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CRISPR/Cas9-mediated generation of AP-1 activity reporter cell line in human embryonic stem cell (WAe007-A-5).
Activator protein 1 (AP-1) is involved in cell fate determination and function. To monitor the AP-1 activity, we cloned a AP-1 binding sites fragment into the upstream of minimal TATA-box promoter, then a luciferase-GFP reporter (LuciGFP) was designated to the downstream of the promoter. With CRISPR/Cas9, the AP-1-LuciGFP reporter was introduced into AAVS1 locus, a safe harbor for gene editing. Thus, this AP-1-LuciGFP reporter cell line could be subjected to monitor the AP-1 activity during the cell differentiation, cell fate transition and disease modeling.
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Homology-Directed Repair by CRISPR-Cas9 Mutagenesis in Xenopus Using Long Single-Stranded Donor DNA Templates via Simple Microinjection of Embryos.
We describe a step-by-step procedure to perform homology-directed repair (HDR)-mediated precise gene editing in Xenopus embryos using long single-stranded DNA (lssDNA) as a donor template for HDR in conjunction with the CRISPR-Cas9 system. A key advantage of this method is that it relies on simple microinjection of fertilized Xenopus eggs, resulting in high yield of healthy founder embryos. These embryos are screened for those animals carrying the precisely mutated locus to then generate homozygous and/or heterozygous mutant lines in the F1 generation. Therefore, we can avoid the more challenging "oocyte host transfer" technique, which is particularly difficult for Xenopus tropicalis, that is required for an alternate HDR approach. Several key points of this protocol are (1) to use efficiently active single-guide RNAs for targeting, (2) to use properly designed lssDNAs, and (3) to use 5'-end phosphorothioate-modification to obtain higher-efficiency HDR.
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"Snip, snip, cure"? Philosophical, legal and biomedical perspectives on novel somatic genomic therapies.
The advent of innovative techniques, such as the CRISPR/Cas system, has opened up a new range of possibilities for modifying the genome, with the potential to address previously unmet therapeutic needs of patients with genetic diseases. These new possibilities have not only raised ethical concerns but also challenged existing classifications of genome modification techniques. While the legal status of some of these new therapies remains uncertain, there is an ongoing debate within philosophy of biology about the information-related metaphors adopted by scientists to describe and classify the genome and its therapeutic modification. Given the continuing advance of new genomic therapies, we show, employing an interdisciplinary approach, that a comprehensive framework for the classification of these technologies is needed to resolve legal and philosophical issues. The first section provides an analysis of the current state of novel genome-modifying techniques in medical genetics. In the second section, we assess the regulatory status of these techniques within the European regulatory framework for advanced therapy medicinal products (ATMPs). Drawing on these results, we argue in the third section from a philosophical perspective that metaphors, such as 'editing' the genome, which are based on a conception of the genome as linear information, cannot adequately capture the breadth of advanced genomic technologies. To accurately categorise these techniques in a manner that meets their diverse applications, we propose introducing the umbrella term 'somatic genomic therapies' (SGTs). Urging an integrative approach to defining and classifying new technologies in medical genetics, we advocate for the development of an integrative concept of SGTs.
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Germline genome editing versus preimplantation genetic diagnosis: Is there a case in favour of germline interventions?
CRISPR is widely considered to be a disruptive technology. However, when it comes to the most controversial topic, germline genome editing (GGE), there is no consensus on whether this technology has any substantial advantages over existing procedures such as embryo selection after in vitro fertilization (IVF) and preimplantation genetic diagnosis (PGD). Answering this question, however, is crucial for evaluating whether the pursuit of further research and development on GGE is justified. This paper explores the question from both a clinical and a moral viewpoint, namely whether GGE has any advantages over existing technologies of selective reproduction and whether GGE could complement or even replace them. In a first step, I review an argument of extended applicability. The paper confirms that there are some scenarios in which only germline intervention allows couples to have (biologically related) healthy offspring, because selection will not avoid disease. In a second step, I examine possible moral arguments in favour of genetic modification, namely that GGE could save some embryos and that GGE would provide certain benefits for a future person that PGD does not. Both arguments for GGE have limitations. With regard to the extended applicability of GGE, however, a weak case in favour of GGE should still be made.
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Fusing T5 exonuclease with Cas9 and Cas12a increases the frequency and size of deletion at target sites.
CRISPR/Cas systems, especially CRISPR/Cas9, generally result in small insertions/deletions, which are unlikely to eliminate the functions of regulatory and other non-coding sequences. To generate larger genomic deletions usually requires the use of pairs of guide RNAs. Here we show that it is possible to create such deletions with a single guide RNA by fusing Cas9 or Cas12a with T5 exonuclease (T5exo). These fusion constructs were found to increase both the frequency and size of deletions at target loci in rice protoplasts and seedlings. Moreover, the genome editing efficiencies of Cas9 and Cas12a were also enhanced by fusion with T5 exonuclease. These T5exo-Cas fusions expand the CRISPR toolbox, and facilitate knockout of regulatory and non-coding DNA sequences. From a wider standpoint, our results suggest a general strategy for producing larger deletions using other Cas nucleases.
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Towards CRISPR/Cas9 Genome Editing in Spodoptera frugiperda: A Proof-of-Concept Targeting Wing and Eye Color Genes.
