CRISPR/Cas9: Principle, Applications, and Delivery through Extracellular Vesicles.
Ontology highlight
ABSTRACT:
SUBMITTER: Horodecka K
PROVIDER: S-EPMC8200053 | biostudies-literature |
REPOSITORIES: biostudies-literature
Similar Datasets
Project description:Therapeutic genome editing has the potential to cure diseases by directly correcting genetic mutations in tissues and cells. Recent progress in the CRISPR-Cas9 systems has led to breakthroughs in gene editing tools because of its high orthogonality, versatility, and efficiency. However, its safe and effective administration to target organs in patients is a major hurdle. Extracellular vesicles (EVs) are endogenous membranous particles secreted spontaneously by all cells. They are key actors in cell-to-cell communication, allowing the exchange of select molecules such as proteins, lipids, and RNAs to induce functional changes in the recipient cells. Recently, EVs have displayed their potential for trafficking the CRISPR-Cas9 system during or after their formation. In this review, we highlight recent developments in EV loading, surface functionalization, and strategies for increasing the efficiency of delivering CRISPR-Cas9 to tissues, organs, and cells for eventual use in gene therapies.
| S-EPMC10571031 | biostudies-literature
Project description:Clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein (Cas) systems can precisely manipulate DNA sequences to change the characteristics of cells and organs, which has potential in the mechanistic research on genes and the treatment of diseases. However, clinical applications are restricted by the lack of safe, targeted and effective delivery vectors. Extracellular vesicles (EVs) are an attractive delivery platform for CRISPR/Cas9. Compared with viral and other vectors, EVs present several advantages, including safety, protection, capacity, penetrating ability, targeting ability and potential for modification. Consequently, EVs are profitably used to deliver the CRISPR/Cas9 in vivo. In this review, the advantages and disadvantages of the delivery form and vectors of the CRISPR/Cas9 are concluded. The favorable traits of EVs as vectors, such as the innate characteristics, physiological and pathological functions, safety and targeting ability of EVs, are summarized. Furthermore, in terms of the delivery of the CRISPR/Cas9 by EVs, EV sources and isolation strategies, the delivery form and loading methods of the CRISPR/Cas9 and applications have been concluded and discussed. Finally, this review provides future directions of EVs as vectors of the CRISPR/Cas9 system in clinical applications, such as the safety, capacity, consistent quality, yield and targeting ability of EVs.
| S-EPMC10053467 | biostudies-literature
Project description:The method of delivery of CRISPR-Cas9 into target cells is a strong determinant of efficacy and specificity in genome editing. Even though high efficiency of Cas9 delivery is necessary for optimal editing, its long-term and high levels of expression correlate with increased off-target activity. We developed vesicles (VEsiCas) carrying CRISPR-SpCas9 ribonucleoprotein complexes (RNPs) that are efficiently delivered into target cells through the fusogenic glycoprotein of the vesicular stomatitis virus (VSV-G). A crucial step for VEsiCas production is the synthesis of the single guide RNA (sgRNA) mediated by the T7 RNA polymerase in the cytoplasm of producing cells as opposed to canonical U6-driven Pol III nuclear transcription. In VEsiCas, the absence of DNA encoding SpCas9 and sgRNA allows rapid clearance of the nuclease components in target cells, which correlates with reduced genome-wide off-target cleavages. Compared with SpCas9 RNPs electroporation, which is currently the method of choice to obtain transient SpCas9 activity, VEsiCas deliver the nuclease with higher efficiency and lower toxicity. We show that a wide variety of cells can be edited through VEsiCas, including a variety of transformed cells, induced pluripotent stem cells (iPSCs), and cardiomyocytes, in vivo. VEsiCas is a traceless CRISPR-Cas9 delivery tool for efficient and safe genome editing that represents a further advancement toward the therapeutic use of the CRISPR-Cas9 technology.
| S-EPMC6041463 | biostudies-literature
Project description:The clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) systems have emerged as a robust and versatile genome editing platform for gene correction, transcriptional regulation, disease modeling, and nucleic acids imaging. However, the insufficient transfection and off-target risks have seriously hampered the potential biomedical applications of CRISPR/Cas9 technology. Herein, we review the recent progress towards CRISPR/Cas9 system delivery based on viral and non-viral vectors. We summarize the CRISPR/Cas9-inspired clinical trials and analyze the CRISPR/Cas9 delivery technology applied in the trials. The rational-designed non-viral vectors for delivering three typical forms of CRISPR/Cas9 system, including plasmid DNA (pDNA), mRNA, and ribonucleoprotein (RNP, Cas9 protein complexed with gRNA) were highlighted in this review. The vector-derived strategies to tackle the off-target concerns were further discussed. Moreover, we consider the challenges and prospects to realize the clinical potential of CRISPR/Cas9-based genome editing.
