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[Application of CRISPR/Cas9 gene editing technology in edible fungi: a review].
The CRISPR/Cas9 gene editing system is a versatile technology for modifying gene, playing a crucial role in the study of functional genes and genetic breeding of plants, animals, fungi, and microorganisms. This review provides a comprehensive analysis of the application of this technology in gene research and genetic breeding of edible fungi. The review covers various aspects, including the delivery and expression strategies of Cas9 and sgRNA, genetic transformation methods, mutant screening, and repair strategies for target sites following DNA double-strand breaks. Additionally, the review summarizes the main challenges and optimization strategies associated with the application of this technology in edible fungi. Lastly, the future application potential of this technology in edible fungi research is discussed, drawing from the authors' personal research background.
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CRISPR/Cas gene editing and delivery systems for cancer therapy.
CRISPR/Cas systems stand out because of simplicity, efficiency, and other superiorities, thus becoming attractive and brilliant gene-editing tools in biomedical field including cancer therapy. CRISPR/Cas systems bring promises for cancer therapy through manipulating and engineering on tumor cells or immune cells. However, there have been concerns about how to overcome the numerous physiological barriers and deliver CRISPR components to target cells efficiently and accurately. In this review, we introduced the mechanisms of CRISPR/Cas systems, summarized the current delivery strategies of CRISPR/Cas systems by physical methods, viral vectors, and nonviral vectors, and presented the current application of CRISPR/Cas systems in cancer clinical treatment. Furthermore, we discussed prospects related to delivery approaches of CRISPR/Cas systems. This article is categorized under: Therapeutic Approaches and Drug Discovery > Emerging Technologies Therapeutic Approaches and Drug Discovery > Nanomedicine for Oncologic Disease.
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The Gene Editing Juggernaut is Picking Up Speed.
CRISPR-Cas9, the tool for editing genes by precisely cutting DNA and letting the body's natural DNA repair processes take over, deservedly led to Nobel prizes in 2020 for its pioneers, Emmanuelle Charpentier and Jennifer Doudna. Since their breakthroughs in 2012, the technology has moved forward in leaps and bounds, and techniques to manipulate genes that were once the realm of science fiction are becoming very much science "fact."
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Utilizing CRISPR-based genetic modification for precise control of seed dormancy: progress, obstacles, and potential directions.
Seed dormancy, a complex trait that is influenced by both nuclear and cytoplasmic factors, poses a significant challenge to agricultural productivity. Conventional dormancy-breaking techniques, including mechanical, physiological, and chemical methods, often yield inconsistent results, impair seed quality, and lack precision. This has necessitated exploration of more targeted and efficient approaches. CRISPR-based gene editing has emerged as a promising tool for the precise regulation of seed dormancy without compromising seed viability or sustainability. Although CRISPR has been successfully applied to modify genes that govern physiological traits in various crops, its use in dormancy regulation remains in the early stages. This review examines recent advancements in CRISPR-based approaches for modulating seed dormancy and discusses key gene targets, modification techniques, and the resulting effects. We also consider the future potential of CRISPR to enhance dormancy control across diverse crop species.
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Implications of CRISPR-Cas9 in Developing Next Generation Biofuel: A Mini-review.
The major drawbacks of biofuel production at the commercial level are its low yield, nonavailability of feedstock, feedback inhibition, presence of inhibitory pathways in various organisms, and biofuel intolerance of organisms. The present review focuses on the implications of the CRISPRCas9 mediated gene editing tool to alter the genome of bacteria, algae, fungi, and higher plants for efficient biofuel production. Gene knockout and gene cassette insertions employing CRISPR-Cas9 in Saccharomyces cerevisiae and Kluyveromyces marxianus have resulted in enhanced production of bioethanol and 2-Phenyl ethanol in these organisms, respectively. Genomes of several bacterial strains were also modified to enhance ethanol and butanol production in them. CRISPR-Cas9 modification of microalgae has demonstrated improved total lipid content, a prerequisite for biofuel production. All over, CRISPR-Cas9 has emerged as a tool of choice for engineering the genome and metabolic pathways of organisms for producing industrial biofuel. In plant-based biofuel production, the biosynthetic pathways of lignin interfere with the satisfactory release of fermentable sugars thus hampering efficient biofuel production. CRISPR-Cas9 has shown a promising role in reducing lignin content in various plants including barley, switchgrass, and rice straw.
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CRISPR/Gal4BD-Cas donor adapting systems based on miniaturized Cas proteins for improved gene editing.
