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Rani N, Kumari K, Hooda V. The role of nanoparticles in transforming plant genetic engineering: advancements, challenges and future prospects. Funct Integr Genomics 2025; 25:23. [PMID: 39841261 DOI: 10.1007/s10142-025-01528-x] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/02/2024] [Revised: 12/26/2024] [Accepted: 01/03/2025] [Indexed: 01/23/2025]
Abstract
Despite years of progress in biotechnology, altering the genetic makeup of many plant species, especially their plastids, remains challenging. The existence of a cell wall poses a significant obstacle to the effectual transportation of biomolecules. Developing efficient methods to introduce genes into plant cells and organelles without causing harm is an ongoing area of research. Traditional approaches like Agrobacterium-mediated transformation, biolistic particle delivery, electroporation and polyethylene glycol (PEG) transformation have shown some success but come with limitations like laborious, time-consuming and causing tissue damage. For instance, the Agrobacterium method can be applied only to the restricted host range, while PEG transformation and biolistic particle delivery are not very efficient. In contrast, nanotechnology made an appearance in the field of genetic engineering. Nanoparticles act as delivery vehicles for many cargos in animals. However, in plants, the application of nanocarriers for the delivery of biomolecules is still in its infant stage. Nonetheless, it holds immense potential for the future of plant biotechnology and genome editing.
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Affiliation(s)
- Neelam Rani
- Department of Botany, Maharshi Dayanand University, Rohtak, 124001, India
| | - Kusum Kumari
- Department of Botany, Maharshi Dayanand University, Rohtak, 124001, India
| | - Vinita Hooda
- Department of Botany, Maharshi Dayanand University, Rohtak, 124001, India.
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2
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Zhu J, Lee H, Huang R, Zhou J, Zhang J, Yang X, Zhou W, Jiang W, Chen S. Harnessing nanotechnology for cancer treatment. Front Bioeng Biotechnol 2025; 12:1514890. [PMID: 39902172 PMCID: PMC11788409 DOI: 10.3389/fbioe.2024.1514890] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/21/2024] [Accepted: 12/30/2024] [Indexed: 02/05/2025] Open
Abstract
Nanotechnology has become a groundbreaking innovation force in cancer therapy, offering innovative solutions to the limitations of conventional treatments such as chemotherapy and radiation. By manipulating materials at the nanoscale, researchers have developed nanocarriers capable of targeted drug delivery, improving therapeutic efficacy while reducing systemic toxicity. Nanoparticles like liposomes, dendrimers, and polymeric nanomaterials have shown significant promise in delivering chemotherapeutic agents directly to tumor sites, enhancing drug bioavailability and minimizing damage to healthy tissues. In addition to drug delivery, with the utilization of tools such as quantum dots and nanosensors that enables more precise identification of cancer biomarkers, nanotechnology is also playing a pivotal role in early cancer detection and diagnosis. Furthermore, nanotechnology-based therapeutic strategies, including photothermal therapy, gene therapy and immunotherapy are offering novel ways to combat cancer by selectively targeting tumor cells and enhancing the immune response. Nevertheless, despite these progressions, obstacles still persist, particularly in the clinical translation of these technologies. Issues such as nanoparticle toxicity, biocompatibility, and the complexity of regulatory approval hinder the widespread adoption of nanomedicine in oncology. This review discusses different applications of nanotechnology in cancer therapy, highlighting its potential and the hurdles to its clinical implementation. Future research needs to concentrate on addressing these obstacles to unlock the full potential of nanotechnology in providing personalized, effective, and minimally invasive cancer treatments.
