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[Experimental teaching design of CRISPR/Cas9 technology in rice breeding application].
The CRISPR/Cas9 gene editing technology has proven to be valuable in crop breeding applications. Understanding and mastering this technology will provide a strong foundation for students majoring in biology, agronomy, and related fields to engage in scientific research and work. To incorporate CRISPR/Cas9 technology into experimental teaching courses at colleges, an innovative teaching experiment entitled "Enhancing the resistance of rice plants to bacterial blight disease using CRISPR/Cas9 technology" was designed. The experiment allows students to deepen their understanding of the basic principles of CRISPR/Cas technology, acquire proficiency in its protocol, and learn to apply the technology for targeted molecular breeding of rice. It not only expands students' knowledge and skills, but also promotes the reform and innovation of experimental teaching methods.
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En bloc and segmental deletions of human XIST reveal X chromosome inactivation-involving RNA elements.
The XIST RNA is a non-coding RNA that induces X chromosome inactivation (XCI). Unlike the mouse Xist RNA, how the human XIST RNA controls XCI in female cells is less well characterized, and its functional motifs remain unclear. To systematically decipher the XCI-involving elements of XIST RNA, 11 smaller XIST segments, including repeats A, D and E; human-specific repeat elements; the promoter; and non-repetitive exons, as well as the entire XIST gene, were homozygously deleted in K562 cells using the Cas9 nuclease and paired guide RNAs at high efficiencies, followed by high-throughput RNA sequencing and RNA fluorescence in situ hybridization experiments. Clones containing en bloc and promoter deletions that consistently displayed no XIST RNAs and a global up-regulation of X-linked genes confirmed that the deletion of XIST reactivates the inactive X chromosome. Systematic analyses of segmental deletions delineated that exon 5 harboring the non-repeat element is important for X-inactivation maintenance, whereas exons 2, 3 and 4 as well as the other repeats in exon 1 are less important, a different situation from that of mouse Xist. This Cas9-assisted dissection of XIST allowed us to understand the unique functional domains within the human XIST RNA.
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Nanoparticle Carriers: A New Era of Precise CRISPR/Cas9 Gene Editing.
The revolutionary CRISPR/Cas9 gene editing technology holds immense potential for treating genetic diseases and tackling conditions like cancer. However, efficient delivery remains a significant challenge. This is where nanoparticles come into play, emerging as powerful allies in the realm of drug delivery. Nanoparticles can accommodate larger insertion sizes, enabling the incorporation of larger Cas9 enzymes and complex guide RNAs, thus opening up the possibility of editing previously inaccessible genetic regions. Their relatively straightforward and scalable production processes make them cost-effective options for wider applications. Notably, nanoparticles excel in vivo, demonstrating efficient tissue penetration and targeted delivery, which are crucial for maximizing therapeutic impact while minimizing side effects. This review aims to explore the potential of nanoparticle-based delivery systems for CRISPR/Cas9, highlighting their advantages and challenges in gene editing applications. The diverse range of nanoparticles further bolsters their potential. Polymeric nanoparticles, for instance, offer tunable properties for customization and controlled release of the CRISPR cargo. Lipid-based nanoparticles facilitate efficient cellular uptake and endosomal escape, ensuring the CRISPR components reach the target DNA. Even gold nanoparticles, known for their unique biocompatibility and photothermal properties, hold promise in light-activated editing strategies. Non-viral delivery systems, particularly those based on nanoparticles, stand out due to their inherent advantages. Collectively, the evidence paints a promising picture: nanoparticles are not merely passive carriers but active participants in the CRISPR/Cas9 delivery landscape. Their versatility, efficiency, and safety position them as key enablers of a future where gene editing can revolutionize drug development, offering personalized and targeted therapies for a wide range of diseases.
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The technical risks of human gene editing.
A recent report from Dr He Jiankui concerning the birth of twin girls harbouring mutations engineered by CRISPR/Cas nucleases has been met with international condemnation. Beside the serious ethical concerns, there are known technical risks associated with CRISPR/Cas gene editing which further raise questions about how these events could have been allowed to occur. Numerous studies have reported unexpected genomic mutation and mosaicism following the use of CRISPR/Cas nucleases, and it is currently unclear how prevalent these disadvantageous events are and how robust and sensitive the strategies to detect these unwanted events may be. Although Dr Jiankui's study appears to have involved certain checks to ascertain these risks, the decision to implant the manipulated embryos, given these unknowns, must nonetheless be considered reckless. Here I review the technical concerns surrounding genome editing and consider the available data from Dr Jiankui in this context. Although the data remains unpublished, preventing a thorough assessment of what was performed, it seems clear that the rationale behind the undertaking was seriously flawed; the procedures involved substantial technical risks which, when added to the serious ethical concerns, fully justify the widespread criticism that the events have received.
