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The BBSome assembly is spatially controlled by BBS1 and BBS4 in human cells.
Bardet-Biedl syndrome (BBS) is a pleiotropic ciliopathy caused by dysfunction of primary cilia. More than half of BBS patients carry mutations in one of eight genes encoding for subunits of a protein complex, the BBSome, which mediates trafficking of ciliary cargoes. In this study, we elucidated the mechanisms of the BBSome assembly in living cells and how this process is spatially regulated. We generated a large library of human cell lines deficient in a particular BBSome subunit and expressing another subunit tagged with a fluorescent protein. We analyzed these cell lines utilizing biochemical assays, conventional and expansion microscopy, and quantitative fluorescence microscopy techniques: fluorescence recovery after photobleaching and fluorescence correlation spectroscopy. Our data revealed that the BBSome formation is a sequential process. We show that the pre-BBSome is nucleated by BBS4 and assembled at pericentriolar satellites, followed by the translocation of the BBSome into the ciliary base mediated by BBS1. Our results provide a framework for elucidating how BBS-causative mutations interfere with the biogenesis of the BBSome.
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New frontiers in the therapy of primary immunodeficiency: From gene addition to gene editing.
The most severe primary immune deficiency diseases (PIDs) have been successfully treated with allogeneic hematopoietic stem cell transplantation for more than 4 decades. However, such transplantations have the best outcomes when there is a well-matched donor available because immune complications, such as graft-versus-host disease, are greater without a matched sibling donor. Gene therapy has been developed as a method to perform autologous transplantations of a patient's own stem cells that are genetically corrected. Through an iterative bench-to-bedside-and-back process, methods to efficiently add new copies of the relevant gene to hematopoietic stem cells have led to safe and effective treatments for several PIDs, including forms of severe combined immune deficiency, Wiskott-Aldrich syndrome, and chronic granulomatous disease. New methods for gene editing might allow additional PIDs to be treated by gene therapy because they will allow the endogenous gene to be repaired and expressed under its native regulatory elements, which are essential for genes involved in cell processes of signaling, activation, and proliferation. Gene therapy is providing exciting new treatment options for patients with PIDs, and advances are sure to continue.
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DNAJC14 gene-edited pigs are resistant to classical pestiviruses.
Infectious diseases remain a major impediment to livestock production, negatively impacting both productivity and welfare. Where key interactions between viruses and host proteins have been identified, it is possible to rationally devise intervention strategies. In vitro studies have identified the host protein DNAJC14 as a core component of the replicative cycle of classical pestiviruses. Outbreaks caused by this group of viruses cause enormous losses in stock farming due to culling and export restrictions. Using CRISPR/Cas9 gene editing, we produced a cohort of pigs with altered DNAJC14. Primary cells from these animals did not support replication of either classical swine fever virus (CSFV) or bovine viral diarrhoea virus (BVDV) in vitro. In vivo challenge with CSFV revealed that the edited pigs displayed complete resistance to infection. This establishes gene editing as an additional strategy that can contribute to the control of classical pestiviruses.
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CRISPR/Cas9: Regulations and challenges for law enforcement to combat its dual-use.
For many years, scientists have aspired to edit any DNA segment of living organisms precisely, and with the discovery of CRISPR/Cas9, they have moved closer to achieving their objective. Using this tool, researchers can knock out or knock-in a gene into or from any part of the genome. The CRISPR-Cas9 system is a highly effective and accurate tool and has replaced previous genome editing technologies such as ZFN and TALEN having lesser precision and accuracy. Advances in CRISPR/Cas technology have been remarkable in recent years, with significant applications in various fields of life sciences research. Despite the diverse applications of the CRISPR system, this tool also poses a threat to living organisms and the environment. The intentional misuse of CRISPR could be a disaster to society and the environment. Different countries have imposed laws and guidelines to regulate genome editing through multiple agencies. However, in some countries, these rules and regulations are still under development or don't exist, leading to higher chances of the technology getting misused. Dual-use of this technology could be a threat to biosecurity and biodefense. This article summarizes the potential applications of the CRISPR system in human and plants, and also evaluate the laws and regulations imposed by different countries to keep genome editing technology under check.
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Development of fragrant broomcorn millet (Panicum miliaceum L.) via CRISPR/Cas12i.3-mediated genome editing.
Co-editing the broomcorn millet PmBADH2a and PmBADH2b genes using CRISPR/Cas12i.3 generated double mutants with significantly increased 2-acetyl-1-pyrroline content, producing fragrant broomcorn millet without compromising major agronomic traits.
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CRISPR editing of miR-33 restores ApoE lipidation and amyloid-β metabolism in ApoE4 sporadic Alzheimer's disease.
Sporadic Alzheimer's disease (sAD) is marked by dysregulated lipid metabolism, prominently involving apolipoprotein E (ApoE). MicroRNA-33 (miR-33) has emerged as a key regulator of lipid homeostasis, yet its role in sAD remains unclear. This study investigated miR-33 dysregulation in APOE ε4 allele (ApoE4)-associated sAD and explored its therapeutic potential using clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated gene editing. Elevated miR-33 expression was observed in both AD patients, particularly those with ApoE4-associated sAD, and in the ApoE4 mouse model, implicating its role in AD pathology. Using CRISPR/Cas9, we modulated miR-33 expression in astrocytes to regulate ApoE lipidation and ameliorate AD-related pathology. Our results show that targeted miR-33 regulation in astrocytes via CRISPR/Cas9 restores ApoE lipidation and mitigates AD pathology in both in vitro and in vivo AD mice. Additionally, applying this gene therapy approach in ApoE4 sAD patient cell lines highlights its translational potential for therapeutic intervention. In conclusion, our findings elucidate miR-33's role in AD pathogenesis and underscore the therapeutic promise of CRISPR-mediated miR-33 targeting for restoring lipid homeostasis and ameliorating AD pathology. This study provides valuable insights into developing miRNA-based gene therapy strategies for treating sAD.