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Affiliation(s)
- Jiajun Zhu
- Department of Laboratory Medicine, Huashan Hospital, Fudan University, Shanghai, China
- Shanghai Medical College, Fudan University, Shanghai, China
| | - HaeJu Lee
- Department of Laboratory Medicine, Huashan Hospital, Fudan University, Shanghai, China
- Shanghai Medical College, Fudan University, Shanghai, China
| | - Ruotong Huang
- Department of Laboratory Medicine, Huashan Hospital, Fudan University, Shanghai, China
- Shanghai Medical College, Fudan University, Shanghai, China
| | - Jianming Zhou
- Shanghai Medical College, Fudan University, Shanghai, China
| | - Jingjun Zhang
- Department of Rehabilitation Medicine, The Sixth People’s Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai, China
| | - Xiaoyi Yang
- Shanghai Medical College, Fudan University, Shanghai, China
| | - Wenhan Zhou
- Shanghai Medical College, Fudan University, Shanghai, China
| | - Wangqing Jiang
- Department of Laboratory Medicine, Huashan Hospital, Fudan University, Shanghai, China
| | - Shuying Chen
- Department of Laboratory Medicine, Huashan Hospital, Fudan University, Shanghai, China
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3
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Fatima H, Singh D, Muhammad H, Acharya S, Aziz MA. Improving the use of CRISPR/Cas9 gene editing machinery as a cancer therapeutic tool with the help of nanomedicine. 3 Biotech 2025; 15:17. [PMID: 39711922 PMCID: PMC11656010 DOI: 10.1007/s13205-024-04186-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/02/2024] [Accepted: 11/22/2024] [Indexed: 12/24/2024] Open
Abstract
CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-associated protein 9) has revolutionized gene editing tools and paved the way for innovations in medical research for disease diagnosis and treatment. However, better specificity and efficient delivery of this gene machinery make it challenging to successfully edit genes for treating various diseases. This is mainly due to cellular barriers, instability in biological environments, and various off-target effects that prohibit safe and efficient delivery under in vivo conditions. This review examines several delivery modes [plasmid, mRNA, RNP (ribonucleoprotein)] and methods for the CRISPR-Cas9 system delivery, focusing on its potential applications in cancer therapy. Biocompatibility and cytotoxicity are crucial factors determining their safe and effective use. Various nanomaterials have been reviewed for their biocompatibility, limitations, and challenges in treating cancer. Among the reviewed nanoparticles, lipid nanoparticles (LNPs) stand out for their biocompatibility due to their biomimetic lipid bilayer that effectively delivers CRISPR/Cas9 cargoes while reducing toxicity. We discuss challenges in in vivo delivery and associated findings such as encapsulation, target delivery, controlled release, and endosomal escape. Future directions involve addressing limitations and adapting CRISPR-Cas9 for clinical trials, ensuring its safe and effective use.
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Affiliation(s)
- Hina Fatima
- Polymer and Process Engineering Department, Indian Institute of Technology Roorkee, Uttarakhand, 247001 India
- College of Medicine, Alfaisal University, 11533 Riyadh, Saudi Arabia
| | - Dimple Singh
- Department of Paper Technology, Indian Institute of Technology, Roorkee, Uttarakhand 247001 India
| | - Huzaifa Muhammad
- College of Medicine, Alfaisal University, 11533 Riyadh, Saudi Arabia
| | - Swati Acharya
- Cancer Nanomedicine Lab, Interdisciplinary Nanotechnology Center, Aligarh Muslim University, Aligarh, UP 202002 India
| | - Mohammad Azhar Aziz
- Cancer Nanomedicine Lab, Interdisciplinary Nanotechnology Center, Aligarh Muslim University, Aligarh, UP 202002 India
- Cancer Nanomedicine Consortium, Aligarh Muslim University, Aligarh, UP 202002 India
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Guzman Gonzalez V, Grunenberger A, Nicoud O, Czuba E, Vollaire J, Josserand V, Le Guével X, Desai N, Coll JL, Divita G, Faure V. Enhanced CRISPR-Cas9 RNA system delivery using cell penetrating peptides-based nanoparticles for efficient in vitro and in vivo applications. J Control Release 2024; 376:1160-1175. [PMID: 39521064 DOI: 10.1016/j.jconrel.2024.11.008] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/27/2024] [Revised: 10/31/2024] [Accepted: 11/05/2024] [Indexed: 11/16/2024]
Abstract
CRISPR-Cas9 system has emerged as a revolutionary gene-editing tool with huge therapeutic potential for addressing the underlying genetic causes of various diseases, including cancer. However, there are challenges such as the delivery method that must be overcome for its clinical application. In addition to the risk of nuclease degradation and rapid clearance of the CRISPR-Cas9 system by macrophages, the large size of Cas9, the high anionic charge density and hydrophilic nature of the RNA hinder their intracellular delivery and overall gene transfection efficiency. In this study, we engineered a novel Peptide-Based Nanoparticles ADGN for the delivery of long RNA. ADGN peptides can form stable self-assembled nanoparticles with CRISPR-Cas9 RNA. They have the ability to cross the cell membrane of various cell types, exhibiting a preference for cancer cells that overexpress laminin receptor and safeguard RNA prior their delivery into the cytoplasm. We demonstrate that ADGN peptides significantly promote CRISPR-Cas9 mediated knockout of the luciferase gene in vitro achieving 60 % efficiency with a preference for G insertion at the targeted site of luciferase gene. Moreover, we have provided evidence that these nanoparticles can also be systemically intravenously administrated in vivo in mice to deliver a functional CRISPR-Cas9 system to tumoral lung cells orthotopically implanted in the mouse, resulting in an effective gene knockout in mice. We also demonstrated that the in vivo distribution of ADGN-RNA is influenced by its peptides to RNA molar ratio. This study introduces a promising new Peptide-Based Nanoparticles for delivering CRISPR-Cas9 system in its RNA form applicable in both in vitro and in vivo models.
