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Generation of two induced pluripotent stem cell lines (TMOi001-A-5, TMOi001-A-6) carrying variants in DISC1 exon 2 using CRISPR/Cas9 gene editing.
DISC1 is a scaffold protein involved in key developmental processes such as neuronal migration, differentiation and neurogenesis. Genetic variants of the DISC1 gene have been linked to neuropsychiatric disorders like schizophrenia, bipolar disorder and major depression. Here, we generated two isogenic iPSC lines carrying mutations in DISC1 exon 2 using CRISPR/Cas9 gene editing. Both lines express pluripotency markers, can be differentiated into the three germ layers and present a normal karyotype. The generated iPSC lines can be used to study the implications of DISC1 mutations in the context of neuropsychiatric diseases in vitro.
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CRISPR/Cas9-based epigenome editing: An overview of dCas9-based tools with special emphasis on off-target activity.
Molecular tools for gene regulation and epigenome editing consist of two main parts: the targeting moiety binding a specific genomic locus and the effector domain performing the editing or regulatory function. The advent of CRISPR-Cas9 technology enabled easy and flexible targeting of almost any locus by co-expression of a small sgRNA molecule, which is complementary to the target sequence and forms a complex with Cas9, directing it to that particular target. Here, we review strategies for recruitment of effector domains, used in gene regulation and epigenome editing, to the dCas9 DNA-targeting protein. To date, the most important CRISPR-Cas9 applications in gene regulation are CRISPR activation or interference, while epigenome editing focuses on targeted changes in DNA methylation and histone modifications. Several strategies for signal amplification by recruitment of multiple effector domains deserve special focus. While some approaches rely on altering the sgRNA molecule and extending it with aptamers for effector domain recruitment, others use modifications to the Cas9 protein by direct fusions with effector domains or by addition of an epitope tag, which also has the ability to bind multiple effector domains. A major barrier to the widespread use of CRISPR-Cas9 technology for therapeutic purposes is its off-target effect. We review efforts to enhance CRISPR-Cas9 specificity by selection of Cas9 orthologs from various bacterial species and their further refinement by introduction of beneficial mutations. The molecular tools available today enable a researcher to choose the best balance of targeting flexibility, activity amplification, delivery method and specificity.
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Gene editing of clock components in Solanum lycopersicum: Effects on gene expression, development, and productivity.
The circadian clock plays a crucial role in regulating key biological processes, including growth and development. While studies in the model plant Arabidopsis thaliana have significantly advanced our understanding of circadian function, recent research has also focused on crop species for improved yield and quality. In this study, we examined the rhythmic behavior and regulatory function of circadian clock components in tomato (Solanum lycopersicum). Time course analyses of gene expression over the circadian cycle revealed robust rhythmic oscillations in tomato leaves under free-running conditions. Comparative analyses showed similar peak phases for several clock genes in Arabidopsis and tomato, suggesting functional conservation. Rhythms in tomato fruits, however, showed reduced amplitude, slight phase changes, or arrhythmia, indicating organ-specific circadian variations. By using CRISPR-Cas9 gene editing strategies (clockcrispr), we also showed that proper clock gene expression is essential for setting the phase in tomato plants. Leaf movement analyses also showed a phase change in the clockcrispr lines, correlating with shorter or longer periods. The clockcrispr lines also displayed distinct growth and developmental phenotypes that differ from those reported in the Arabidopsis clock mutant counterparts. Our transcriptomic analyses identified species-specific regulation of key target genes. The results offer mechanistic insights into the conserved and divergent molecular pathways governing circadian phenotypic variations between Arabidopsis and tomato plants.
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Therapeutic Germline Editing: Sense and Sensibility.
Safe and effective heritable editing of the human genome is years away from the clinic because of formidable technical, statutory, regulatory, and societal challenges. In particular, we note the fledgling state of the science, the imperatives of editing efficiency, specificity, and uniformity, and the extant legal roadblock.
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CRISPR/Cas9 facilitates rapid generation of constitutive forms of transcription factors in Aspergillus niger through specific on-site genomic mutations resulting in increased saccharification of plant biomass.
The CRISPR/Cas9 system has been successfully applied for gene editing in filamentous fungi. Previous studies reported that single stranded oligonucleotides can be used as repair templates to induce point mutations in some filamentous fungi belonging to genus Aspergillus. In Aspergillus niger, extensive research has been performed on regulation of plant biomass degradation, addressing transcription factors such as XlnR or GaaR, involved in (hemi-)cellulose and pectin utilization, respectively. Single nucleotide mutations leading to constitutively active forms of XlnR and GaaR have been previously reported. However, the mutations were performed by the introduction of versions obtained through site-directed or UV-mutagenesis into the genome. Here we report a more time- and cost-efficient approach to obtaining constitutively active versions by application of the CRISPR/Cas9 system to generate the desired mutation on-site in the A. niger genome. This was also achieved using only 60-mer single stranded oligonucleotides, shorter than the previously reported 90-mer strands. In this study, we show that CRISPR/Cas9 can also be used to efficiently change functional properties of the proteins encoded by the target gene by on-site genomic mutations in A. niger. The obtained strains with constitutively active XlnR and GaaR versions resulted in increased production of plant biomass degrading enzymes and improved release of d-xylose and l-arabinose from wheat bran, and d-galacturonic acid from sugar beet pulp.
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[Will the CRISPR / CAS9 system change the genome of humanity?].
