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[Advances of Cas9/sgRNA delivery system for gene editing].
In the application of CRISPR genome editing, direct cellular delivery of non-replicable Cas9/sgRNA may reduce unwanted gene targeting and integrational mutagenesis, thus offering greater specificity and safety. Cas9/sgRNA delivery system holds great potential for treating genetic diseases. This review summarizes the advances of Cas9/sgRNA delivery systems and its therapeutic applications, providing new understandings and inspirations for vector design and future clinical applications.
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Design of SaCas9-HF for In Vivo Gene Therapy.
Genome alteration results in several diseases for which therapeutics are limited. Gene editing provides a strong and potential alternative for the treatment of rare and genetic diseases. CRISPR-Cas9-based system is now being envisaged as a potential tool for the cure of genetic diseases. The RNA-guided nuclease, SaCas9 enzyme, along with its HF versions is widely employed for in vivo gene editing because of its small size and high efficiency. The current work summarizes the widely used and improved methods for in vivo manipulation of genes. The potential of CRISPR-Cas9-based systems can be harnessed to treat genetic diseases and holds great promise for therapeutic interventions in gene therapy. The in vivo gene editing poses a caveat in the form of delivery systems, the tissue in question, and several other factors. This work describes the methods which have been optimized to offer high efficiency, delivery, and gene editing in vivo.
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Towards a CRISPR view of early human development: applications, limitations and ethical concerns of genome editing in human embryos.
Developmental biologists have become increasingly aware that the wealth of knowledge generated through genetic studies of pre-implantation mouse development might not easily be translated to the human embryo. Comparative studies have been fueled by recent technological advances in single-cell analysis, allowing in-depth analysis of the human embryo. This field could shortly gain more momentum as novel genome editing technologies might, for the first time, also allow functional genetic studies in the human embryo. In this Spotlight article, we summarize the CRISPR-Cas9 genome editing system and discuss its potential applications and limitations in human pre-implantation embryos, and the ethical considerations thereof.
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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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Metal-organic frameworks for CRISPR/Cas9 gene editing delivery: Innovations in therapeutic and diagnostic applications.
CRISPR/Cas gene editing technology demonstrates significant promise in the treatment of various diseases, and a precise, efficient and safe delivery system is a key to realize gene therapy. Although traditional viral vectors can achieve superior transfection efficiency, viruses suffer from low reproduction efficiency and the risk of random gene integration, further limiting their wide application. Notably, metal-organic frameworks (MOFs), with tunable pore structure, easy surface chemical modification, good biocompatibility and physiological stability, have drawn much attention in the domain of targeted delivery of gene editing systems. Compared to lipid nanoparticles (LNPs) and extracellular vesicles (EVs), MOFs offer superior cargo loading (>80 % for proteins) and protect nucleic acids from degradation, while their stimuli-responsive degradation enables controlled release. This review focus on the cutting-edge advances of intelligent-responsive MOFs in delivering gene editing systems to against diseases, including endogenous responses (e.g., ATP, pH, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Additionally, we further summarize the research of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination. This study will help facilitate the clinical translation of MOFs-based CRISPR/Cas delivery systems in the field of therapy and detection of diseases. STATEMENT OF SIGNIFICANCE: This article reviews the cutting-edge advances of intelligent-responsive MOFs in delivering CRISPR/Cas systems to against diseases, including endogenous responses (e.g., pH, ATP, redox microenvironment) and exogenous stimulus responses (e.g., photothermal, ultrasound) in the disease microenvironment, as well as systematically summarize the synergistic therapy of gene editing therapy combined with chemotherapy, chemodynamic therapy, photodynamic therapy, and sonodynamic therapy based on the delivery systems of MOFs. Importantly, the potential applications of MOFs-based CRISPR/Cas delivery system as a bio-probe for viral, nucleic acid and RNA examination also have been discussed. This study will provide insights for the development of MOFs-based CRISPR/Cas delivery systems in the therapy and detection of clinical diseases.
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GEEP Method: An Optimized Electroporation-Mediated Gene Editing Approach for Establishment of Knockout Pig Lines.
Pigs are excellent large animal models owing to their several physiological and anatomical similarities to humans. Somatic cell nuclear transfer using gene-modified cells is the mainstream approach for generating genetically modified pigs. Recent advances in improving gene editors such as the CRISPR/Cas9 system have enabled direct gene modification in zygotes/embryos. Here, we describe the gene editing by electroporation of Cas9 protein (GEEP) method, an optimized electroporation-mediated method for the introduction of CRISPR/Cas9 into porcine zygotes/embryos. The simplicity and micromanipulation-free procedures are the major advantages of this method.
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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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Increasing the efficiency of gene editing with CRISPR-Cas9 via concurrent expression of the Beta protein.
Escherichia coli has emerged as an important host for the production of biopharmaceuticals or other industrially relevant molecules. An efficient gene editing tool is indispensable for ensuring high production levels and optimal release of target products. However, in Escherichia coli, the CRISPR-Cas9 system has been shown to achieve gene modifications with relatively low frequency. Large-scale PCR screening is required, hindering the identification of positive clones. The beta protein, which weakly binds to single-stranded DNA but tightly associates with complementary strand annealing products, offers a promising solution to this issue. In the present study, we describe a targeted and continuous gene editing strategy for the Escherichia coli genome. This strategy involves the coexpression of the beta protein alongside the CRISPR-Cas9 system, enabling a variety of genome modifications such as gene deletion and insertion with an efficiency exceeding 80 %. The integrity of beta proteins is essential for the CRISPR-Cas9/Beta-based gene editing system. In this work, the deletion of either the N- or C-terminal domain significantly impaired system efficiency. Overall, our findings established the CRISPR-Cas9/Beta system as a suitable gene editing tool for various applications in Escherichia coli.
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Huntington disease: Selective deactivation of Huntington disease mutant allele by CRISPR-Cas9 gene editing.
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Efficient Gene Editing of Human Induced Pluripotent Stem Cells Using CRISPR/Cas9.
The generation of targeted mutants is a crucial step toward studying the biomedical effect of genes of interest. The generation of such mutants in human induced pluripotent stem cells (iPSCs) is of an utmost importance as these cells carry the potential to be differentiated into any cell lineage. Using the CRISPR/Cas9 nuclease system for induction of targeted double-strand breaks, gene editing of target loci in iPSCs can be achieved with high efficiency. This chapter covers protocols for the preparation of reagents to target loci of interest, the transfection, and for the genotyping of single cell-derived iPSC clones. Furthermore, we provide a protocol for the convenient generation of plasmids enabling multiplex gene targeting.
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