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Affiliation(s)
- Veronica Guzman Gonzalez
- University Grenoble Alpes, INSERM U 1209, CNRS UMR 5309, Cancer Targets and Experimental Therapeutics Team, Institute for Advanced Biosciences, 38000 Grenoble, France.; DivinCell SAS, Nimes, France
| | | | - Olivier Nicoud
- University Grenoble Alpes, INSERM U 1209, CNRS UMR 5309, Cancer Targets and Experimental Therapeutics Team, Institute for Advanced Biosciences, 38000 Grenoble, France
| | | | - Julien Vollaire
- University Grenoble Alpes, INSERM U 1209, CNRS UMR 5309, Cancer Targets and Experimental Therapeutics Team, Institute for Advanced Biosciences, 38000 Grenoble, France
| | - Véronique Josserand
- University Grenoble Alpes, INSERM U 1209, CNRS UMR 5309, Cancer Targets and Experimental Therapeutics Team, Institute for Advanced Biosciences, 38000 Grenoble, France
| | - Xavier Le Guével
- University Grenoble Alpes, INSERM U 1209, CNRS UMR 5309, Cancer Targets and Experimental Therapeutics Team, Institute for Advanced Biosciences, 38000 Grenoble, France
| | - Nail Desai
- Aadigen, LLC, Pacific Palisades, CA, USA
| | - Jean-Luc Coll
- University Grenoble Alpes, INSERM U 1209, CNRS UMR 5309, Cancer Targets and Experimental Therapeutics Team, Institute for Advanced Biosciences, 38000 Grenoble, France
| | - Gilles Divita
- DivinCell SAS, Nimes, France; Aadigen, LLC, Pacific Palisades, CA, USA
| | - Virginie Faure
- University Grenoble Alpes, INSERM U 1209, CNRS UMR 5309, Cancer Targets and Experimental Therapeutics Team, Institute for Advanced Biosciences, 38000 Grenoble, France..
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Goolab S, Scholefield J. Making gene editing accessible in resource limited environments: recommendations to guide a first-time user. Front Genome Ed 2024; 6:1464531. [PMID: 39386178 PMCID: PMC11461239 DOI: 10.3389/fgeed.2024.1464531] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/14/2024] [Accepted: 09/05/2024] [Indexed: 10/12/2024] Open
Abstract
The designer nuclease, CRISPR-Cas9 system has advanced the field of genome engineering owing to its programmability and ease of use. The application of these molecular scissors for genome engineering earned the developing researchers the Nobel prize in Chemistry in the year 2020. At present, the potential of this technology to improve global challenges continues to grow exponentially. CRISPR-Cas9 shows promise in the recent advances made in the Global North such as the FDA-approved gene therapy for the treatment of sickle cell anaemia and β-thalassemia and the gene editing of porcine kidney for xenotransplantation into humans affected by end-stage kidney failure. Limited resources, low government investment with an allocation of 1% of gross domestic production to research and development including a shortage of skilled professionals and lack of knowledge may preclude the use of this revolutionary technology in the Global South where the countries involved have reduced science and technology budgets. Focusing on the practical application of genome engineering, successful genetic manipulation is not easily accomplishable and is influenced by the chromatin landscape of the target locus, guide RNA selection, the experimental design including the profiling of the gene edited cells, which impacts the overall outcome achieved. Our assessment primarily delves into economical approaches of performing efficient genome engineering to support the first-time user restricted by limited resources with the aim of democratizing the use of the technology across low- and middle-income countries. Here we provide a comprehensive overview on existing experimental techniques, the significance for target locus analysis and current pitfalls such as the underrepresentation of global genetic diversity. Several perspectives of genome engineering approaches are outlined, which can be adopted in a resource limited setting to enable a higher success rate of genome editing-based innovations in low- and middle-income countries.
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Affiliation(s)
- Shivani Goolab
- Bioengineering and Integrated Genomics Group, Future Production Chemicals Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa
| | - Janine Scholefield
- Bioengineering and Integrated Genomics Group, Future Production Chemicals Cluster, Council for Scientific and Industrial Research, Pretoria, South Africa
- Department of Human Biology, Faculty of Health Sciences, University of Cape Town, Cape Town, South Africa
- Division of Human Genetics, Faculty of Health Sciences, University of the Witwatersrand, Johannesburg, South Africa
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6
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Kim M, Hwang Y, Lim S, Jang HK, Kim HO. Advances in Nanoparticles as Non-Viral Vectors for Efficient Delivery of CRISPR/Cas9. Pharmaceutics 2024; 16:1197. [PMID: 39339233 PMCID: PMC11434874 DOI: 10.3390/pharmaceutics16091197] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/16/2024] [Revised: 09/05/2024] [Accepted: 09/09/2024] [Indexed: 09/30/2024] Open
Abstract
The clustered regularly interspaced short palindromic repeat (CRISPR)/Cas9 system is a gene-editing technology. Nanoparticle delivery systems have attracted attention because of the limitations of conventional viral vectors. In this review, we assess the efficiency of various nanoparticles, including lipid-based, polymer-based, inorganic, and extracellular vesicle-based systems, as non-viral vectors for CRISPR/Cas9 delivery. We discuss their advantages, limitations, and current challenges. By summarizing recent advancements and highlighting key strategies, this review aims to provide a comprehensive overview of the role of non-viral delivery systems in advancing CRISPR/Cas9 technology for clinical applications and gene therapy.
