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Utilization and Potential of RNA-Based Therapies in Cardiovascular Disease.
Cardiovascular disease (CVD) remains the largest cause of mortality worldwide. The development of new effective therapeutics is a major unmet need. The current review focuses broadly on the concept of nucleic acid (NA)-based therapies, considering the use of various forms of NAs, including mRNAs, miRNAs, siRNA, and guide RNAs, the latter specifically for the purpose of CRISPR-Cas directed gene editing. We describe the current state-of-the-art of RNA target discovery and development, the status of RNA therapeutics in the context of CVD, and some of the challenges and hurdles to be overcome.
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Advances in plant molecular biology: towards new challenges.
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Genome Editing for Global Food Security.
Global food security is increasingly challenging in light of population increase, the impact of climate change on crop production, and limited land available for agricultural expansion. Here we outline how genome editing provides excellent and timely methods to optimize crop plants, and argue the urgency for societal acceptance and support.
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Fast and cloning-free CRISPR/Cas9-mediated genomic editing in mammalian cells.
CHoP-In (CRISPR/Cas9-mediated Homology-independent PCR-product integration) is a fast, non-homologous end-joining based, strategy for genomic editing in mammalian cells. There is no requirement for cloning in generation of the integration donor, instead the desired integration donor is produced as a polymerase chain reaction (PCR) product, flanked by the Cas9 recognition sequences of the target locus. When co-transfected with the cognate Cas9 and guide RNA, double strand breaks are introduced at the target genomic locus and at both ends of the PCR product. This allows incorporation into the genomic locus via hon-homologous end joining. The approach is versatile, allowing N-terminal, C-terminal or internal tag integration and gives predictable genomic integrations, as demonstrated for a selection of well characterised membrane trafficking proteins. The lack of donor vectors offers advantages over existing methods in terms of both speed and hands-on time. As such this approach will be a useful addition to the genome editing toolkit of those working in mammalian cell systems.
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Integrating Omics and Gene Editing Tools for Rapid Improvement of Traditional Food Plants for Diversified and Sustainable Food Security.
Indigenous communities across the globe, especially in rural areas, consume locally available plants known as Traditional Food Plants (TFPs) for their nutritional and health-related needs. Recent research shows that many TFPs are highly nutritious as they contain health beneficial metabolites, vitamins, mineral elements and other nutrients. Excessive reliance on the mainstream staple crops has its own disadvantages. Traditional food plants are nowadays considered important crops of the future and can act as supplementary foods for the burgeoning global population. They can also act as emergency foods in situations such as COVID-19 and in times of other pandemics. The current situation necessitates locally available alternative nutritious TFPs for sustainable food production. To increase the cultivation or improve the traits in TFPs, it is essential to understand the molecular basis of the genes that regulate some important traits such as nutritional components and resilience to biotic and abiotic stresses. The integrated use of modern omics and gene editing technologies provide great opportunities to better understand the genetic and molecular basis of superior nutrient content, climate-resilient traits and adaptation to local agroclimatic zones. Recently, realizing the importance and benefits of TFPs, scientists have shown interest in the prospection and sequencing of TFPs for their improvements, cultivation and mainstreaming. Integrated omics such as genomics, transcriptomics, proteomics, metabolomics and ionomics are successfully used in plants and have provided a comprehensive understanding of gene-protein-metabolite networks. Combined use of omics and editing tools has led to successful editing of beneficial traits in several TFPs. This suggests that there is ample scope for improvement of TFPs for sustainable food production. In this article, we highlight the importance, scope and progress towards improvement of TFPs for valuable traits by integrated use of omics and gene editing techniques.
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Exploring the dynamic nature of divalent metal ions involved in DNA cleavage by CRISPR-Cas12a.
CRISPR-Cas12a has been widely used in genome editing and nucleic acid detection. In both of these applications, Cas12a cleaves target DNA in a divalent metal ion-dependent manner. However, when and how metal ions contribute to the cleavage reaction is unclear. Here, using a single-molecule FRET assay, we reveal that these metal ions are necessary for stabilising cleavage-competent conformations and that they are easily exchangeable, suggesting that they are dynamically coordinated.
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Gene editing of CD3 epsilon to redirect regulatory T cells for adoptive T cell transfer.
Adoptive transfer of antigen-specific regulatory T cells (Tregs) is a promising strategy to combat immunopathologies in transplantation and autoimmune diseases. However, their low frequency in peripheral blood poses challenges for both manufacturing and clinical application. Chimeric antigen receptors have been used to redirect the specificity of Tregs, using retroviral vectors. However, retroviral gene transfer is costly, time consuming, and raises safety issues. Here, we explored non-viral CRISPR-Cas12a gene editing to redirect Tregs, using human leukocyte antigen (HLA)-A2-specific constructs for proof-of-concept studies in transplantation models. Knock-in of an antigen-binding domain into the N terminus of CD3 epsilon (CD3ε) gene generates Tregs expressing a chimeric CD3ε-T cell receptor fusion construct (TRuC) protein that integrates into the endogenous TCR/CD3 complex. These CD3ε-TRuC Tregs exhibit potent antigen-dependent activation while maintaining responsiveness to TCR/CD3 stimulation. This enables preferential enrichment of TRuC-redirected Tregs over CD3ε knockout Tregs via repetitive CD3/CD28 stimulation in a good manufacturing practice-compatible expansion system. CD3ε-TRuC Tregs retained their phenotypic, epigenetic, and functional identity. In a humanized mouse model, HLA-A2-specific CD3ε-TRuC Tregs demonstrate superior protection of allogeneic HLA-A2+ skin grafts from rejection compared with polyclonal Tregs. This approach provides a pathway for developing clinical-grade CD3ε-TRuC-based Treg cell products for transplantation immunotherapy and other immunopathologies.
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Inventive steps: the CRISPR patent dispute and scientific progress: The recent patent decisions about CRISPR tell us a lot about how advances in biology are actually made-and how they are not.
Inventions in scientific research and inventions under patent law do not necessarily mean the same—in particular in molecular biology. [Image: see text]
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Exploiting the Endogenous Type II-A CRISPR-Cas System for Functional Engineering of Probiotic Lacticaseibacillus rhamnosus GG.
Lacticaseibacillus rhamnosus GG (LGG) is one of the most extensively studied probiotic strains, widely used in food and health applications. However, the absence of efficient, precise genome editing methods has limited its broader potential and functional versatility. Here, we present an endogenous type II-A CRISPR-Cas genome editing workflow for LGG designed for functional strain construction. Using a plasmid interference assay together with single-nucleotide substitutions, we confirm the precise PAM requirement as 5'-NGAAA-3'. We pair a synthetic sgRNA cassette with homology-directed repair donors to enable targeted deletions and insertions across multiple loci, achieving modest but practically relevant editing efficiencies (11.1-25.0% of recovered transformants) that support routine strain construction. Using this optimised and precise genome engineering method, we generated a β-glucuronidase (GUS)-expressing LGG strain for robust strain tracking within complex microbial communities. This work removes barriers to LGG engineering, expands the probiotic CRISPR toolkit, and provides broadly applicable strategies for designing next-generation probiotics with applications in food biotechnology and microbial therapeutics.
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Genetic Analysis Based on CRISPR/Cas9 Technology in Plants.
Genome-editing technology is a type of genetic engineering in which DNA is inserted into, replaced in, or deleted from the genome using artificially engineered nucleases or genetic scissors [...].
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