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Advances in Modeling Polyglutamine Diseases Using Genome Editing Tools.
Polyglutamine (polyQ) diseases, including Huntington's disease, are a group of late-onset progressive neurological disorders caused by CAG repeat expansions. Although recently, many studies have investigated the pathological features and development of polyQ diseases, many questions remain unanswered. The advancement of new gene-editing technologies, especially the CRISPR-Cas9 technique, has undeniable value for the generation of relevant polyQ models, which substantially support the research process. Here, we review how these tools have been used to correct disease-causing mutations or create isogenic cell lines with different numbers of CAG repeats. We characterize various cellular models such as HEK 293 cells, patient-derived fibroblasts, human embryonic stem cells (hESCs), induced pluripotent stem cells (iPSCs) and animal models generated with the use of genome-editing technology.
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CRISPR/Cas9-Mediated triple gene editing of Pi21, Bsr-d1, and Xa5 confers broad-spectrum disease resistance in elite early-season rice without yield compromise.
Rice blast and bacterial blight are devastating diseases that severely threaten global rice production. To develop durable resistance without compromising yield, we utilized CRISPR/Cas9 gene editing to simultaneously knock out three negative immune regulators, Pi21, Bsr-d1, and Xa5, in two elite early-rice cultivars (ZZU53 and ZZU100). We successfully obtained transgene-free homozygous triple mutants that exhibited significantly enhanced resistance to both Magnaporthe oryzae (M. oryzae) and Xanthomonas oryzae pv. oryzae (Xoo). This broad-spectrum resistance correlates with the constitutive upregulation of key defense genes in the salicylic acid (SA) pathway (OsPR1a, OsPR1b, and OsWRKY45) and jasmonic acid (JA) pathways (OsPR4). Comprehensive agronomic evaluation demonstrated that critical agricultural traits such as thousand-grain weight, panicle architecture, heading date, and tiller number remained comparable to wild-type plants, indicating no substantial yield penalty. This approach provides an efficient, transgene-free pathway for developing disease-resistant early-rice cultivars. Multiple environment field trials are now required to evaluate its long-term agricultural viability.
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Protocol for genetic engineering in Drosophila suzukii using microinjection.
The spotted wing Drosophila (Drosophila suzukii Matsumura) is recognized globally as a significant economic pest. Here, we present a protocol for genetic engineering in D. suzukii using microinjection. We describe steps for genetic engineering techniques, including transposon-mediated germline transformation, recombinase-mediated genome targeting, and CRISPR-mediated gene editing. This protocol can significantly expand the toolkit for functional genomics and genetic control studies of this pest. For complete details on the use and execution of this protocol, please refer to Schetelig and Handler,1 Schetelig et al.2 Yan et al.,3 and Yan et al.4.
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Robust miniature Cas-based transcriptional modulation by engineering Un1Cas12f1 and tethering Sso7d.
The miniature V-F CRISPR-Cas12f system has been repurposed for gene editing and transcription modulation. The small size of Cas12f satisfies the packaging capacity of adeno-associated virus (AAV) for gene therapy. However, the efficiency of Cas12f-mediated transcriptional activation varies among different target sites. Here, we developed a robust miniature Cas-based transcriptional activation or silencing system using Un1Cas12f1. We engineered Un1Cas12f1 and the cognate guide RNA and generated miniCRa, which led to a 1,319-fold increase in the activation of the ASCL1 gene. The activity can be further increased by tethering DNA-binding protein Sso7d to miniCRa and generating SminiCRa, which reached a 5,628-fold activation of the ASCL1 gene and at least hundreds-fold activation at other genes examined. We adopted these mutations of Un1Cas12f1 for transcriptional repression and generated miniCRi or SminiCRi, which led to the repression of ∼80% on average of eight genes. We generated an all-in-one AAV vector AIOminiCRi used to silence the disease-related gene SERPINA1. AIOminiCRi AAVs led to the 70% repression of the SERPINA1 gene in the Huh-7 cells. In summary, miniCRa, SminiCRa, miniCRi, and SminiCRi are robust miniature transcriptional modulators with high specificity that expand the toolbox for biomedical research and therapeutic applications.
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Precise correction of G6PD Viangchan mutation in iPSCs by prime editing strategy.
Individuals with glucose-6-phosphate dehydrogenase (G6PD) deficiency hold a significant risk of severe hemolytic crises under oxidative stress. Currently, the definitive and curative treatment for the disorder has not been developed. Among over 200 G6PD variants, G6PD Viangchan (c.871 G > A) is the most prevalent and has been extensively studied in Southeast Asia. This study assessed the effectiveness of prime editing for correcting the G6PD Viangchan mutation in an established mutant HEK293T cell line and G6PD-deficient induced pluripotent stem cells (iPSCs). Using optimized modalities, prime editing achieved a high correction efficiency of over 25% in the HEK293T cells. In iPSCs, this gene editing tool yielded satisfactory correction outcomes, with approximately 5% corrected alleles. Our findings indicate that prime editing provides high precision, producing minimal by-products below baseline and showing undetectable off-target effects. Overall, prime editing has the potential to correct the G6PD Viangchan mutation, providing a valuable approach for future therapeutic strategies and the generation of isogenic cell lines to promote extensive studies in drug discovery and the pathogenesis of the G6PD variant.
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Significant enhancement of fatty acid composition in seeds of the allohexaploid, Camelina sativa, using CRISPR/Cas9 gene editing.
