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CRISPR/Cas9-Mediated Knockout of the White Gene in Agasicles hygrophila.
Agasicles hygrophila is the most effective natural enemy for the control of the invasive weed Alternanthera philoxeroides (Mart.) Griseb. However, research on the gene function and potential genetic improvement of A. hygrophila is limited due to a lack of effective genetic tools. In this study, we employed the A. hygrophila white (AhW) gene as a target gene to develop a CRISPR/Cas9-based gene editing method applicable to A. hygrophila. We showed that injection of Cas9/sgRNA ribonucleoprotein complexes (RNPs) of the AhW gene into pre-blastoderm eggs induced genetic insertion and deletion mutations, leading to white eyes. Our results demonstrate that CRISPR/Cas9-mediated gene editing is possible in A. hygrophila, offering a valuable tool for studies of functional genomics and genetic improvement of A. hygrophila, which could potentially lead to more effective control of invasive weeds through the development of improved strains of this biocontrol agent. In addition, the white-eyed mutant strain we developed could potentially be useful for other transgenic research studies on this species.
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Walk the Line: Debating a Germline Editing Moratorium.
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Cas9-PE: a robust multiplex gene editing tool for simultaneous precise editing and site-specific random mutation in rice.
In molecular design breeding, the simultaneous introduction of desired functional genes through specific nucleotide modifications and the elimination of genes regulating undesired phenotypic traits or agronomic components require advanced gene editing tools. Due to limited editing efficiency, even with the use of highly precise editing tools, such as prime editing (PE), simultaneous editing of multiple mutation types poses a challenge. Here, we replaced Cas9 nickase (nCas9) with Cas9 to construct a Cas9-mediated PE (Cas9-PE) system in rice. This system not only enables precise editing, but also allows for site-specific random mutation. Moreover, leveraging the precision of Cas9-PE, we established a transgene-free multiplex gene editing system using a co-editing strategy. This strategy involved the Agrobacterium-mediated transient expression of the precise editing rice endogenous acetolactate synthase gene ALSS627I to confer herbicide bispyribac-sodium (BS) resistance as a selection marker. This study provides a versatile and efficient multiplex gene editing tool for molecular design breeding.
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From gene editing to tumor eradication: The CRISPR revolution in cancer therapy.
Cancer continues to be a significant worldwide health concern, characterized by high rates of occurrence and death. Unfortunately, existing treatments frequently fall short of delivering satisfying therapeutic outcomes. Immunotherapy has ushered in a new era in the treatment of solid tumors, yet its effectiveness is still constrained and comes with unwanted side effects. The advancement of cutting-edge technology, propelled by gene analysis and manipulation at the molecular scale, shows potential for enhancing these therapies. The advent of genome editing technologies, including CRISPR-Cas9, can greatly augment the efficacy of cancer immunotherapy. This review explores the mechanism of CRISPR-Cas9-mediated genome editing and its wide range of tools. The study focuses on analyzing the effects of CRISPR-induced double-strand breaks (DSBs) on cancer immunotherapy, specifically by gene knockdown or knockin. In addition, the study emphasizes the utilization of CRISPR-Cas9-based genome-wide screening to identify targets, the potential of spatial CRISPR genomics, and the extensive applications and difficulties of CRISPR-Cas9 in fundamental research, translational medicine, and clinical environments.
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Can the Thought of Teilhard de Chardin Carry Us Past Current Contentious Discussions of Gene Editing Technologies?
The advent of CRISPR-Cas9 technology has increased attention, and contention, regarding the use and regulation of genome editing technologies. Public discussions continue to give evidence of this debate falling back into the previous polarized positions of technological enthusiasts versus those who are more cautious in their approach. One response to this contentious relapse could be to view this promising and problematic new technology from a radically different perspective that embraces both the excitement of this technological advance and the prudence necessary to use it well. The thought of Teilhard de Chardin provides this desired perspective, and some insights that may help carry forward public discussions to achieve widely accepted uses and regulations.
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Impacts of oocyte/zygote timing for in vitro fertilization and gene editing in the dog.
Previously, we reported the first live births of dogs using in vitro fertilization (IVF), embryo cryopreservation, and transfer. These techniques have potential applications in the conservation of endangered canids, and development of gene editing/repair technologies that could improve animal welfare by restoring normal gene function and removing predisposition to disease. Here, we used IVF as a springboard for initial attempts at genetic modification through gene editing/repair using the Clustered Regularly-Interspaced Short Palindromic Repeat (CRISPR)-CRISPR-associated endonuclease (Cas9) system. We showed previously that timing is critical for successful IVF in that the canine oocyte must be exposed to the oviductal environment beyond simply reaching metaphase II. Others have shown that timing of injection of CRISPR-Cas9 constructs is critical in gene editing, influencing the extent of genetic mosaicism. Therefore, we investigated whether timing of injection of the gene editing/repair constructs might influence the success of embryo production and gene editing in the dog. We achieved similar IVF success to our prior report in generating 2-cell control embryos, and found equally reduced embryo production whether injection was performed in oocytes prior to fertilization, or in presumptive single-cell zygotes already exposed to sperm. We had no success at generating offspring with precise single-nucleotide changes in KRT71 via homology-directed repair (HDR), but did identify mutation of FGF5 using non-homologous end joining (NHEJ). These findings underscore the difficulties inherent to gene repair, but represent important progress on reproducibility of canine IVF, improved techniques of oocyte/embryo handling, and impact of timing of injections on embryo development.
