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Adenoviral Vector System: A Comprehensive Overview of Constructions, Therapeutic Applications and Host Responses.
Adenoviral vectors are crucial for gene therapy and vaccine development, offering a platform for gene delivery into host cells. Since the discovery of adenoviruses, first-generation vectors with limited capacity have evolved to third-generation vectors flacking viral coding sequences, balancing safety and gene-carrying capacity. The applications of adenoviral vectors for gene therapy and anti-viral treatments have expanded through the use of in vitro ligation and homologous recombination, along with gene editing advancements such as CRISPR-Cas9. Current research aims to maintain the efficacy and safety of adenoviral vectors by addressing challenges such as pre-existing immunity against adenoviral vectors and developing new adenoviral vectors from rare adenovirus types and non-human species. In summary, adenoviral vectors have great potential in gene therapy and vaccine development. Through continuous research and technological advancements, these vectors are expected to lead to the development of safer and more effective treatments.
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Optimization of the Genome Editing CRISPR-Cas9 Technology in Scedosporium apiospermum.
Scedosporium species are opportunistic pathogens causing a large variety of human infections. To date, there is limited information on the pathogenic mechanisms of these fungi, partly because of the limited number of genetic tools available. Here, the CRISPR-Cas9 technology, which provided promising results for functional genomic studies in filamentous fungi, was optimized for Scedosporium species using in vitro assembled Cas9 ribonucleoprotein (RNP) complexes. In these fungi, functional genomic studies are particularly complex in a wild-type strain, because of the high frequency of non-homologous recombination. Prior disruption of the KU70 gene encoding one of the components of the non-homologous end joining system is required, which necessitates the use of a first selection marker. The cleavage of the target gene at each end using a dual RNA-guided Cas9 complex, followed by recombination with a repair template containing the hygromycin resistance gene, allowed disruption of the target gene in the ΔKU70 mutant. Four genes encoding dioxygenases, catalyzing the critical ring-opening step in aromatic hydrocarbons, were successfully disrupted, and the optimum efficiency was observed using 5 μg of the HygR repair cassette. Alternatively, in the wild-type strain, the exclusive use of two Cas9 RNP complexes was enough to achieve an efficient deletion method; one dioxygenase gene was successfully deleted in up to 20% of the obtained colonies. These last experimental conditions path the way to multiple gene deletions and complementation experiments, which cannot be reached using our first procedure since only two selection markers are available for Scedosporium species.
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Metarhizium anisopliae engineering mediated by a CRISPR/Cas9 recyclable system.
The advent of CRISPR/Cas technology has revolutionized genome editing, offering simplicity, precision, and cost-effectiveness. While its application in biological control fungi has been limited, including the cosmopolitan fungus Metarhizium anisopliae, recent advancements show promise. However, integrating cas9 and selection-marker genes into fungal genomes poses challenges, including reduced efficiency, toxicity, and off-target effects. Besides, marker-free genetic engineering through a CRISPR recyclable system presents a viable solution, enabling efficient mutant generation without compromising fitness and virulence. This study pioneers the construction of marker-free strains of M. anisopliae using a CRISPR/Cas9 recyclable system. Precise deletion of albA and ku70, alongside gfp cassette insertion, confirms the system efficiency. This innovative approach holds significant potential for facilitating in-depth molecular studies, understanding their ecological roles in agricultural systems, and enhancing biocontrol efficacy against insect pests through genetic improvement.
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Reducible Branched Ester-Amine Quadpolymers (rBEAQs) Codelivering Plasmid DNA and RNA Oligonucleotides Enable CRISPR/Cas9 Genome Editing.
Functional codelivery of plasmid DNA and RNA oligonucleotides in the same nanoparticle system is challenging due to differences in their physical properties as well as their intracellular locations of function. In this study, we synthesized a series of reducible branched ester-amine quadpolymers (rBEAQs) and investigated their ability to coencapsulate and deliver DNA plasmids and RNA oligos. The rBEAQs are designed to leverage polymer branching, reducibility, and hydrophobicity to successfully cocomplex DNA and RNA in nanoparticles at low polymer to nucleic acid w/w ratios and enable high delivery efficiency. We validate the synthesis of this new class of biodegradable polymers, characterize the self-assembled nanoparticles that these polymers form with diverse nucleic acids, and demonstrate that the nanoparticles enable safe, effective, and efficient DNA-siRNA codelivery as well as nonviral CRISPR-mediated gene editing utilizing Cas9 DNA and sgRNA codelivery.
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Precision Breeding for a Global Staple Food: A Systematic Review with a Strategic Framework for CRISPR-Cas Applications in Rice (Oryza sativa L.).
Background: Rice is one of the world's main staple crops, and improving its productivity and resilience is important to achieving food security under varying climatic conditions. Objectives: This systematic review synthesizes the existing evidence on the application, technical limitations, and potential of the development of genome editing technologies (CRISPR-Cas) in rice (Oryza sativa L.), as well as presents a novel approach called the CRISPR Trait Prioritization and Readiness Framework (CTPRF). Methods: Peer-reviewed articles that reported applications of genome editing based on the CRISPR-Cas system in the genome of rice for trait improvement or functional genomics were identified through searches fromPubMed, Scopus, Web of Science, and Google Scholar with studies published between 2012 and 2025. Studies were screened on predefined inclusion criteria related to experimental validation, reporting of editing efficiency, and clear phenotypic results. Data on CRISPR systems, target genes, methods of delivery, traits modified, and phenotypic results were extracted and synthesized by comparative analysis. Results: A wide variety of different CRISPR systems have been used in rice, and our results indicate that NHEJ-mediated knockouts are effective in average genotypes with editing efficiencies in the range of 70-90%, but HDR and prime editing are still under 10%. The CTPRF is being introduced as a strategic decision support tool to evaluate traits from four dimensions: technical feasibility, phenotypic predictability, impact potential, and regulatory pathway. We use this framework for case studies in pioneering countries (USA, Japan, China) and show how it can be useful for guiding research investment and policy. Conclusions: CRISPR-Cas technologies have transformed rice breeding, but their introduction requires overcoming genotype-dependent barriers to transformation and negotiating patchwork regulatory environments. The CTPRF offers a roadmap for the acceleration of the development of climate-resilient and nutritious rice varieties for the action plan.
