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Enhanced immunogenicity of swine coronavirus PEDV by engineering transcriptional regulatory sequences.
Porcine epidemic diarrhea virus (PEDV) is a porcine enteropathogenic coronavirus that can cause severe diarrhea and high mortality in neonatal piglets. Therefore, the development of safe and effective vaccines remains a top priority for controlling PEDV infection. Previous studies have found that the transcription regulatory sequence (TRS) of the viral M gene has the strongest regulatory ability for the expression of exogenous gene. In order to investigate whether the transcription level of the S gene is increased after swapping body TRS of the S gene (TRS-S) with the body-TRS of M gene (TRS-M), CRISPR/Cas9 gene editing technology were used to replace the TRS-S of PEDV with TRS-M, and found that replacing TRS-S by TRS-M, as well as the downstream bases of TRS-M, can effectively promote the expression of S protein. Subsequently, the recombinant strain with high expression of S protein was prepared into an inactivated vaccine, which induced higher levels of S-specific immunoglobulin G (IgG), IgA, and neutralizing antibodies in immunized pregnant sows. The newborn spiking pigs also obtained high levels of antibodies through breastfeeding, and after being challenged, it was found that clinical symptoms and microscopic lesions were significantly reduced. In conclusion, these data suggest that replacing TRS-S with the transcriptionally efficient TRS-M effectively promotes PEDV S protein expression, and this modified strain exhibits improved immunogenicity and better protection against viral challenge, which provides new method for the research and development of PEDV vaccines.
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Advances in gene editing strategies for epidermolysis bullosa.
Epidermolysis bullosa represents a monogenetic disease comprising a variety of heterogeneous mutations in at least 16 genes encoding structural proteins crucial for skin integrity. Due to well-defined mutations but still lacking causal treatment options for the disease, epidermolysis bullosa represents an ideal candidate for gene therapeutic interventions. Recent developments and improvements in the genome editing field have paved the way for the translation of various gene repair strategies into the clinic. With the ability to accurately predict and monitor targeting events within the human genome, the translation might soon be possible. Here, we describe current advancements in the genome editing field for epidermolysis bullosa, along with a discussion of aspects and strategies for precise and personalized gene editing-based medicine, in order to develop efficient and safe ex vivo as well as in vivo genome editing therapies for epidermolysis bullosa patients in the future.
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Rapid genome editing by CRISPR-Cas9-POLD3 fusion.
Precision CRISPR gene editing relies on the cellular homology-directed DNA repair (HDR) to introduce custom DNA sequences to target sites. The HDR editing efficiency varies between cell types and genomic sites, and the sources of this variation are incompletely understood. Here, we have studied the effect of 450 DNA repair protein-Cas9 fusions on CRISPR genome editing outcomes. We find the majority of fusions to improve precision genome editing only modestly in a locus- and cell-type specific manner. We identify Cas9-POLD3 fusion that enhances editing by speeding up the initiation of DNA repair. We conclude that while DNA repair protein fusions to Cas9 can improve HDR CRISPR editing, most need to be optimized to the cell type and genomic site, highlighting the diversity of factors contributing to locus-specific genome editing outcomes.
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CRISPR-Cpf1-mediated genome editing and gene regulation in human cells.
Clustered regularly interspaced short palindromic repeat (CRISPR) system is being championed as a robust and flexible tool for genome editing. Compared with CRISPR associated protein 9 (Cas9), the CRISPR from Prevotella and Francisella 1 (Cpf1) protein has some distinct characteristics, including RNase activity, T-rich protospacer adjacent motif (PAM) preference and generation of sticky cutting ends. The extremely low propensity of off-target effects and relatively high editing efficiency represent prominent advantages of Cpf1 over Cas9. CRISPR-Cpf1, alone or fused with function domains, has broadly expanded the applications such as multiplex gene knockout, transcriptional repression or activation and epigenome editing in a drug controlled way. Meanwhile, the modification of CRISPR RNAs (crRNAs) with aptamer RNA achieves great promotion on genome editing. Moreover, disease-associated gene manipulation in mice, tumor mutation detection in patients with cancers, and more yet to come, represent growing demands of CRISPR-Cpf1 in clinical genome therapy. In this review, we summarized the unique properties of Cpf1 and the molecular mechanisms underlying CRISPR-Cpf1 on gene editing and regulation in human cells.
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Label-free Quantitative Analysis of Protein Expression Alterations in miR-26a-Knockout HeLa Cells using SWATH-MS Technology.