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Improved Genome Editing in the Ascidian Ciona with CRISPR/Cas9 and TALEN.
The ascidian Ciona intestinalis type A (or Ciona robusta) is an important organism for elucidating the mechanisms that make the chordate body plan. CRISPR/Cas9 and TAL effector nuclease (TALEN) are widely used to quickly address genetic functions in Ciona. Our previously reported method of CRISPR/Cas9-mediated mutagenesis in this animal has inferior mutation rates compared to those of TALENs. We here describe an updated way to effectively mutate genes with CRISPR/Cas9 in Ciona. Although the construction of TALENs is much more laborious than that of CRISPR/Cas9, this technique is useful for tissue-specific knockouts that are not easy even by the optimized CRISPR/Cas9 method.
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Construction of a series of episomal plasmids and their application in the development of an efficient CRISPR/Cas9 system in Pichia pastoris.
The methylotrophic yeast Pichia pastoris is widely used in recombinant expression of eukaryotic proteins owing to the ability of post-translational modification, tightly regulated promoters, and high cell density fermentation. However, episomal plasmids for heterologous gene expression and the CRISPR/Cas9 system for genome editing have not been well developed in P. pastoris. In the present study, a panel of episomal plasmids containing various autonomously replicating sequences (ARSs) were constructed and their performance in transformation efficiency, copy numbers, and propagation stability were systematically compared. Among the five ARSs with different origins, panARS isolated from Kluyveromyces lactis was determined to have the best performance and used to develop an efficient CRISPR/Cas9 based genome editing system. Compared with a previously reported system using the endogenous and most commonly used ARS (PARS1), the CRISPR/Cas9 genome editing efficiency was increased for more than tenfold. Owing to the higher plasmid stability with panARS, efficient CRISPR/Cas9-mediated genome editing with a type III promoter (i.e. SER promoter) to drive the expression of the single guide RNA (sgRNA) was achieved for the first time. The constructed episomal plasmids and developed CRISPR/Cas9 system will be important synthetic biology tools for both fundamental studies and industrial applications of P. pastoris.
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Dual-vector nanoengineered mRNA-CRISPR delivery systems for precision gene therapy in choroidal melanoma.
Choroidal melanoma is the most common primary intraocular malignancy, and effective gene therapy remains limited by ocular barriers, tumor heterogeneity, and inefficient nucleic acid delivery. This review critically analyses the molecular rationale, engineering principles, emerging nanoarchitectures, biological validation, and translational prospects of dual-vector nanoengineered mRNA-CRISPR delivery systems for precision gene therapy in choroidal melanoma. The manuscript also highlights key challenges and future strategies for clinical implementation. Current evidence indicates that dual-vector platforms enable independent optimization of mRNA and CRISPR cargos while maintaining coordinated spatiotemporal therapeutic activity. We conducted a literature search in PubMed, Scopus, and Web of Science for relevant studies published from January 2015 to September 2026. Functional cargo partitioning, vector modularity, programmable intracellular trafficking, and ocular-targeted nanoengineering improve delivery precision and reduce biological compromise associated with co-encapsulation. Lipid, polymeric, hybrid, biomimetic, and viral-nonviral architectures each provide distinct engineering advantages depending on therapeutic objectives. Preclinical studies support enhanced genome editing, transient protein restoration, improved tumor suppression, and reduced off-target effects. Nevertheless, manufacturing scalability, regulatory standardization, long-term safety, and clinical validation remain major barriers to translation. Dual-vector nanoengineered mRNA-CRISPR systems represent a promising next-generation platform for precision gene therapy in choroidal melanoma by enabling coordinated and programmable therapeutic delivery. Continued advances in modular nanoengineering, personalized cargo selection, and clinically scalable manufacturing will be essential for successful translation into precision ocular oncology.
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Dual CRISPR-Cas9 Cleavage Mediated Gene Excision and Targeted Integration in Yarrowia lipolytica.
CRISPR-Cas9 technology has been successfully applied in Yarrowia lipolytica for targeted genomic editing including gene disruption and integration; however, disruptions by existing methods typically result from small frameshift mutations caused by indels within the coding region, which usually resulted in unnatural protein. In this study, a dual cleavage strategy directed by paired sgRNAs is developed for gene knockout. This method allows fast and robust gene excision, demonstrated on six genes of interest. The targeted regions for excision vary in length from 0.3 kb up to 3.5 kb and contain both non-coding and coding regions. The majority of the gene excisions are repaired by perfect nonhomologous end-joining without indel. Based on this dual cleavage system, two targeted markerless integration methods are developed by providing repair templates. While both strategies are effective, homology mediated end joining (HMEJ) based method are twice as efficient as homology recombination (HR) based method. In both cases, dual cleavage leads to similar or improved gene integration efficiencies compared to gene excision without integration. This dual cleavage strategy will be useful for not only generating more predictable and robust gene knockout, but also for efficient targeted markerless integration, and simultaneous knockout and integration in Y. lipolytica.
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