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Affiliation(s)
- Minse Kim
- Division of Chemical Engineering and Bioengineering, College of Art, Culture and Engineering, Kangwon National University, Chuncheon 24341, Republic of Korea
- Department of Smart Health Science and Technology, Kangwon National University, Chuncheon 24341, Republic of Korea
| | - Youngwoo Hwang
- Department of Smart Health Science and Technology, Kangwon National University, Chuncheon 24341, Republic of Korea
- Department of Systems Immunology, Division of Biomedical Convergence, College of Biomedical Science, Kangwon National University, Chuncheon 24341, Republic of Korea
| | - Seongyu Lim
- Division of Chemical Engineering and Bioengineering, College of Art, Culture and Engineering, Kangwon National University, Chuncheon 24341, Republic of Korea
- Department of Smart Health Science and Technology, Kangwon National University, Chuncheon 24341, Republic of Korea
| | - Hyeon-Ki Jang
- Division of Chemical Engineering and Bioengineering, College of Art, Culture and Engineering, Kangwon National University, Chuncheon 24341, Republic of Korea
- Department of Smart Health Science and Technology, Kangwon National University, Chuncheon 24341, Republic of Korea
- Multidimensional Genomics Research Center, Kangwon National University, Chuncheon 24341, Republic of Korea
| | - Hyun-Ouk Kim
- Division of Chemical Engineering and Bioengineering, College of Art, Culture and Engineering, Kangwon National University, Chuncheon 24341, Republic of Korea
- Department of Smart Health Science and Technology, Kangwon National University, Chuncheon 24341, Republic of Korea
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Chaiyawat P, Sangkhathat S, Chiangjong W, Wongtrakoongate P, Hongeng S, Pruksakorn D, Chutipongtanate S. Targeting pediatric solid tumors in the new era of RNA therapeutics. Crit Rev Oncol Hematol 2024; 200:104406. [PMID: 38834094 DOI: 10.1016/j.critrevonc.2024.104406] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/31/2023] [Revised: 04/26/2024] [Accepted: 05/29/2024] [Indexed: 06/06/2024] Open
Abstract
Despite substantial progress in pediatric cancer treatment, poor prognosis remained for patients with recurrent or metastatic disease, given the limitations of approved targeted treatments and immunotherapies. RNA therapeutics offer significant potential for addressing a broad spectrum of diseases, including cancer. Advances in manufacturing and delivery systems are paving the way for the rapid development of therapeutic RNAs for clinical applications. This review summarizes therapeutic RNA classifications and the mechanisms of action, highlighting their potential in manipulating major cancer-related pathways and biological effects. We also focus on the pre-clinical investigation of RNA molecules with efficient delivery systems for their therapeutic potential targeting pediatric solid tumors.
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Affiliation(s)
- Parunya Chaiyawat
- Musculoskeletal Science and Translational Research Center, Department of Orthopedics, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; Center of Multidisciplinary Technology for Advanced Medicine (CMUTEAM), Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; Extracellular Vesicle Working Group, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
| | - Surasak Sangkhathat
- Department of Biomedical Science, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand; Department of Surgery, Faculty of Medicine, Prince of Songkla University, Songkhla, Thailand; Extracellular Vesicle Working Group, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
| | - Wararat Chiangjong
- Pediatric Translational Research Unit, Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; Extracellular Vesicle Working Group, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
| | - Patompon Wongtrakoongate
- Department of Biochemistry, Faculty of Science, Mahidol University, Bangkok 10400, Thailand; Extracellular Vesicle Working Group, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
| | - Suradej Hongeng
- Division of Hematology and Oncology, Department of Pediatrics, Faculty of Medicine Ra-mathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; Extracellular Vesicle Working Group, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA
| | - Dumnoensun Pruksakorn
- Musculoskeletal Science and Translational Research Center, Department of Orthopedics, Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; Center of Multidisciplinary Technology for Advanced Medicine (CMUTEAM), Faculty of Medicine, Chiang Mai University, Chiang Mai 50200, Thailand; Extracellular Vesicle Working Group, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
| | - Somchai Chutipongtanate
- Pediatric Translational Research Unit, Department of Pediatrics, Faculty of Medicine Ramathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; Division of Hematology and Oncology, Department of Pediatrics, Faculty of Medicine Ra-mathibodi Hospital, Mahidol University, Bangkok 10400, Thailand; MILCH and Novel Therapeutics Lab, Division of Epidemiology, Department of Environmental and Public Health Sciences, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA; Extracellular Vesicle Working Group, University of Cincinnati College of Medicine, Cincinnati, OH 45267, USA.
