, and face persistent challenges in genetic transformation. Clustered regularly interspaced short palindromic repeats (CRISPR)/Cas-based genome editing has emerged as a precise and efficient tool for rapid trait improvement in soybean. While many existing reviews of soybean have been published, this literature review addresses some fundamental challenges with respect to genome editing. This study summarizes recent advancements in soybean genome editing, offering insights into the practical informatics of soybean-specific genomic databases that are required for successful construct design. It also discusses emerging technologies such as base editing, prime editing, and the increasingly important role of artificial intelligence in accelerating target identification and construct design. It highlights applications in nutritional enhancement, abiotic and biotic stress tolerance, and improvement of key agronomic traits. Beyond the conventional topics, it addresses the current regulatory landscape that researchers must navigate for releasing genome-edited varieties and provides a comprehensive perspective on practical solutions to overcome major constraints in soybean genetic engineering.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1821910"},"PeriodicalIF":5.9,"publicationDate":"2026-09-17","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13629178/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149119289","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}

{"title":"Overcoming delivery barriers of large all-in-one CRISPR/Cas9 plasmids using lysine-based lipid nanoparticles.","authors":"Wenhan Sung, Tianshu Li, Shinji Takeoka","doi":"10.3389/fgeed.2026.1888079","DOIUrl":"https://doi.org/10.3389/fgeed.2026.1888079","url":null,"abstract":"<p><strong>Introduction: </strong>Lipid nanoparticles (LNPs) are widely used as drug delivery systems (DDS) for the intracellular delivery of gene-editing tools such as the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system. Previously, a lysine-headgroup cationic lipid (K3C16) was reported to exhibit high biocompatibility and cellular internalization via plasma membrane fusion when formulated as liposomes, suggesting its potential as a low-toxicity gene delivery material. However, the translation of this lipid into a multi-component lipid nanoparticle (LNP) matrix via microfluidic engineering for large cargo encapsulation has not been explored. In this study, K3C16 was successfully engineered for the first time as the main lipid component of LNPs to deliver large plasmid DNA encoding the CRISPR/Cas9 system.</p><p><strong>Methods: </strong>First, 1,2-dioleoyl-sn-glycero-3-phosphoethanolamine (DOPE) and 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) were evaluated as helper phospholipids to identify an optimal lipid composition. LNPs containing DOPE exhibited significantly higher transfection efficiency than those containing DOPC and were therefore selected for subsequent experiments. Next, LNPs formulated with different cationic or ionizable lipids were prepared and evaluated in HEK293 cells for transfection efficiency, cytotoxicity, and gene-editing efficiency.</p><p><strong>Results: </strong>LNPs containing the commercial ionizable lipid SM-102 showed higher transfection and gene-editing efficiencies than the lysine-headgroup cationic lipids represent a promising and biocompatible platform for CRISPR/Cas9 plasmid delivery; however, increased cytotoxicity was observed in highly transfected cell populations. In contrast, the lysine-headgroup cationic LMPs achieved effective delivery of large plasmid DNA and CRISPR/Cas9-mediated gene editing while maintaining superior biocompatibility.</p><p><strong>Conclusion: </strong>These results demonstrate that lysine-headgroup cationic lipids represent a promising and biocompatible platform for CRISPR/Cas9 plasmid delivery. Further optimization of lipid composition and molar ratios may enhance transfection and gene-editing efficiencies, advancing the development of safer nonviral gene delivery systems.</p>","PeriodicalId":73086,"journal":{"name":"Frontiers in genome editing","volume":"8 ","pages":"1888079"},"PeriodicalIF":5.9,"publicationDate":"2026-09-16","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC13624556/pdf/","citationCount":null,"resultStr":null,"platform":"Semanticscholar","paperid":"149119318","PeriodicalName":null,"FirstCategoryId":null,"ListUrlMain":null,"RegionNum":0,"RegionCategory":"","ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":"OA","EPubDate":null,"PubModel":null,"JCR":null,"JCRName":null,"Score":null,"Total":0}