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CRISPR/Cas9 Application for Gene Copy Fate Survey of Polyploid Vertebrates.
Polyploidization occurs widely in eukaryotes, and especially in plants. Polyploid plants and some fishes have been commercialized. Typically, severe genomic perturbations immediately follow polyploidization and little is known about how polyploid offspring survives the genetic and epigenetic changes. Investigations into this require the identification of genes related to polyploidization and the discrimination of dosage-balance from paternal and maternal copies, and regardless of the mechanism being either autopolyploidization or allopolyploidization. New approaches and technologies may discern the mosaic of novel gene functions gained through the recombination of paternal and maternal genes in allopolyploidization. Modifications of Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR) with CRISPR-associated system (Cas) protein 9 (CRISPR/Cas9) have been employed in studies of polyploidization of plants. However, the approach has seldom been applied to polyploidization in vertebrates. Herein, we use CRISPR/Cas9 to trace gene-fate in tetraploid goldfish, and specifically to identify the functional differentiation of two divergent copies of fgf20a, which are expressed differently throughout embryonic development. We expect this gene editing system will be applicable to studies of polyploids and the genetic improvement of polyploid livestock.
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Genome Editing in Mice.
Programmable nucleases like CRISPR/Cas9 enable to edit the mouse genome directly in the zygote. Several methods have been successfully used for this. Here we describe injection into one of the pronuclei of the zygote and electroporation of zygotes. Alternative methods will be mentioned.
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Efficient genome engineering in Agrobacterium tumefaciens C58 using recombineering assisted by CRISPR/Cas9.
Recombineering, a technique derived from phage-encoded homologous recombination, has emerged as a vital approach for bacterial genome engineering. Agrobacterium tumefaciens is extensively utilized to transfer DNA into the host plant genomes. To facilitate the transformation of various plant species, particularly those of considerable economic value, genetic modifications of Agrobacterium strains are essential. Our previous studies established an Agrobacterium-specific phage-encoded homologous recombination system for Agrobacterium species. Yet, recent investigations have indicated that there is a substantial variability in the recombination efficiency of these recombineering systems for gene editing across different genome loci in A. tumefaciens. In this work, we present the development of an efficient genome engineering tool for A. tumefaciens by integrating recombineering with CRISPR/Cas9 technology. Initially, we found that lengthening the homology arms significantly enhanced genome editing efficiency. Nevertheless, at certain genomic sites, even when the length of the homology arms was increased, the editing efficiency remained suboptimal. Subsequently, combination of the Agrobacterium-specific recombineering system with the CRISPR/Cas9 system markedly enhanced the genome engineering efficiency. This study offers an enhanced and efficient genome engineering tool for A. tumefaciens, which could potentially be applied to other species within the Agrobacterium genus.
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Red fruit, orange fruit, orange fruit, red fruit: genome editing in tomato.
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Polymeric micellar nanoparticles for effective CRISPR/Cas9 genome editing in cancer.
The clustered regularly interspaced short palindromic repeat (CRISPR)-associated protein 9 (Cas9) gene editing has attracted extensive attentions in various fields, however, its clinical application is hindered by the lack of effective and safe delivery system. Herein, we reported a cationic micelle nanoparticle composed of cholesterol-modified branched small molecular PEI (PEI-CHO) and biodegradable PEG-b-polycarbonate block copolymer (PEG-PC), denoted as PEG-PC/PEI-CHO/pCas9, for the CRISPR/Cas9 delivery to realize genomic editing in cancer. Specifically, PEI-CHO condensed pCas9 into nanocomplexes, which were further encapsulated into PEG-PC nanoparticles (PEG-PC/PEI-CHO/pCas9). PEG-PC/PEI-CHO/pCas9 had a PEG shell, protecting DNA from degradation by nucleases. Enhanced cellular uptake of PEG-PC/PEI-CHO/pCas9 nanoparticles was observed as compared to that mediated by Lipo2k/pCas9 nanoparticles, thus leading to significantly elevated transfection efficiency after escaping from endosomes via the proton sponge effect of PEI. In addition, the presence of PEG shell greatly improved biocompatibility, and significantly enhanced the in vivo tumor retention of pCas9 compared to PEI-CHO/pCas9. Notably, apparent downregulation of GFP expression could be achieved both in vitro and in vivo by using PEG-PC/PEI-CHO/pCas9-sgGFP nanoparticles. Furthermore, PEG-PC/PEI-CHO/pCas9-sgMcl1 induced effective apoptosis and tumor suppression in a HeLa tumor xenograft mouse model by downregulating Mcl1 expression. This work may provide an alternative paradigm for the efficient and safe genome editing in cancer.
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What Do We Really Think About Human Germline Genome Editing, and What Does It Mean for Medicine?
