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Evaluation of CRISPR/Cas9 Constructs in Wheat Cell Suspension Cultures.
Despite intensive optimization efforts, developing an efficient sequence-specific CRISPR/Cas-mediated genome editing method remains a challenge, especially in polyploid cereal species such as wheat. Validating the efficacy of nuclease constructs prior to using them in planta is, thus, a major step of every editing experiment. Several construct evaluation strategies were proposed, with PEG-mediated plasmid transfection of seedling-derived protoplasts becoming the most popular. However, the usefulness of this approach is affected by associated construct copy number bias and chromatin relaxation, both influencing the outcome. Therefore, to achieve a reliable evaluation of CRISPR/Cas9 constructs, we proposed a system based on an Agrobacterium-mediated transformation of established wheat cell suspension cultures. This system was used for the evaluation of a CRISPR/Cas9 construct designed to target the ABA 8'-hydroxylase 1 gene. The efficiency of editing was verified by cost-effective means of Sanger sequencing and bioinformatic analysis. We discuss advantages and potential future developments of this method in contrast to other in vitro approaches.
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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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Genome Editing During Development Using the CRISPR-Cas Technology.
Over the years, the study of gene function during development involved the implementation of sophisticated transgenic strategies to visualize how organisms change during their lifetime. These strategies are diverse and extremely useful and allowed the discovery of some of the fundamental mechanisms governing organism's development. Such strategies can be time-consuming, in some cases expensive, and require complex infrastructure. With the advent of the genome editing CRISPR-Cas9 RNA-guided DNA endonuclease system a tremendous progress has been achieved in manipulating diverse organisms and cell types. In recent years this system has contributed importantly to the design of novel experimental strategies to further understand developmental processes, to generate genetically modified animal models, and develop disease models. Here we highlight examples in which the genome editing CRISPR-Cas9 system has been employed to understand the mechanisms controlling embryonic development and disease.
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Genome editing in the mushroom-forming basidiomycete Coprinopsis cinerea, optimized by a high-throughput transformation system.
Mushroom-forming basidiomycetes produce a wide range of metabolites and have great value not only as food but also as an important global natural resource. Here, we demonstrate CRISPR/Cas9-based genome editing in the model species Coprinopsis cinerea. Using a high-throughput reporter assay with cryopreserved protoplasts, we identified a novel promoter, CcDED1 pro , with seven times stronger activity in this assay than the conventional promoter GPD2. To develop highly efficient genome editing using CRISPR/Cas9 in C. cinerea, we used the CcDED1 pro to express Cas9 and a U6-snRNA promoter from C. cinerea to express gRNA. Finally, CRISPR/Cas9-mediated GFP mutagenesis was performed in a stable GFP expression line. Individual genome-edited lines were isolated, and loss of GFP function was detected in hyphae and fruiting body primordia. This novel method of high-throughput CRISPR/Cas9-based genome editing using cryopreserved protoplasts should be a powerful tool in the study of edible mushrooms.
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Precision Recovery After Spinal Cord Injury: Integrating CRISPR Technologies, AI-Driven Therapeutics, Single-Cell Omics, and System Neuroregeneration.
Spinal cord injury (SCI) remains one of the toughest obstacles in neuroscience and regenerative medicine due to both severe functional loss and limited healing ability. This article aims to provide a key integrative, mechanism-focused review of the molecular landscape of SCI and the new disruptive therapy technologies that are now evolving in the SCI arena. Our goal is to unify a fundamental pathophysiology of neuroinflammation, ferroptosis, glial scarring, and oxidative stress with the translation of precision treatment approaches driven by artificial intelligence (AI), CRISPR-mediated gene editing, and regenerative bioengineering. Drawing upon advances in single-cell omics, systems biology, and smart biomaterials, we will discuss the potential for reprogramming the spinal cord at multiple levels, from transcriptional programming to biomechanical scaffolds, to change the course from an irreversible degeneration toward a directed regenerative pathway. We will place special emphasis on using AI to improve diagnostic/prognostic and inferred responses, gene and cell therapies enabled by genomic editing, and bioelectronics capable of rehabilitating functional connectivity. Although many of the technologies described below are still in development, they are becoming increasingly disruptive capabilities of what it may mean to recover from an SCI. Instead of prescribing a particular therapeutic fix, we provide a future-looking synthesis of interrelated biological, computational, and bioengineering approaches that conjointly chart a course toward adaptive, personalized neuroregeneration. Our intent is to inspire a paradigm shift to resolve paralysis through precision recovery and to be grounded in a spirit of humility, rigor, and an interdisciplinary approach.
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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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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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Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope.
TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.
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Multiplex Single-Nucleotide Microbial Genome Editing Achieved by CRISPR-Cas9 Using 5'-End-Truncated sgRNAs.
Multiplex genome editing with CRISPR-Cas9 offers a cost-effective solution for time and labor savings. However, achieving high accuracy remains a challenge. In an Escherichia coli model system, we achieved highly efficient single-nucleotide level simultaneous editing of the galK and xylB genes using the 5'-end-truncated single-molecular guide RNA (sgRNA) method. Furthermore, we successfully demonstrated the simultaneous editing of three genes (galK, xylB, and srlD) at single-nucleotide resolution. To showcase practical application, we targeted the cI857 and ilvG genes in the genome of E. coli. While untruncated sgRNAs failed to produce any edited cells, the use of truncated sgRNAs allowed us to achieve simultaneous and accurate editing of these two genes with an efficiency of 30%. This enabled the edited cells to retain their lysogenic state at 42 °C and effectively alleviated l-valine toxicity. These results suggest that our truncated sgRNA method holds significant potential for widespread and practical use in synthetic biology.
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