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Qu S, Yi C, Zhao Q, Ni Y, Ouyang S, Qi H, Cheng GJ, Zhang Y. Single-Cell Synchro-Subtractive-Additive Nanoscale Surgery with Femtosecond Lasers. NANO LETTERS 2024; 24:8801-8808. [PMID: 38989671 DOI: 10.1021/acs.nanolett.4c00970] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 07/12/2024]
Abstract
Herein, an in situ "synchro-subtractive-additive" technique of femtosecond laser single-cell surgery (FLSS) is presented to address the inadequacies of existing surgical methods for single-cell manipulation. This process is enabled by synchronized nanoscale three-dimensional (3D) subtractive and additive manufacturing with ultrahigh precision on various parts of the cells, in that the precise removal and modification of a single-cell structure are realized by nonthermal ablation, with synchronously ultrafast solidification of the specially designed hydrogel by two photopolymerizations. FLSS is a minimally invasive technique with a post-operative survival rate of 70% and stable proliferation. It opens avenues for bottom-up synthetic biology, offering new methods for artificially synthesizing organelle-like 3D structures and modifying the physiological activities of cells.
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Affiliation(s)
- Shuyuan Qu
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan 430079, China
- Medical Research Institute, School of Medicine, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan 430071, China
| | - Chenqi Yi
- Institute of Technological Sciences, Wuhan University, Wuhan 430072, China
| | - Qin Zhao
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan 430079, China
- Medical Research Institute, School of Medicine, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan 430071, China
| | - Yueqi Ni
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan 430079, China
- Medical Research Institute, School of Medicine, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan 430071, China
| | - Simin Ouyang
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan 430079, China
- Medical Research Institute, School of Medicine, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan 430071, China
| | - Haoning Qi
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan 430079, China
- Medical Research Institute, School of Medicine, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan 430071, China
| | - Gary J Cheng
- School of Industrial Engineering, Purdue University, West Lafayette, Indiana 47906, United States
- School of Materials Engineering, Purdue University, West Lafayette, Indiana 47906, United States
| | - Yufeng Zhang
- State Key Laboratory of Oral & Maxillofacial Reconstruction and Regeneration, Key Laboratory of Oral Biomedicine Ministry of Education, Hubei Key Laboratory of Stomatology, School & Hospital of Stomatology, Taikang Center for Life and Medical Sciences, Wuhan University, Wuhan 430079, China
- Medical Research Institute, School of Medicine, Frontier Science Center for Immunology and Metabolism, Wuhan University, Wuhan 430071, China
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Taghdiri M, Mussolino C. Viral and Non-Viral Systems to Deliver Gene Therapeutics to Clinical Targets. Int J Mol Sci 2024; 25:7333. [PMID: 39000440 PMCID: PMC11242246 DOI: 10.3390/ijms25137333] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/29/2024] [Revised: 06/10/2024] [Accepted: 06/24/2024] [Indexed: 07/16/2024] Open
Abstract
Clustered regularly interspersed short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) technology has revolutionized the field of gene therapy as it has enabled precise genome editing with unprecedented accuracy and efficiency, paving the way for clinical applications to treat otherwise incurable genetic disorders. Typically, precise genome editing requires the delivery of multiple components to the target cells that, depending on the editing platform used, may include messenger RNA (mRNA), protein complexes, and DNA fragments. For clinical purposes, these have to be efficiently delivered into transplantable cells, such as primary T lymphocytes or hematopoietic stem and progenitor cells that are typically sensitive to exogenous substances. This challenge has limited the broad applicability of precise gene therapy applications to those strategies for which efficient delivery methods are available. Electroporation-based methodologies have been generally applied for gene editing applications, but procedure-associated toxicity has represented a major burden. With the advent of novel and less disruptive methodologies to deliver genetic cargo to transplantable cells, it is now possible to safely and efficiently deliver multiple components for precise genome editing, thus expanding the applicability of these strategies. In this review, we describe the different delivery systems available for genome editing components, including viral and non-viral systems, highlighting their advantages, limitations, and recent clinical applications. Recent improvements to these delivery methods to achieve cell specificity represent a critical development that may enable in vivo targeting in the future and will certainly play a pivotal role in the gene therapy field.