Targeted precise point editing and knock-in can be achieved by homology-directed repair(HDR) based gene editing strategies in mammalian cells. However, the inefficiency of HDR strategies seriously restricts their application in precision medicine and molecular design breeding. In view of the problem that exogenous donor DNA cannot be efficiently recruited autonomously at double-stranded breaks(DSBs) when using HDR strategies for gene editing, the concept of donor adapting system(DAS) was proposed and the CRISPR/Cas9-Gal4BD DAS was developed previously. Due to the large size of SpCas9 protein, its fusion with the Gal4BD adaptor is inconvenient for protein expression, virus vector packaging and in vivo delivery. In this study, two novel CRISPR/Gal4BD-SlugCas9 and CRISPR/Gal4BD-AsCas12a DASs were further developed, using two miniaturized Cas proteins, namely SlugCas9-HF derived from Staphylococcus lugdunensis and AsCas12a derived from Acidaminococcus sp. Firstly, the SSA reporter assay was used to assess the targeting activity of different Cas-Gal4BD fusions, and the results showed that the fusion of Gal4BD with SlugCas9 and AsCas12a N-terminals had minimal distraction on their activities. Secondly, the HDR efficiency reporter assay was conducted for the functional verification of the two DASs and the corresponding donor patterns were optimized simultaneously. The results demonstrated that the fusion of the Gal4BD adaptor binding sequence at the 5'-end of intent dsDNA template (BS-dsDNA) was better for the CRISPR/Gal4BD-AsCas12a DAS, while for the CRISPR/Gal4BD-SlugCas9 DAS, the dsDNA-BS donor pattern was recommended. Finally, CRISPR/Gal4BD-SlugCas9 DAS was used to achieve gene editing efficiency of 24%, 37% and 31% respectively for EMX1, NUDT5 and AAVS1 gene loci in HEK293T cells, which was significantly increased compared with the controls. In conclusion, this study provides a reference for the subsequent optimization of the donor adapting systems, and expands the gene editing technical toolbox for the researches on animal molecular design breeding.
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A CRISPR/Cas9 based polymeric nanoparticles to treat/inhibit microbial infections.
The latest breakthrough towards the adequate and decisive methods of gene editing tools provided by CRISPR/Cas9 (Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR Associated System), has been repurposed into a tool for genetically engineering eukaryotic cells and now considered as the major innovation in gene-related disorders. Nanotechnology has provided an alternate way to overcome the conventional problems where methods to deliver therapeutic agents have failed. The use of nanotechnology has the potential to safe-side the CRISPR/Cas9 components delivery by using customized polymeric nanoparticles for safety and efficacy. The pairing of two (CRISPR/Cas9 and nanotechnology) has the potential for opening new avenues in therapeutic use. In this review, we will discuss the most recent advances in developing nanoparticle-based CRISPR/Cas9 gene editing cargo delivery with a focus on several polymeric nanoparticles including fabrication proposals to combat microbial infections.
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Rewriting CAR-T cell fate: CRISPR/Cas gene editing for solid tumor therapy.
Although chimeric antigen receptor T (CAR-T) cell therapy has achieved remarkable success in hematological malignancies, its therapeutic efficacy in solid tumors remains limited by several challenges, including insufficient tumor infiltration, T cell exhaustion and the immunosuppressive tumor microenvironment (TME). CRISPR/Cas, a third-generation gene editing technology developed in recent years, is characterized by its simplicity and high efficiency. This technology has demonstrated broad application potential across multiple fields and has emerged as a powerful tool for improving CAR-T cell therapy. In this review, we summarize recent advances in the application of CRISPR/Cas gene editing technology to enhance the antitumor activity of CAR-T cells against solid tumors. We also discuss the key challenges currently faced and systematically propose potential strategies for overcoming the limitations.
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Genome Editing: CRISPR-Cas9.
In the present chapter, we present the protocols and guidelines to facilitate implementation of CRISPR-Cas9 technology in fungi where few or no genetic tools are in place. Hence, we firstly explain how to identify dominant markers for genetic transformation. Secondly, we provide a guide for construction of Cas9/sgRNA episomal expression vectors. Thirdly, we present how to mutagenize reporter genes to explore the efficiency of CRISPR-Cas9 in the relevant fungus and to ease subsequent CRISPR-mediated genetic engineering. Lastly, we describe how to make CRISPR-mediated marker-dependent and marker-free gene targeting.
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A status-quo review on CRISPR-Cas9 gene editing applications in tomato.
Epigenetic changes are emancipated in horticultural crops including tomato due to a variety of environmental factors. These modifications rely on plant phenotypes mediated by genetic architecture consequently resulting in hereditary epigenetic memory. Genome editing strategies like CRISPR (Clustered Regularly Interspaced Short Palindromic Repeat)/CRISPR-associated protein 9 (Cas9) technologies have revolutionized plants biology foreseeing stable inheritance of epigenetic modifications. CRISPR/Cas9 strategy poses as explicit advancement in providing precise genome editing with minimal off-target mutations, ease of experimental design, higher efficiency, versatility, and cost-effectiveness. Dicot crops, especially tomato remain an ideal candidate for CRISPR/Cas9 based gene modulations thereby augmenting productivity and yields. In the present review, key questions on CRISPR/Cas9 applications aid in enhanced growth based on optimal gene discovery, de novo modification, trait improvement, and biotic/abiotic stress management are discussed. In addition, comparative scenario in tomato and similar horticultural crops are adequately summarized for the pros and cons. Further, limitations hampering potential benefits and success phenomena of the lab to field transition of gene editing alterations are discussed collaterally in addressing futuristic optimization for CRISPR/Cas9 research in tomato.
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