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Off-target effects in CRISPR/Cas9 gene editing.
Gene editing stands for the methods to precisely make changes to a specific nucleic acid sequence. With the recent development of the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, gene editing has become efficient, convenient and programmable, leading to promising translational studies and clinical trials for both genetic and non-genetic diseases. A major concern in the applications of the CRISPR/Cas9 system is about its off-target effects, namely the deposition of unexpected, unwanted, or even adverse alterations to the genome. To date, many methods have been developed to nominate or detect the off-target sites of CRISPR/Cas9, which laid the basis for the successful upgrades of CRISPR/Cas9 derivatives with enhanced precision. In this review, we summarize these technological advancements and discuss about the current challenges in the management of off-target effects for future gene therapy.
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Utilization of CRISPR/Cas9 gene editing in cellular therapies for lymphoid malignancies.
The ability to change the genetic information of immune cells is a powerful tool for basic and clinical settings. CRISPR/Cas9 gene editing technology by providing an efficient approach has accelerated immune cell therapy of cancers. Lymphoid cancers comprise a wide array of disease including lymphoma, multiple myeloma and lymphocytic leukemia. Here, we review therapeutic applications of the CRISPR/Cas9 technology for immune cell therapy of common lymphoid malignancies. We describe current and future therapeutic application of CRISPR/Cas9 technology with the focus on the production and applications of engineered hematopoietic and immune cells against lymphoid malignancies. Furthermore, we provide an overview of the possible challenges and optimization of CRISPR/Cas9 system for ex- and in vivo applications within recent years.
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Frankenstein Cas9: engineering improved gene editing systems.
The discovery of CRISPR-Cas9 and its widespread use has revolutionised and propelled research in biological sciences. Although the ability to target Cas9's nuclease activity to specific sites via an easily designed guide RNA (gRNA) has made it an adaptable gene editing system, it has many characteristics that could be improved for use in biotechnology. Cas9 exhibits significant off-target activity and low on-target nuclease activity in certain contexts. Scientists have undertaken ambitious protein engineering campaigns to bypass these limitations, producing several promising variants of Cas9. Cas9 variants with improved and alternative activities provide exciting new tools to expand the scope and fidelity of future CRISPR applications.
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Advancements of the CRISPR/Cas9 System in the Treatment of Liver Cancer.
In recent years, the CRISPR/Cas9 system has become a rapidly advancing gene editing technology with significant advantages in various fields, particularly biomedicine. Liver cancer is a severe malignancy that threatens human health and is primarily treated with surgery, radiotherapy, and chemotherapy. However, surgery may not be suitable for advanced cases of liver cancer with distant metastases. Moreover, radiotherapy and chemotherapy have low specificity and numerous side effects that limit their effectiveness; therefore, more effective and safer treatments are required. With the advancement of the biomolecular mechanism of cancer, CRISPR/Cas9 gene editing technology has been widely used in the study of liver cancer to gain insights into gene functions, establish tumor models, screen tumor phenotype-related genes, and perform gene therapy. This review outlines the research progress of CRISPR/Cas9 gene editing technology in the treatment of liver cancer and provides a relevant theoretical basis for its research and application in the treatment of liver cancer.
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A Conformational Restriction Strategy for the Control of CRISPR/Cas Gene Editing with Photoactivatable Guide RNAs.
The CRISPR/Cas system is one of the most powerful tools for gene editing. However, approaches for precise control of genome editing and regulatory events are still desirable. Here, we report the spatiotemporal and efficient control of CRISPR/Cas9- and Cas12a-mediated editing with conformationally restricted guide RNAs (gRNAs). This approach relied on only two or three pre-installed photo-labile substituents followed by an intramolecular cyclization, representing a robust synthetic method in comparison to the heavily modified linear gRNAs that often require extensive screening and time-consuming optimization. This tactic could direct the precise cleavage of the genes encoding green fluorescent protein (GFP) and the vascular endothelial growth factor A (VEGFA) protein within a predefined cutting region without notable editing leakage in live cells. We also achieved light-mediated myostatin (MSTN) gene editing in embryos, wherein a new bow-knot-type gRNA was constructed with excellent OFF/ON switch efficiency. Overall, our work provides a significant new strategy in CRISPR/Cas editing with modified circular gRNAs to precisely manipulate where and when genes are edited.
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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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