The CRISPR/Cas9 nuclease system is a powerful and flexible tool for genome editing, and novel applications of this system are being developed rapidly. Here, we used CRISPR/Cas9 to target the FAD2 gene in Arabidopsis thaliana and in the closely related emerging oil seed plant, Camelina sativa, with the goal of improving seed oil composition. We successfully obtained Camelina seeds in which oleic acid content was increased from 16% to over 50% of the fatty acid composition. These increases were associated with significant decreases in the less desirable polyunsaturated fatty acids, linoleic acid (i.e. a decrease from ~16% to <4%) and linolenic acid (a decrease from ~35% to <10%). These changes result in oils that are superior on multiple levels: they are healthier, more oxidatively stable and better suited for production of certain commercial chemicals, including biofuels. As expected, A. thaliana T2 and T3 generation seeds exhibiting these types of altered fatty acid profiles were homozygous for disrupted FAD2 alleles. In the allohexaploid, Camelina, guide RNAs were designed that simultaneously targeted all three homoeologous FAD2 genes. This strategy that significantly enhanced oil composition in T3 and T4 generation Camelina seeds was associated with a combination of germ-line mutations and somatic cell mutations in FAD2 genes in each of the three Camelina subgenomes.
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ALPINE: a scalable pipeline for comprehensive classification of gene-editing outcomes from long-read amplicon sequencing.
SUMMARY: CRISPR genome editing has enabled precise genetic modification for gene and cell therapies, but edits often produce heterogeneous on-target outcomes, including homology-directed repair (HDR) knock-ins, DNA repair template integrations, and structural variants. Existing tools are frequently limited to short reads or lack viral vector-specific integration categories needed for therapeutic development. Here, we present ALPINE (Amplicon Long-read Pipeline for INtegration Evaluation), a scalable and reproducible pipeline for classifying and quantifying gene-editing outcomes from long-read amplicon sequencing supporting both PacBio HiFi and Oxford Nanopore platforms. ALPINE classifies reads into 10+ categories, including DNA repair vector integration subtypes, and performs variant calling near the gene-edited site with batch, multi-sample reporting. Uniquely, ALPINE can distinguish between cells treated with multiple DNA repair vectors and identify distinct molecular features, such as inverted terminal repeats (ITRs), enabling comprehensive characterization of complex gene editing outcomes. Dual-target benchmarking on simulated datasets demonstrated high accuracy for transgene integration events. Independent validation on public crosslinked-HDR dataset confirmed ALPINE's integration detection capabilities, and application to edited T cell samples demonstrated comprehensive gene-editing outcome profiling. AVAILABILITY: ALPINE is available under MIT license at https://github.com/Maggi-Chen/ALPINE and https://doi.org/10.5281/zenodo.20272510. All analysis scripts and visualization code used in this manuscript are available at https://github.com/Maggi-Chen/ALPINE-manuscript-analysis. Simulated datasets are deposited at Zenodo (https://doi.org/10.5281/zenodo.20260865). Public dataset PRJNA913199 is available through NCBI SRA.
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CRISPR/Cas9 Editing Sites Identification and Multi-Elements Association Analysis in Camellia sinensis.
CRISPR/Cas9 is an efficient genome-editing tool, and the identification of editing sites and potential influences in the Camellia sinensis genome have not been investigated. In this study, bioinformatics methods were used to characterise the Camellia sinensis genome including editing sites, simple sequence repeats (SSRs), G-quadruplexes (GQ), gene density, and their relationships. A total of 248,134,838 potential editing sites were identified in the genome, and five PAM types, AGG, TGG, CGG, GGG, and NGG, were observed, of which 66,665,912 were found to be specific, and they were present in all structural elements of the genes. The characteristic region of high GC content, GQ density, and PAM density in contrast to low gene density and SSR density was identified in the chromosomes in the joint analysis, and it was associated with secondary metabolites and amino acid biosynthesis pathways. CRISPR/Cas9, as a technology to drive crop improvement, with the identified editing sites and effector elements, provides valuable tools for functional studies and molecular breeding in Camellia sinensis.
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Genome-edited rabbits: Unleashing the potential of a promising experimental animal model across diverse diseases.
Animal models are extensively used in all aspects of biomedical research, with substantial contributions to our understanding of diseases, the development of pharmaceuticals, and the exploration of gene functions. The field of genome modification in rabbits has progressed slowly. However, recent advancements, particularly in CRISPR/Cas9-related technologies, have catalyzed the successful development of various genome-edited rabbit models to mimic diverse diseases, including cardiovascular disorders, immunodeficiencies, aging-related ailments, neurological diseases, and ophthalmic pathologies. These models hold great promise in advancing biomedical research due to their closer physiological and biochemical resemblance to humans compared to mice. This review aims to summarize the novel gene-editing approaches currently available for rabbits and present the applications and prospects of such models in biomedicine, underscoring their impact and future potential in translational medicine.
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Efficient CRISPR/Cas9-mediated knockin of reporter genes in rats at ROSA26 by pronuclear microinjection.
The genetic modification of rats is a key technology for advancing biomedical research on human diseases. CRISPR/Cas9-mediated genome editing enables the generation of knockout rats in a single step, without the need for embryonic stem cells, by directly injecting genome editing components into zygotes. This simplifies the process, reduces costs, and accelerates gene function analysis in rats. However, the insertion of a gene cassette into a target site has remained inefficient, limiting the generation of knockin (KI) rats. To overcome this issue, we developed an optimized method that covers the entire process from zygote harvesting with superovulation to timed microinjection, ensuring the consistent generation of KI rats. We successfully generated four different fluorescent reporter lines at the ROSA26 locus in rats. Our study provides detailed, step-by-step protocols for donor vector design, zygote collection, microinjection, founder screening, and cryopreservation in rats.
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