| S-EPMC8538656 | biostudies-literature
Project description:Since its mechanism discovery in 2012 and the first application for mammalian genome editing in 2013, CRISPR-Cas9 has revolutionized the genome engineering field and created countless opportunities in both basic science and translational medicine. The first clinical trial of CRISPR therapeutics was initiated in 2016, which employed ex vivo CRISPR-Cas9 edited PD-1 knockout T cells for the treatment of non-small cell lung cancer. So far there have been dozens of clinical trials registered on ClinicalTrials.gov in regard to using the CRISPR-Cas9 genome editing as the main intervention for therapeutic applications; however, most of these studies use ex vivo genome editing approach, and only a few apply the in vivo editing strategy. Compared to ex vivo editing, in vivo genome editing bypasses tedious procedures related to cell isolation, maintenance, selection, and transplantation. It is also applicable to a wide range of diseases and disorders. The main obstacles to the successful translation of in vivo therapeutic genome editing include the lack of safe and efficient delivery system and safety concerns resulting from the off-target effects. In this review, we highlight the therapeutic applications of in vivo genome editing mediated by the CRISPR-Cas9 system. Following a brief introduction of the history, biology, and functionality of CRISPR-Cas9, we showcase a series of exemplary studies in regard to the design and implementation of in vivo genome editing systems that target the brain, inner ear, eye, heart, liver, lung, muscle, skin, immune system, and tumor. Current challenges and opportunities in the field of CRISPR-enabled therapeutic in vivo genome editing are also discussed.
| S-EPMC10018374 | biostudies-literature
Project description:Extracellular vesicles (EVs) are membranous nanovesicles secreted from living cells, shuttling macromolecules in intercellular communication and potentially possessing intrinsic therapeutic activity. Due to their stability, low immunogenicity, and inherent interaction with recipient cells, EVs also hold great promise as drug delivery vehicles. Indeed, they have been used to deliver nucleic acids, proteins, and small molecules in preclinical investigations. Furthermore, EV-based drugs have entered early clinical trials for cancer or neurodegenerative diseases. Despite their appeal as delivery vectors, however, EV-based drug delivery progress has been hampered by heterogeneity of sample types and methods as well as a persistent lack of standardization, validation, and comprehensive reporting. This review highlights specific requirements for EVs in drug delivery and describes the most pertinent approaches for separation and characterization. Despite residual uncertainties related to pharmacodynamics, pharmacokinetics, and potential off-target effects, clinical-grade, high-potency EV drugs might be achievable through GMP-compliant workflows in a highly standardized environment.
| S-EPMC8217305 | biostudies-literature
Project description:The term 'extracellular vesicles' refers to a heterogeneous population of vesicular bodies of cellular origin that derive either from the endosomal compartment (exosomes) or as a result of shedding from the plasma membrane (microvesicles, oncosomes and apoptotic bodies). Extracellular vesicles carry a variety of cargo, including RNAs, proteins, lipids and DNA, which can be taken up by other cells, both in the direct vicinity of the source cell and at distant sites in the body via biofluids, and elicit a variety of phenotypic responses. Owing to their unique biology and roles in cell-cell communication, extracellular vesicles have attracted strong interest, which is further enhanced by their potential clinical utility. Because extracellular vesicles derive their cargo from the contents of the cells that produce them, they are attractive sources of biomarkers for a variety of diseases. Furthermore, studies demonstrating phenotypic effects of specific extracellular vesicle-associated cargo on target cells have stoked interest in extracellular vesicles as therapeutic vehicles. There is particularly strong evidence that the RNA cargo of extracellular vesicles can alter recipient cell gene expression and function. During the past decade, extracellular vesicles and their RNA cargo have become better defined, but many aspects of extracellular vesicle biology remain to be elucidated. These include selective cargo loading resulting in substantial differences between the composition of extracellular vesicles and source cells; heterogeneity in extracellular vesicle size and composition; and undefined mechanisms for the uptake of extracellular vesicles into recipient cells and the fates of their cargo. Further progress in unravelling the basic mechanisms of extracellular vesicle biogenesis, transport, and cargo delivery and function is needed for successful clinical implementation. This Review focuses on the current state of knowledge pertaining to packaging, transport and function of RNAs in extracellular vesicles and outlines the progress made thus far towards their clinical applications.