Fall armyworm (Spodoptera frugiperda), a globally significant destructive lepidopteran invasive pest, has recently invaded Africa and Asia, threatening food security. Conventional method of management, including chemical insecticides, are often ineffective due to various reasons compelling the need to explore alternative strategies. In this regard, CRISPR/Cas9 based genome editing has emerged as a powerful tool for functional genomics in insects, enabling to introduce site-specific mutations for various purposes. In this study, we applied multiplex CRISPR/Cas9 ribonucleoprotein (RNP) injections to disrupt two key genes in S. frugiperda: the spalt (Sfspalt), which regulates wing patterning, and tryptophan 2,3-dioxygenase (Sfto/vermillion) gene, involved in eye pigmentation. Microinjection of sgRNA/Cas9 ribonucleoprotein complex into freshly laid eggs resulted in distinct phenotypic alterations, including altered wing pigmentation and modified eyespot patterns, as well as golden-yellow eye color mutants. Genotyping and ICE analysis confirmed the presence of frameshift mutations in the target loci, supporting the phenotypic changes. Notably, while mutations were detected, only a single individual was confirmed to carry mutations in both genes simultaneously. These results demonstrate the technical feasibility of multiplex CRISPR/Cas9 editing in S. frugiperda, but also reveal a low frequency of confirmed events under the present experimental conditions. Therefore, this study is considered a proof of concept establishing a preliminary workflow in multiplex platform. The findings provide foundational insights for further optimization of genome editing strategies targeting sex related genes in this agriculturally important pest.
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Application and development of genome editing technologies to the Solanaceae plants.
Genome editing technology using artificial nucleases, including zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regulatory interspaced short palindromic repeats (CRISPR)-Cas9, can mutagenize the target sites of genes of interest. This technology has been successfully applied in several crops, including the Solanaceae plants, such as tomato, potato, tobacco, and petunia. Among the three nucleases, CRISPR-Cas9 is the best for breeding, crop improvement, and the functional analysis of genes of interest, because of its simplicity and high efficiency. Although the technology is useful for reverse genetics, its use in plants is limited due to a lack of regeneration protocols and sequence information. In this review, the present status of genome editing technology in Solanaceae plants is described, and techniques that may improve genome editing technologies are discussed.
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Status and advancement of root-knot nematode management strategies and the emerging CRISPR/Cas biotechnology application.
Root-knot nematodes (RKNs), Meloidogyne spp., exhibit a broad host range, threatening more than 3000 species of plants, including agriculturally important crops such as cotton (Gossypium hirsutum), tomato (Lycopersicon esculentum) and rice (Oryza sativa). Among the over 90 RKN species, the four most prevalent are M. incognita, M. arenaria, M. javanica, and M. hapla, with M. incognita being the most damaging. This paper reviewed the current RKN management strategies, including chemical nematicides, biological control, crop rotation, and resistant varieties, with a focus on the application of the revolutionary CRISPR/Cas genome editing tool in developing RKN resistance in plants. CRISPR/Cas has been widely utilized for improving crop traits due to its specificity, streamline, and inheritability. Recent progress has demonstrated the simplicity and robustness of CRISPR/Cas technology in improving plant traits. Among these, the development of nematode resistance by CRISPR/Cas knocking out of plant compatibility factors in model and commercial plants, has achieved significant progress. This review summarizes the RKN parasitism mechanisms and plant compatibility factors that would be promising CRISPR/Cas targets. The fundamentals and key aspects of CRISPR/Cas genome editing technology are addressed and discussed, and an example experimental pipeline for developing nematode resistance in cotton is described.
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CRISPR/Cas13a-based genome editing for establishing the detection method of H9N2 subtype avian influenza virus.
Avian influenza virus (AIV) subtype H9N2 has significantly threatened the poultry business in recent years by having become the predominant subtype in flocks of chickens, ducks, and pigeons. In addition, the public health aspects of H9N2 AIV pose a significant threat to humans. Early and rapid diagnosis of H9N2 AIV is therefore of great importance. In this study, a new method for the detection of H9N2 AIV based on fluorescence intensity was successfully established using CRISPR/Cas13a technology. The Cas13a protein was first expressed in a prokaryotic system and purified using nickel ion affinity chromatography, resulting in a high-purity Cas13a protein. The best RPA (recombinase polymerase amplification) primer pairs and crRNA were designed and screened, successfully constructing the detection of H9N2 AIV based on CRISPR/Cas13a technology. Optimal concentration of Cas13a and crRNA was determined to optimize the constructed assay. The sensitivity of the optimized detection system is excellent, with a minimum detection limit of 10° copies/μL and didn't react with other avian susceptible viruses, with excellent specificity. The detection method provides the basis for the field detection of the H9N2 AIV.
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Transgenerationally Transmitted DNA Demethylation of a Spontaneous Epialleles Using CRISPR/dCas9-TET1cd Targeted Epigenetic Editing in Arabidopsis.
CRISPR/dCas9 is an important DNA modification tool in which a disarmed Cas9 protein with no nuclease activity is fused with a specific DNA modifying enzyme. A previous study reported that overexpression of the TET1 catalytic domain (TET1cd) reduces genome-wide methylation in Arabidopsis. A spontaneous naturally occurring methylation region (NMR19-4) was identified in the promoter region of the PPH (Pheophytin Pheophorbide Hydrolase) gene, which encodes an enzyme that can degrade chlorophyll and accelerate leaf senescence. The methylation status of NMR19-4 is associated with PPH expression and leaf senescence in Arabidopsis natural accessions. In this study, we show that the CRISPR/dCas9-TET1cd system can be used to target the methylation of hypermethylated NMR19-4 region to reduce the level of methylation, thereby increasing the expression of PPH and accelerating leaf senescence. Furthermore, hybridization between transgenic demethylated plants and hypermethylated ecotypes showed that the demethylation status of edited NMR19-4, along with the enhanced PPH expression and accelerated leaf senescence, showed Mendelian inheritance in F1 and F2 progeny, indicating that spontaneous epialleles are stably transmitted trans-generationally after demethylation editing. Our results provide a rational approach for future editing of spontaneously mutated epialleles and provide insights into the epigenetic mechanisms that control plant leaf senescence.
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