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Affiliation(s)
- Maryam Taghdiri
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, 79106 Freiburg, Germany
- Ph.D. Program, Faculty of Biology, University of Freiburg, 79106 Freiburg, Germany
| | - Claudio Mussolino
- Institute for Transfusion Medicine and Gene Therapy, Medical Center-University of Freiburg, 79106 Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Medical Center-University of Freiburg, 79106 Freiburg, Germany
- Faculty of Medicine, University of Freiburg, 79106 Freiburg, Germany
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10
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Khoshandam M, Soltaninejad H, Hamidieh AA, Hosseinkhani S. CRISPR, CAR-T, and NK: Current applications and future perspectives. Genes Dis 2024; 11:101121. [PMID: 38545126 PMCID: PMC10966184 DOI: 10.1016/j.gendis.2023.101121] [Citation(s) in RCA: 1] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/02/2022] [Accepted: 08/16/2023] [Indexed: 11/11/2024] Open
Abstract
Chimeric antigen receptor T (CAR-T) cell therapy represents a breakthrough in personalized cancer treatments. In this regard, synthetic receptors comprised of antigen recognition domains, signaling, and stimulatory domains are used to reprogram T-cells to target tum or cells and destroy them. Despite the success of this approach in refractory B-cell malignancies, the optimal potency of CAR T-cell therapy for many other cancers, particularly solid tumors, has not been validated. Natural killer cells are powerful cytotoxic lymphocytes specialized in recognizing and dispensing the tumor cells in coordination with other anti-tumor immunity cells. Based on these studies, many investigations are focused on the accurate designing of CAR T-cells with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system or other novel gene editing tools that can induce hereditary changes with or without the presence of a double-stranded break into the genome. These methodologies can be specifically focused on negative controllers of T-cells, induce modifications to a particular gene, and produce reproducible, safe, and powerful allogeneic CAR T-cells for on-demand cancer immunotherapy. The improvement of the CRISPR/Cas9 innovation offers an adaptable and proficient gene-editing capability in activating different pathways to help natural killer cells interact with novel CARs to particularly target tumor cells. Novel achievements and future challenges of combining next-generation CRISPR-Cas9 gene editing tools to optimize CAR T-cell and natural killer cell treatment for future clinical trials toward the foundation of modern cancer treatments have been assessed in this review.
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Affiliation(s)
- Mohadeseh Khoshandam
- Department of Reproductive Biology, Academic Center for Education, Culture, and Research (ACECR), Qom branch 3716986466, Iran
- National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 14965/161, Iran
| | - Hossein Soltaninejad
- Department of stem cells technology and Tissue Regeneration, Faculty of Interdisciplinary Science and Technologies, Tarbiat Modares University, Tehran 15614, Iran
- Pediatric Cell Therapy and Gene Therapy Research Center, Gene, Cell & Tissue Research Institute, Tehran University of Medical Sciences, Tehran 1417935840, Iran
| | - Amir Ali Hamidieh
- Pediatric Cell Therapy and Gene Therapy Research Center, Gene, Cell & Tissue Research Institute, Tehran University of Medical Sciences, Tehran 1417935840, Iran
| | - Saman Hosseinkhani
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 15614, Iran
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11
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Allemailem KS, Almatroudi A, Rahmani AH, Alrumaihi F, Alradhi AE, Alsubaiyel AM, Algahtani M, Almousa RM, Mahzari A, Sindi AAA, Dobie G, Khan AA. Recent Updates of the CRISPR/Cas9 Genome Editing System: Novel Approaches to Regulate Its Spatiotemporal Control by Genetic and Physicochemical Strategies. Int J Nanomedicine 2024; 19:5335-5363. [PMID: 38859956 PMCID: PMC11164216 DOI: 10.2147/ijn.s455574] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/29/2023] [Accepted: 05/30/2024] [Indexed: 06/12/2024] Open
Abstract
The genome editing approach by clustered regularly interspaced short palindromic repeats (CRISPR)/associated protein 9 (CRISPR/Cas9) is a revolutionary advancement in genetic engineering. Owing to its simple design and powerful genome-editing capability, it offers a promising strategy for the treatment of different infectious, metabolic, and genetic diseases. The crystal structure of Streptococcus pyogenes Cas9 (SpCas9) in complex with sgRNA and its target DNA at 2.5 Å resolution reveals a groove accommodating sgRNA:DNA heteroduplex within a bilobate architecture with target recognition (REC) and nuclease (NUC) domains. The presence of a PAM is significantly required for target recognition, R-loop formation, and strand scission. Recently, the spatiotemporal control of CRISPR/Cas9 genome editing has been considerably improved by genetic, chemical, and physical regulatory strategies. The use of genetic modifiers anti-CRISPR proteins, cell-specific promoters, and histone acetyl transferases has uplifted the application of CRISPR/Cas9 as a future-generation genome editing tool. In addition, interventions by chemical control, small-molecule activators, oligonucleotide conjugates and bioresponsive delivery carriers have improved its application in other areas of biological fields. Furthermore, the intermediation of physical control by using heat-, light-, magnetism-, and ultrasound-responsive elements attached to this molecular tool has revolutionized genome editing further. These strategies significantly reduce CRISPR/Cas9's undesirable off-target effects. However, other undesirable effects still offer some challenges for comprehensive clinical translation using this genome-editing approach. In this review, we summarize recent advances in CRISPR/Cas9 structure, mechanistic action, and the role of small-molecule activators, inhibitors, promoters, and physical approaches. Finally, off-target measurement approaches, challenges, future prospects, and clinical applications are discussed.
