Bettina Nagy, Aysha Jameel, Gábor V. Horváth, Katalin Török, Ferhan Ayaydin, Nikolett László, Divya Teja Dondapati, Sándor Mórocz, Zoltán Zombori, Györgyi Ferenc, Dénes Dudits
Given the growing emphasis on the efficient application of CRISPR/Cas9 in plant genome editing, there is a need for methodologies that enhance targeted mutagenesis efficiency. In the present study, we utilized stable transgenic maize callus tissues harboring active CRISPR/Cas9 vector systems targeting either the abnormal cytokinin response 1 (ARE1) gene or the potassium channel gene (KAT2). The presented DNA sequencing data indicate that the KAT2 -targeting vector, which utilizes a 3′-end truncated guide RNA, retained mutagenic activity, albeit at a notably low frequency. We quantified mutagenic events following exposure to elevated temperatures (37 °C for 48 h) and various chromatin-modifying compounds, including sodium butyrate (NaBt), nicotinamide (NA), and 5-azacytidine (AZA). High-temperature treatment alone increased the frequency of nucleotide deletions (5.8-fold relative to the untreated samples) in the ARE1 target gene. Conversely, in the KAT2 gene, both deletions and insertions were stimulated (6.4 and 6.2-fold, respectively). As a novel approach, we evaluated the mutagenic efficacy of chromatin-modifying compounds administered in conjunction with heat treatment. The combined application of NaBt and AZA with elevated temperature was particularly effective in increasing insertion frequencies; in the ARE1 (with NaBt + heat: 17.3-fold) and KAT2 (with AZA + heat: 21.4-fold) genes. Cell cycle analysis of suspension-cultured cells revealed that chromatin-modifying treatments altered cell cycle progression, evidenced by an accumulation of cells in the G2 phase and a significant reduction in the number of mitotic cells. Subsequent DNA sequencing demonstrated a differential increase in the induction of deletions and insertions, alongside an expansion of the deleted regions.
Katariina Mamia, Anniken Solveig Matheson Sollano, Vanja Lundberg, Svetlana Vakkilainen, Eliisa Kekäläinen, Camilla Schalin-Jäntti, Eira Leinonen, Børre Fevang, Juha Grönholm, Timi Martelius, Shiva Dahal-Koirala, Emma Maria Haapaniemi
CRISPR/Cas9 enables precision gene editing via homology-directed repair (HDR) for mutation correction and disease modelling. Here we present a customizable T cell single nucleotide variant (SNV) correction platform based on non-viral HDR, previously described in Mamia et al. Precision T Cell Correction Platform for Inborn Errors of Immunity. Molecular Therapy, 2025. To enable reproducible application, we provide a comprehensive, step-by-step 8-day HDR workflow for editing primary T cells, covering CRISPR/Cas9 reagent design, custom genomic editing from start to finish, optimization and validation strategies, on-target detection, and flow cytometric assessment of T cell phenotype and exhaustion. The platform separates locus-specific reagent optimization from a fixed cellular workflow, enabling implementation of new genomic targets without modifying the downstream T cell editing pipeline. Using this workflow, we have achieved up to 80% editing efficiency across multiple loci and donors. We have further optimized the T cell culture platform for PBMC stimulation to promote CD4 + and CD8 + T cell activation and proliferation. We validated this culture platform in 33 patients with Inborn Errors of Immunity using flow cytometry and observed that the defined culture conditions maintain low T cell exhaustion and support persistence of memory T cell populations. The protocol was developed under research-grade conditions but supports scalability and transition to preclinical and clinical GMP workflows. Overall, this adaptable platform is a broadly applicable framework for precision T cell engineering across diverse genomic targets offering opportunities for both in vitro disease modeling and therapeutic genome correction.
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.
Wei Jin, Xiaobo Chen, Weifeng Jin, Hai Liao, Li Yu
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. 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. 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 128 3 resolution). 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.
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
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. 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. 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. 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.