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Targeted Gene Editing in Wheat During Haploid Production via Wide Hybridization with Transgenic Maize Expressing Cas9 and Guide RNA.
The clustered regularly interspersed short palindromic repeats (CRISPR)/Cas9 system is an efficient and versatile genome engineering tool, which has been widely used for targeted mutagenesis and gene functional characterization in various organisms. This system is simple because it only requires a Cas9 enzyme serving as a nuclease and guide RNA (gRNA) containing a 20-nt sequence matching the target gene. Delivery of a vector expressing Cas9 and gRNA or the preassembled Cas9/gRNA complex as a ribonucleoprotein (RNP) into plant cells for gene targeting are usually via the biolistic- or Agrobacterium-mediated approach. However, most wheat genotypes suffer from low efficiency of callus induction and plant regeneration from explants receiving the vector or RNP delivered by the biolistic- or Agrobacterium-mediated transformation method, limiting the application of genome editing systems in many commercially grown wheat varieties. Here, we describe a stepwise protocol for targeted gene editing in wheat via wide hybridization with transgenic maize expressing Cas9 and gRNA. A binary vector expressing Cas9 and gRNA is constructed and used for Agrobacterium-mediated transformation to generate transgenic maize plants, which are used to pollinate emasculated spikes of wheat varieties. After fertilization, the maize chromosomes enter the transient hybrid zygote and the transgene (T-DNA) on a maize chromosome expresses the Cas9 enzyme and gRNA, which forms an RNP complex to edit the target gene in wheat genome. After several cell divisions, maize chromosomes in the hybrid zygote are eliminated, resulting in formation of haploid wheat embryos with the target gene edited, which can be rescued by embryo culture technique to produce haploid plants. Doubled haploid (DH) wheat plants with homozygous gene mutations are developed by chromosome doubling through colchicine treatment of the haploid plants. The wheat × maize hybridization combined with the CRISPR/Cas9 system provides a one-step approach for generating DH lines with the target gene edited from any wheat genotypes of interest.
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Application of CRISPR/Cas9 system in breeding of new antiviral plant germplasm.
With the development and improvement of CRISPR/Cas9 system in genomic editing technology, the system has been applied to the prevention and control of animal viral infectious diseases, which has made considerable achievements. It has also been applied to the study of highly efficient gene targeting editing in plant virus genomes. The CRISPR/Cas9-mediated targeted gene modification has not only achieved the genome editing of plant DNA virus, but also showed the genome editing potential of plant RNA virus. In addition, the CRISPR/Cas9 system functions at the gene transcriptional and post-transcriptional level, indicating that the system could regulate the replication of plant viruses through different ways. Compared with other plant viral disease control strategies, this system is more accurate in genome editing, more stable in gene expression regulation, and has broader spectrum of resistance to virus disease. In this review, we summarized the advantages, main problems and development tendency of CRISPR/cas9 system in breeding of new antiviral plant germplasms.
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Navigating gene editing in porcine embryos: Methods, challenges, and future perspectives.
Gene editing technologies, particularly CRISPR/Cas9, have emerged as transformative tools in genetic modification, significantly advancing the use of porcine embryos in biomedical and agricultural research. This review comprehensively examines the various methodologies for gene editing and delivery methods, such as somatic cell nuclear transfer (SCNT), microinjection, electroporation, and lipofection. This review, focuses on the advantages or limitations of using different biological sources (in vivo- vs. in vitro oocytes/embryos). Male germ cell manipulation using sperm-mediated gene transfer (SMGT) and testis-mediated gene transfer (TMGT) represent innovative approaches for producing genetically modified animals. Although these technologies have revolutionized the genetic engineering field, all these strategies face challenges, including high rates of off-target events and mosaicism. This review emphasizes the need to refine these methods, with a focus on reducing mosaicism and improving editing accuracy. Further advancements are essential to unlocking the full potential of gene editing for both agricultural applications and biomedical innovations.
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CRISPR/Cas9-Mediated Genome Editing for Pseudomonas fulva, a Novel Pseudomonas Species with Clinical, Animal, and Plant-Associated Isolates.
As one of the most widespread groups of Gram-negative bacteria, Pseudomonas bacteria are prevalent in almost all natural environments, where they have developed intimate associations with plants and animals. Pseudomonas fulva is a novel species of Pseudomonas with clinical, animal, and plant-associated isolates, closely related to human and animal health, plant growth, and bioremediation. Although genetic manipulations have been proven as powerful tools for understanding bacterial biological and biochemical characteristics and the evolutionary origins, native isolates are often difficult to genetically manipulate, thereby making it a time-consuming and laborious endeavor. Here, by using the CRISPR-Cas system, a versatile gene-editing tool with a two-plasmid strategy was developed for a native P. fulva strain isolated from the model organism silkworm (Bombyx mori) gut. We harmonized and detailed the experimental setup and clarified the optimal conditions for bacteria transformation, competent cell preparation, and higher editing efficiency. Furthermore, we provided some case studies, testing and validating this approach. An antibiotic-related gene, oqxB, was knocked out, resulting in the slow growth of the P. fulva deletion mutant in LB containing chloramphenicol. Fusion constructs with knocked-in gfp exhibited intense fluorescence. Altogether, the successful construction and application of new genetic editing approaches gave us more powerful tools to investigate the functionalities of the novel Pseudomonas species.
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