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Engineering of the endogenous HBD promoter increases HbA2.
The β-hemoglobinopathies, such as sickle cell disease and β-thalassemia, are one of the most common genetic diseases worldwide and are caused by mutations affecting the structure or production of β-globin subunits in adult hemoglobin. Many gene editing efforts to treat the β-hemoglobinopathies attempt to correct β-globin mutations or increase γ-globin for fetal hemoglobin production. δ-globin, the subunit of adult hemoglobin A2, has high homology to β-globin and is already pan-cellularly expressed at low levels in adult red blood cells. However, upregulation of δ-globin is a relatively unexplored avenue to increase the amount of functional hemoglobin. Here, we use CRISPR-Cas9 to repair non-functional transcriptional elements in the endogenous promoter region of δ-globin to increase overall expression of adult hemoglobin 2 (HbA2). We find that insertion of a KLF1 site alone is insufficient to upregulate δ-globin. Instead, multiple transcription factor elements are necessary for robust upregulation of δ-globin from the endogenous locus. Promoter edited HUDEP-2 immortalized erythroid progenitor cells exhibit striking increases of HBD transcript, from less than 5% to over 20% of total β-like globins in clonal populations. Edited CD34 +hematopoietic stem and progenitors (HSPCs) differentiated to primary human erythroblasts express up to 46% HBD in clonal populations. These findings add mechanistic insight to globin gene regulation and offer a new therapeutic avenue to treat β-hemoglobinopathies.
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Cell-free screening of CRISPR-Cas activity by microfluidics-assisted in vitro compartmentalization.
CRISPR-Cas systems are responsible for antiviral immunity of prokaryotic cells and have been repurposed as powerful genome-editing tools. Cell-free gene expression has been applied for the rapid characterization of CRISPR-Cas systems in microtiter plates. In vitro compartmentalization makes use of artificial microcompartments that individually act as bioreactors. Here, we performed cell-free reactions of CRISPR-Cas activity into microtiter plates, which we proceeded to encapsulate into double emulsion (DE) droplets generated by on-chip microfluidics. Emulsion droplets were screened for CRISPR-Cas activity based on relative fluorescence levels using a common cell sorter, and enrichment for the expected guide (g)RNA genotype was observed. Encapsulation of single gene copies per droplet is an important prerequisite for applying this technique to complex gene libraries. We show a proof-of-principle assay for efficient, compartmentalized gene amplification using magnetic microbeads. In conclusion, we demonstrate the feasibility of microfluidics-based, high-throughput, cell-free screening of CRISPR-Cas activity.
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Genome Editing in Zebrafish Using High-Fidelity Cas9 Nucleases: Choosing the Right Nuclease for the Task.
Shortly after the development of the CRISPR/Cas9 system, it was recognized that it is prone to induce off-target mutations at significant frequencies. Therefore, there is a strong motivation to develop Cas9 enzymes with reduced off-target activity. Multiple rational design or selection approaches have been applied to develop several Cas9 versions with reduced off-target activities (high fidelity). To make these high-fidelity Cas9s available for model systems other than human cells and bacterial strains, as, for example, in zebrafish, new specialized expression vectors need to be developed. In this chapter, we focused on the HypaCas9 and HiFi Cas9 high-fidelity enzymes and incorporated the mutations of these Cas9 versions into a codon-optimized zebrafish Cas9 vector. This optimized vector was further improved by introducing an artificial polyadenine insert (A71) since polyadenylation is known to enhance mRNA translational efficiency. The Hypa-nCas9n and HiFi-nCas9n vectors were produced by single-site mutagenesis from pT3TS-nCas9n-A71 vector. We then tested the polyadenylated mRNAs for nCas9n, Hypa-nCas9n, HiFi-nCas9n, and HiFi-Cas9 protein for editing efficiency in five genome editing strategies and found that these high-fidelity Cas9 versions had different performances ranging from activity at 2-4 sites, where the wild-type nCas9n is active, indicating that these Cas9 versions have different sgRNA preferences. In summary, the developed new high-fidelity Cas9 vectors will enable researchers to perform much more accurate genome editing.
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Protocol for efficient CRISPR-Cas9-mediated fluorescent tag knockin in hard-to-transfect erythroid cell lines.
Precise insertion of fluorescent tags by CRISPR-Cas9-mediated homologous recombination (HR) in mammalian genes is a powerful tool allowing to study gene function and protein gene products. Here, we present a protocol for efficient HR-mediated targeted insertion of fluorescent markers in the genome of hard-to-transfect erythroid cell lines MEL (mouse erythroleukemic) and MEDEP (mouse ES cell-derived erythroid progenitor line). We describe steps for plasmid construction, electroporation, amplification, and verification of genome editing. We then detail procedures for isolating positive clones and validating knockin clones. For complete details on the use and execution of this protocol, please refer to Deleuze et al.1.
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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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