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Advances in and Perspectives on Transgenic Technology and CRISPR-Cas9 Gene Editing in Broccoli.
Broccoli, a popular international Brassica oleracea crop, is an important export vegetable in China. Broccoli is not only rich in protein, vitamins, and minerals but also has anticancer and antiviral activities. Recently, an Agrobacterium-mediated transformation system has been established and optimized in broccoli, and transgenic transformation and CRISPR-Cas9 gene editing techniques have been applied to improve broccoli quality, postharvest shelf life, glucoraphanin accumulation, and disease and stress resistance, among other factors. The construction and application of genetic transformation technology systems have led to rapid development in broccoli worldwide, which is also good for functional gene identification of some potential traits in broccoli. This review comprehensively summarizes the progress in transgenic technology and CRISPR-Cas9 gene editing for broccoli over the past four decades. Moreover, it explores the potential for future integration of digital and smart technologies into genetic transformation processes, thus demonstrating the promise of even more sophisticated and targeted crop improvements. As the field continues to evolve, these innovations are expected to play a pivotal role in the sustainable production of broccoli and the enhancement of its nutritional and health benefits.
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Glucocorticoid receptor quaternary structure drives chromatin occupancy and transcriptional outcome.
Most transcription factors, including nuclear receptors, are widely modeled as binding regulatory elements as monomers, homodimers, or heterodimers. Recent findings in live cells show that the glucocorticoid receptor NR3C1 (also known as GR) forms tetramers on enhancers, owing to an allosteric alteration induced by DNA binding, and suggest that higher oligomerization states are important for the gene regulatory responses of GR. By using a variant (GRtetra) that mimics this allosteric transition, we performed genome-wide studies using a GR knockout cell line with reintroduced wild-type GR or reintroduced GRtetra. GRtetra acts as a super receptor by binding to response elements not accessible to the wild-type receptor and both induces and represses more genes than GRwt. These results argue that DNA binding induces a structural transition to the tetrameric state, forming a transient higher-order structure that drives both the activating and repressive actions of glucocorticoids.
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Recent advances in spatiotemporal control of the CRISPR/Cas9 system.
The CRISPR/Cas9 gene-editing technology, derived from the adaptive immune mechanisms of bacteria, has demonstrated remarkable advantages in fields such as gene function research and the treatment of genetic diseases due to its simplicity in design, precise targeting, and ease of use. Despite challenges such as off-target effects and cytotoxicity, effective spatiotemporal control strategies have been achieved for the CRISPR/Cas9 system through precise regulation of Cas9 protein activity as well as engineering of guide RNAs (gRNAs). This review provides a comprehensive analysis of the core components and functional mechanisms underlying the CRISPR/Cas9 system, highlights recent advancements in spatiotemporal control strategies, and discusses future directions for development.
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Miniaturized CRISPR: Ultra Compact Systems for In Vivo Delivery and Portable Diagnostics.
Reduced-size CRISPR systems have become a possible remedy to the delivery and size constraints of the traditional SpCas9 (~ 1368 Å). Recently described small nucleases, including Cas12f (400-700 Å) or CasX (~ 980 Å), along with designed mini-Cas9 versions, can efficiently be used in vivo to edit cells as well as to perform point-of-care diagnostics because of their lower molecular weight and less complex structures. This review will sum up progress in compact Cas protein engineering, guide RNA optimization, and delivery vector miniaturization, and point to their influence in therapeutic gene editing and portable diagnostic platforms. We additionally cover the contemporary issues of interest, such as off-target activity, delivery barriers and regulatory requirements, and future opportunities provided through AI-assisted protein design and synthetic biology. The miniaturized CRISPR technology is bound to substantially transform the translational arena of gene editing and world diagnostics.
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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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A novel RNA-guided RNA-targeting CRISPR tool.
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CRISPR-Enabled Autonomous Transposable Element (CREATE) for RNA-based gene editing and delivery.
To address a wide range of genetic diseases, genome editing tools that can achieve targeted delivery of large genes without causing double-strand breaks (DSBs) or requiring DNA templates are necessary. Here, we introduce CRISPR-Enabled Autonomous Transposable Element (CREATE), a genome editing system that combines the programmability and precision of CRISPR/Cas9 with the RNA-mediated gene insertion capabilities of the human LINE-1 (L1) element. CREATE employs a modified L1 mRNA to carry a payload gene, and a Cas9 nickase to facilitate targeted editing by L1-mediated reverse transcription and integration without relying on DSBs or DNA templates. Using this system, we demonstrate programmable insertion of a 1.1 kb gene expression cassette into specific genomic loci of human cell lines and primary T cells. Mechanistic studies reveal that CREATE editing is highly specific with no observed off-target events. Together, these findings establish CREATE as a programmable, RNA-based gene delivery technology with broad therapeutic potential.
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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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Using CRISPR/Cas to enhance gene expression for crop trait improvement by editing miRNA targets.
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CRISPR/Cas9 Genome-Editing Technology and Potential Clinical Application in Gastric Cancer.
Gastric cancer is the subject of clinical and basic studies due to its high incidence and mortality rates worldwide. Due to the diagnosis occurring in advanced stages and the classic treatment methodologies such as gastrectomy and chemotherapy, they are extremely aggressive and limit the quality of life of these patients. CRISPR/Cas9 is a tool that allows gene editing and has been used to explore the functions of genes related to gastric cancer, in addition to being used in the treatment of this neoplasm, greatly increasing our understanding of cancer genomics. In this mini-review, we seek the current status of the CRISPR/Cas9 gene-editing technology in gastric cancer research and clinical research.
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