| S-EPMC7249041 | biostudies-literature
Project description:The CRISPR (clustered regularly interspaced short palindromic repeats)/Cas9 genome editing system has been a major technological breakthrough that has brought revolutionary changes to genome editing for therapeutic and diagnostic purposes and precision medicine. With the advent of the CRISPR/Cas9 system, one of the critical limiting factors has been the safe and efficient delivery of this system to cells or tissues of interest. Several approaches have been investigated to find delivery systems that can attain tissue-targeted delivery, lowering the chances of off-target editing. While viral vectors have shown promise for in vitro, in vivo and ex vivo delivery of CRISPR/Cas9, their further clinical applications have been restricted due to shortcomings including limited cargo packaging capacity, difficulties with large-scale production, immunogenicity and insertional mutagenesis. Rapid progress in nonviral delivery vectors, including the use of lipid, polymer, peptides, and inorganic nanoparticle-based delivery systems, has established nonviral delivery approaches as a viable alternative to viral vectors. This review will introduce the molecular mechanisms of the CRISPR/Cas9 gene editing system, current strategies for delivering CRISPR/Cas9-based tools, an overview of strategies for overcoming off-target genome editing, and approaches for improving genome targeting and tissue targeting. We will also highlight current developments and recent clinical trials for the delivery of CRISPR/Cas9. Finally, future directions for overcoming the limitations and adaptation of this technology for clinical trials will be discussed.
| S-EPMC10052255 | biostudies-literature
Project description:Globally, approximately 1 in 4 cancers in women are diagnosed as breast cancer (BC). Despite significant advances in the diagnosis and therapy BCs, many patients develop metastases or relapses. Hence, novel therapeutic strategies are required, that can selectively and efficiently kill malignant cells. Direct targeting of the genetic and epigenetic aberrations that occur in BC development is a promising strategy to overcome the limitations of current therapies, which target the tumour phenotype. The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas system, composed of only an easily modifiable single guide RNA (sgRNA) sequence bound to a Cas9 nuclease, has revolutionised genome editing due to its simplicity and efficiency compared to earlier systems. CRISPR/Cas9 and its associated catalytically inactivated dCas9 variants facilitate the knockout of overexpressed genes, correction of mutations in inactivated genes, and reprogramming of the epigenetic landscape to impair BC growth. To achieve efficient genome editing in vivo, a vector is required to deliver the components to target cells. Gold nanomaterials, including gold nanoparticles and nanoclusters, display many advantageous characteristics that have facilitated their widespread use in theranostics, as delivery vehicles, and imaging and photothermal agents. This review highlights the therapeutic applications of CRISPR/Cas9 in treating BCs, and briefly describes gold nanomaterials and their potential in CRISPR/Cas9 delivery.
| S-EPMC7768851 | biostudies-literature
Project description:Extracellular vesicles (EVs) hold great promise for transporting CRISPR-Cas9 RNA-guided endonucleases (RNP) throughout the body. However, the cell-selective delivery of EVs is still a challenge. Here, we designed valency-controlled tetrahedral DNA nanostructures (TDNs) conjugated with DNA aptamer, and loaded the valency-controlled TDNs on EV surface via cholesterol anchoring for specific cell targeting. The targeting efficacy of different ratios of aptamer/cholesterol from 1:3 to 3:1 in TDNs on decorating EVs was investigated. TDNs with one aptamer and three cholesterol anchors (TDN1) efficiently facilitated the tumor-specific accumulation of the EVs in cultured HepG2 cells and human primary liver cancer-derived organoids, as well as xenograft tumor models. The intracellular delivery of RNP by TDN1-EVs successfully realized its subsequent genome editing, leading to the downregulation of GFP or WNT10B in specific cells. This system was ultimately applied to reduce the protein expression of WNT10B, which presented remarkable tumor growth inhibition in vitro, ex vivo and in vivo, and could be extended to other therapeutic targets. The present study provides a platform for the directional display of aptamer on surface labeling and the EVs-based Cas9 delivery, which provides a meaningful idea for future cell-selective gene editing.
| S-EPMC7498310 | biostudies-literature