Genome editing has captured widespread attention due to its potential therapeutic applications. Early studies with human embryos have established the feasibility of human germline genome editing but raise complex social, ethical, and legal questions. In light of the potential impact of genome editing on the practice of cardiovascular medicine, we surveyed ≈300 attendees at a recent American Heart Association conference to elicit their opinions on somatic and germline genome editing. The results were revealing and highlight the need to broadly engage the public and solicit the opinions of various constituencies before proceeding with clinical germline genome editing.
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Targeted editing of CCL5 with CRISPR-Cas9 nanoparticles enhances breast cancer immunotherapy.
Breast cancer remains one of the leading causes of cancer-related mortality among women worldwide. Immunotherapy, a promising therapeutic approach, often faces challenges due to the immunosuppressive tumor microenvironment. This study explores the innovative use of CRISPR-Cas9 technology in conjunction with FCPCV nanoparticles to target and edit the C-C Motif Chemokine Ligand 5 (CCL5) gene, aiming to improve the efficacy of breast cancer immunotherapy. Single-cell RNA sequencing (scRNA-seq) and TCGA-BRCA data identified CCL5 as a key immune-related gene in breast cancer. Using CRISPR-Cas9, sgRNA targeting CCL5 was designed and delivered to breast cancer cells and humanized mouse models via FCPCV nanoparticles. In vitro experiments demonstrated that FCPCV nanoparticles effectively silenced CCL5, enhanced CD8+ T cell activity, and increased the production of cytokines such as IFN-γ, TNF-α, and GZMB. In vivo studies revealed significant tumor suppression, improved immune microenvironment, and increased CD8+/CD4+ ratios in treated mice, without notable toxic side effects. These findings highlight the potential of CRISPR-Cas9 nanoparticle-mediated gene editing as a novel strategy for enhancing breast cancer immunotherapy, providing a new direction for personalized and effective cancer treatment.
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Methods for CRISPR-Cas as Ribonucleoprotein Complex Delivery In Vivo.
The efficient delivery of CRISPR-Cas components is still a key and unsolved problem. CRISPR-Cas delivery in the form of a Cas protein+sgRNA (ribonucleoprotein complex, RNP complex), has proven to be extremely effective, since it allows to increase on-target activity, while reducing nonspecific activity. The key point for in vivo genome editing is the direct delivery of artificial nucleases and donor DNA molecules into the somatic cells of an adult organism. At the same time, control of the dose of artificial nucleases is impossible, which affects the efficiency of genome editing in the affected cells. Poor delivery efficiency and low editing efficacy reduce the overall potency of the in vivo genome editing process. Here we review how this problem is currently being solved in scientific works and what types of in vivo delivery methods of Cas9/sgRNA RNPs have been developed.
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Gene editing to enhance pesticide resistance in a beneficial predatory mite.
Successful integrated pest management (IPM) often depends on a suite of control strategies that are compatible with one another. The predatory mite Neoseiulus californicus (Acari: Phytoseiidae) is a key biological control agent of spider mites (especially Tetranychus spp.) and other diminutive, yet important arthropod pests of agriculture. However, like many natural enemies, N. californicus is highly sensitive to chemical pesticides, limiting its overall effectiveness in the field. Here, we used CRISPR/Cas9 gene editing and Receptor-Mediated Ovary Transduction of Cargo (ReMOT Control) to create N. californicus harboring loss-of-function mutations in the nicotinic acetylcholine receptor α6 subunit (nAChRα6), the target of the pesticide, spinetoram. The resulting knockout strain (FZ-α6KO) exhibited a 23-fold increase in resistance to spinetoram compared to its parental strain. Inheritance of resistance to spinetoram in FZ-α6KO was autosomal, recessive, and tightly linked with the nAChRα6 gene. We demonstrate that by pairing gene editing with ReMOT Control, extremely small and fragile beneficial arthropods can be genetically manipulated. These results suggest that genetic modification to enhance pesticide resistance in beneficial predatory mites could improve the compatibility between the use of pesticides and biological control organisms.
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CRISPR/Cas-Based Techniques for Live-Cell Imaging and Bioanalysis.
CRISPR/Cas systems have found widespread applications in gene editing due to their high accuracy, high programmability, ease of use, and affordability. Benefiting from the cleavage properties (trans- or cis-) of Cas enzymes, the scope of CRISPR/Cas systems has expanded beyond gene editing and they have been utilized in various fields, particularly in live-cell imaging and bioanalysis. In this review, we summarize some fundamental working mechanisms and concepts of the CRISPR/Cas systems, describe the recent advances and design principles of CRISPR/Cas mediated techniques employed in live-cell imaging and bioanalysis, highlight the main applications in the imaging and biosensing of a wide range of molecular targets, and discuss the challenges and prospects of CRISPR/Cas systems in live-cell imaging and biosensing. By illustrating the imaging and bio-sensing processes, we hope this review will guide the best use of the CRISPR/Cas in imaging and quantifying biological and clinical elements and inspire new ideas for better tool design in live-cell imaging and bioanalysis.
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