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Establishing targeted carp TLR22 gene disruption via homologous recombination using CRISPR/Cas9.
Recent advances in gene editing techniques have not been exploited in farmed fishes. We established a gene targeting technique, using the CRISPR/Cas9 system in Labeo rohita, a farmed carp (known as rohu). We demonstrated that donor DNA was integrated via homologous recombination (HR) at the site of targeted double-stranded nicks created by CRISPR/Cas9 nuclease. This resulted in the successful disruption of rohu Toll-like receptor 22 (TLR22) gene, involved in innate immunity and exclusively present in teleost fishes and amphibians. The null mutant, thus, generated lacked TLR22 mRNA expression. Altogether, this is the first evidence that the CRISPR/Cas9 system is a highly efficient tool for targeted gene disruption via HR in teleosts for generating model large-bodied farmed fishes.
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Virus-induced systemic and heritable gene editing in pepper (Capsicum annuum L.).
Genome editing using the CRISPR/Cas system enables rapid and efficient plant breeding by directly introducing desired traits into elite lines within a short time frame. However, challenges associated with conventional Agrobacterium tumefaciens-mediated transformation and regeneration have limited gene editing in pepper (Capsicum annuum L.). In this study, we applied and optimized a virus-induced gene editing (VIGE) system to overcome these limitations. We inoculated transgenic pepper seedlings already expressing Cas9 with vectors based on tobacco rattle virus 2 (TRV2) expressing single guide RNAs (sgRNAs) targeting Phytoene desaturase (PDS); shoots regenerated from inoculated cotyledons displayed photobleaching phenotypes. To promote sgRNA mobility and maintain its integrity, we modified the pTRV2-sgRNA vector by incorporating a self-cleaving hammerhead ribozyme (HH) sequence to produce an intact sgRNA fused to part of the mobile RNA of FLOWERING LOCUS T. Additionally, we tested alternative mobile elements, such as tRNAIle and tRNAMet. Furthermore, we cultivated plants at the low temperature of 20°C following TRV inoculation to increase TRV persistence and spread. These optimizations, including vector modifications and cultivation conditions, resulted in a systemic editing efficiency of 36.3%, as evidenced by systemic leaves showing photobleaching phenotypes. We determined that 8.5% of progeny from plants inoculated with the pTRV-HH-CaPDS-sgRNA-FT construct were mutated at the CaPDS locus. In addition, we used our VIGE system to successfully edit FASCICULATE, producing mutants whose inflorescences showed a fasciculate phenotype. Direct inoculation with a TRV-based vector expressing a mobile sgRNA to bypass tissue culture, therefore, offers an effective tool for molecular studies and breeding in pepper.
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Human-mediated outdoor genome editing is not possible so therefore poses no risk to the environment.
There is a world-wide re-examination of the regulations that surround genetic technologies, including gene edited organisms. In many countries, crop plants with small gene edits and where no foreign DNA is introduced are exempt from detailed regulatory assessment. This will allow these types of plants to be released, after assessment for benefit and risk, by plant breeders or plant scientists. The full regulatory risk assessment and risk management of novel (including transgenic) plants is well established and focusses on five key criteria (weediness, gene flow, plant pests, non-target impact, biodiversity). However, plants produced by "traditional" plant breeding technologies, many of which have been subject to random mutagenesis or wide crosses that may introduce enormous numbers of DNA changes, are almost never considered novel so are not assessed for risk through a regulatory system. The most targeted, versatile and widely used gene editing technique involves the enzyme-RNA complex, CRISPR-Cas. This method can produce far more precise and targeted changes than "traditional" mutagenesis techniques. Getting the CRISPR-Cas machinery into plant or animal cells requires highly sterile tissue culture and sophisticated delivery tools. Therefore, gene editing, in the open environment by "field spraying", is not currently possible. While other uses of nucleotide chemistry - such as double stranded RNA - have been applied to plants to knock down gene expression, this is not gene editing and produces no DNA change. Suggestions that gene editing using CRISPR-Cas can occur through spraying directly on to plants in the outside environment is fanciful, incorrect and misleading.
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[A modular approach to in vivo gene therapy].
In vivo inactivation of a deleterious gene has been achieved in a small trial, with excellent clinical results. Interestingly, the delivery and editing system is the same as in previous work on a different disease, and the new therapy required simply changing the guide RNA used to target the Cas9 nuclease. This modular approach could be extended to a number of other genetic diseases.
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