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Affiliation(s)
- Khaled S Allemailem
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
| | - Ahmad Almatroudi
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
| | - Arshad Husain Rahmani
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
| | - Faris Alrumaihi
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
| | - Arwa Essa Alradhi
- General Administration for Infectious Disease Control, Ministry of Health, Riyadh 12382, Saudi Arabia
| | - Amal M Alsubaiyel
- Department of Pharmaceutics, College of Pharmacy, Qassim University, Buraydah 51452, Saudi Arabia
| | - Mohammad Algahtani
- Department of Laboratory & Blood Bank, Security Forces Hospital, Mecca 21955, Saudi Arabia
| | - Rand Mohammad Almousa
- Department of Education, General Directorate of Education, Qassim 52361, Saudi Arabia
| | - Ali Mahzari
- Department of Laboratory Medicine, Faculty of Applied Medical Sciences, Al-Baha University, Al-Baha 65527, Saudi Arabia
| | - Abdulmajeed A A Sindi
- Department of Basic Medical Sciences, Faculty of Applied Medical Sciences, Al-Baha University, Al-Baha 65527, Saudi Arabia
| | - Gasim Dobie
- Department of Medical Laboratory Technology, Faculty of Applied Medical Sciences, Jazan University, Gizan 82911, Saudi Arabia
| | - Amjad Ali Khan
- Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
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12
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Singh D. Revolutionizing Lung Cancer Treatment: Innovative CRISPR-Cas9 Delivery Strategies. AAPS PharmSciTech 2024; 25:129. [PMID: 38844700 DOI: 10.1208/s12249-024-02834-6] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/15/2024] [Accepted: 05/08/2024] [Indexed: 06/11/2024] Open
Abstract
Lung carcinoma, including both non-small cell lung cancer (NSCLC) and small cell lung cancer (SCLC), remains a significant global health challenge due to its high morbidity and mortality rates. The objsective of this review is to meticulously examine the current advancements and strategies in the delivery of CRISPR-Cas9 gene-editing technology for the treatment of lung carcinoma. This technology heralds a new era in molecular biology, offering unprecedented precision in genomic modifications. However, its therapeutic potential is contingent upon the development of effective delivery mechanisms that ensure the efficient and specific transport of gene-editing tools to tumor cells. We explore a variety of delivery approaches, such as viral vectors, lipid-based nanoparticles, and physical methods, highlighting their respective advantages, limitations, and recent breakthroughs. This review also delves into the translational and clinical significance of these strategies, discussing preclinical and clinical studies that investigate the feasibility, efficacy, and safety of CRISPR-Cas9 delivery for lung carcinoma. By scrutinizing the landscape of ongoing clinical trials and offering translational perspectives, we aim to elucidate the current state and future directions of this rapidly evolving field. The review is structured to first introduce the problem and significance of lung carcinoma, followed by an overview of CRISPR-Cas9 technology, a detailed examination of delivery strategies, and an analysis of clinical applications and regulatory considerations. Our discussion concludes with future perspectives and challenges, such as optimizing delivery strategies, enhancing specificity, mitigating immunogenicity concerns, and addressing regulatory issues. This comprehensive overview seeks to provide insights into the potential of CRISPR-Cas9 as a revolutionary approach for targeted therapies and personalized medicine in lung carcinoma, emphasizing the importance of delivery strategy development in realizing the full potential of this groundbreaking technology.
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Affiliation(s)
- Dilpreet Singh
- University Institute of Pharma Sciences, Chandigarh University, Gharuan, Mohali, 140413, India.
- University Centre for Research and Development, Chandigarh University, Gharuan, Mohali, 140413, India.
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13
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Lee JH, Han JP. In vivo LNP-CRISPR Approaches for the Treatment of Hemophilia. Mol Diagn Ther 2024; 28:239-248. [PMID: 38538969 PMCID: PMC11068834 DOI: 10.1007/s40291-024-00705-1] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 03/13/2024] [Indexed: 05/04/2024]
Abstract
Hemophilia is a genetic disorder that is caused by mutations in coagulation factor VIII (hemophilia A) or IX (hemophilia B) genes resulting in blood clotting disorders. Despite advances in therapies, such as recombinant proteins and products with extended half-lives, the treatment of hemophilia still faces two major limitations: the short duration of therapeutic effect and production of neutralizing antibodies against clotting factors (inhibitor). To overcome these limitations, new hemophilia treatment strategies have been established such as gene therapy, bispecific antibody, and rebalancing therapy. Although these strategies have shown promising results, it is difficult to achieve a permanent therapeutic effect. Advances in the clustered regularly interspaced short palindromic repeat (CRISPR) technology have allowed sustainable treatment by correcting mutated genes. Since genome editing generates irreversible changes in host genome, safety must be ensured by delivering target organs. Therefore, the delivery tool of the CRISPR system is crucial for safe, accurate, and efficient genome editing. Recently, non-viral vector lipid nanoparticles (LNPs) have emerged as safer tools for delivering CRISPR systems than other viral vectors. Several previous hemophilia pre-clinical studies using LNP-CRISPR showed that sufficient and sustainable therapeutic effects, which means that LNP-CRISPR-mediated genome-editing therapy can be a valid option for the treatment of hemophilia. In this paper, we summarize the latest advancements in the successful treatment of hemophilia and the potential of CRISPR-mediated genome-editing therapy using LNPs.