MicroRNAs (miRNAs) bind to the 3'-untranslated region of target mRNAs in a sequence-specific manner and subsequently repress gene translation. Human miR-26a has been studied extensively, but the target transcripts are far from complete. We first employed the CRISPR-Cas9 system to generate an miR-26a-knockout line in human cervical cancer HeLa cells. The miR26a-knockout line showed increased cell growth and altered proliferation. Proteomics technology of sequential window acquisition of all theoretical mass spectra (SWATH-MS) was utilized to compare the protein abundance between the wild-type and the knockout lines, with an attempt to identify transcripts whose translation was influenced by miR-26a. Functional classification of the proteins with significant changes revealed their function in stress response, proliferation, localization, development, signaling, etc. Several proteins in the cell cycle/proliferation signaling pathway were chosen to be validated by western blot and parallel reaction monitoring (PRM). The satisfactory consistency among the three approaches indicated the reliability of the SWATH-MS quantification. Among the computationally predicted targets, a subset of the targets was directly regulated by miR-26a, as demonstrated by luciferase assays and Western blotting. This study creates an inventory of miR-26a-targeted transcripts in HeLa cells and provides fundamental knowledge to further explore the functions of miR-26a in human cancer.
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Inconclusive studies on possible CRISPR-Cas off-targets should moderate expectations about enzymes that have evolved to be non-specific.
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Public acceptance and stakeholder views of gene edited foods: a global overview.
The increasing popularity of gene editing in plants has prompted research on stakeholder views. Gene edited foods are often more accepted than genetically modified foods, though differences occur within target groups, regions, and products. Nevertheless, marketing challenges related to a lack of familiarity with the technology, labeling, and risk perception remain.
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Inducible CRISPR/Cas systems in precision oncology: Current applications and future perspectives.
BACKGROUND: Inducible CRISPR/Cas systems enable spatiotemporal control of genome editing in response to chemical, optical, biological, or physical stimuli. By restricting genome-editing activity to defined conditions, these systems may reduce off-target exposure and immune burden while improving tumor-selective control, making them attractive tools for precision oncology. MAIN BODY: This review summarizes the molecular mechanisms, design principles, and current applications of inducible CRISPR/Cas systems in cancer research and therapy. These platforms are classified into chemically inducible, optogenetic, tumor microenvironment-responsive, physically triggered, and logic-gated systems. Regulatory strategies are discussed at multiple levels, including transcriptional control, post-translational regulation, guide RNA engineering, and stimulus-responsive delivery. Key applications include functional genomic screening, cancer modeling, therapeutic gene editing, immunotherapy enhancement, and combinatorial treatment strategies. We also examine current delivery approaches, including viral vectors, lipid nanoparticles, stimulus-responsive nanocarriers, and biomimetic platforms. CONCLUSION: Inducible CRISPR/Cas systems represent a promising platform for next-generation precision cancer therapy. However, substantial optimization and rigorous preclinical validation remain necessary to address challenges related to leaky expression, induction efficiency, tissue penetration, immunogenicity, and long-term safety before clinical translation can be realized. KEY POINTS: Inducible CRISPR/Cas systems enable conditional genome editing in precision oncology. Chemical, optical, TME-responsive, physical, and logic-gated systems offer distinct control features. Delivery, leakiness, immunogenicity, and safety remain key translational barriers. Ex vivo immune-cell engineering and locoregional delivery may offer nearer-term clinical routes.
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A new 'mini-CRISPR' flexes its editing power in monkey muscles.
The downsized DNA-slicing machinery may reach more tissues to take aim at more diseases.
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Decoupling the bridge helix of Cas12a results in a reduced trimming activity, increased mismatch sensitivity and impaired conformational transitions.
The widespread and versatile prokaryotic CRISPR-Cas systems (clustered regularly interspaced short palindromic repeats and associated Cas proteins) constitute powerful weapons against foreign nucleic acids. Recently, the single-effector nuclease Cas12a that belongs to the type V CRISPR-Cas system was added to the Cas enzymes repertoire employed for gene editing purposes. Cas12a is a bilobal enzyme composed of the REC and Nuc lobe connected by the wedge, REC1 domain and bridge helix (BH). We generated BH variants and integrated biochemical and single-molecule FRET (smFRET) studies to elucidate the role of the BH for the enzymatic activity and conformational flexibility of Francisella novicida Cas12a. We demonstrate that the BH impacts the trimming activity and mismatch sensitivity of Cas12a resulting in Cas12a variants with improved cleavage accuracy. smFRET measurements reveal the hitherto unknown open and closed state of apo Cas12a. BH variants preferentially adopt the open state. Transition to the closed state of the Cas12a-crRNA complex is inefficient in BH variants but the semi-closed state of the ternary complex can be adopted even if the BH is deleted in its entirety. Taken together, these insights reveal that the BH is a structural element that influences the catalytic activity and impacts conformational transitions of FnCas12a.
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