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Affiliation(s)
- Jeong Hyeon Lee
- Graduate School of International Agricultural Technology, Institute of Green BioScience and Technology, Seoul National University, 1447 Pyeongchang-ro, Daewha, Pyeongchang, 25354, Gangwon, Korea
| | - Jeong Pil Han
- Graduate School of International Agricultural Technology, Institute of Green BioScience and Technology, Seoul National University, 1447 Pyeongchang-ro, Daewha, Pyeongchang, 25354, Gangwon, Korea.
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14
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Xu Z. CRISPR/Cas9-mediated silencing of CD44: unveiling the role of hyaluronic acid-mediated interactions in cancer drug resistance. NAUNYN-SCHMIEDEBERG'S ARCHIVES OF PHARMACOLOGY 2024; 397:2849-2876. [PMID: 37991544 DOI: 10.1007/s00210-023-02840-8] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/13/2023] [Accepted: 11/07/2023] [Indexed: 11/23/2023]
Abstract
A comprehensive overview of CD44 (CD44 Molecule (Indian Blood Group)), a cell surface glycoprotein, and its interaction with hyaluronic acid (HA) in drug resistance mechanisms across various types of cancer is provided, where CRISPR/Cas9 gene editing was utilized to silence CD44 expression and examine its impact on cancer cell behavior, migration, invasion, proliferation, and drug sensitivity. The significance of the HA-CD44 axis in tumor microenvironment (TME) delivery and its implications in specific cancer types, the influence of CD44 variants and the KHDRBS3 (KH RNA Binding Domain Containing, Signal Transduction Associated 3) gene on cancer progression and drug resistance, and the potential of targeting HA-mediated pathways using CRISPR/Cas9 gene editing technology to overcome drug resistance in cancer were also highlighted.
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Affiliation(s)
- Zhujun Xu
- Wuhan No.1 Hospital, Wuhan, 430022, Hubei, China.
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15
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Guri-Lamce I, AlRokh Y, Kim Y, Maeshima R, Graham C, Hart SL, McGrath JA, Jacków-Malinowska J. Topical gene editing therapeutics using lipid nanoparticles: 'gene creams' for genetic skin diseases? Br J Dermatol 2024; 190:617-627. [PMID: 38149939 DOI: 10.1093/bjd/ljad528] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/03/2023] [Revised: 12/12/2023] [Accepted: 12/17/2023] [Indexed: 12/28/2023]
Abstract
Patients living with inherited skin diseases have benefited from recent advances in DNA sequencing technologies that provide new or improved diagnostics. However, developing and delivering new treatments for the 'genodermatoses' remains challenging. The goal of creating topical preparations that can recover the inherent gene pathology remains largely aspirational. However, recent progress in two fields - the chemistry of topical delivery formulations (lipid nanoparticles) and the molecular biology of gene repair (CRISPR-Cas9, base and prime editing) - presents new opportunities to address this unmet need. In this review, we discuss how lipid nanoparticle delivery vehicles could be used to deliver gene-editing tools to formulate topical 'gene creams' suitable for the treatment of genodermatoses. We summarize the historical landscape of topical therapeutics and advances in gene editing that may herald an era of new therapies for patients with inherited skin disorders.
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Affiliation(s)
- Ina Guri-Lamce
- St John's Institute of Dermatology, King's College London, London, UK
| | - Yara AlRokh
- St John's Institute of Dermatology, King's College London, London, UK
| | - Youngah Kim
- St John's Institute of Dermatology, King's College London, London, UK
| | - Ruhina Maeshima
- Genetics and Genomic Medicine Department, UCL Great Ormond Street Institute of Child Health, UCL, London, UK
| | - Carina Graham
- Genetics and Genomic Medicine Department, UCL Great Ormond Street Institute of Child Health, UCL, London, UK
| | - Stephen L Hart
- Genetics and Genomic Medicine Department, UCL Great Ormond Street Institute of Child Health, UCL, London, UK
| | - John A McGrath
- St John's Institute of Dermatology, King's College London, London, UK
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16
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Mariano CG, de Oliveira VC, Ambrósio CE. Gene editing in small and large animals for translational medicine: a review. Anim Reprod 2024; 21:e20230089. [PMID: 38628493 PMCID: PMC11019828 DOI: 10.1590/1984-3143-ar2023-0089] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/05/2023] [Accepted: 02/16/2024] [Indexed: 04/19/2024] Open
Abstract
The CRISPR/Cas9 system is a simpler and more versatile method compared to other engineered nucleases such as Zinc Finger Nucleases (ZFNs) and Transcription Activator-Like Effector Nucleases (TALENs), and since its discovery, the efficiency of CRISPR-based genome editing has increased to the point that multiple and different types of edits can be made simultaneously. These advances in gene editing have revolutionized biotechnology by enabling precise genome editing with greater simplicity and efficacy than ever before. This tool has been successfully applied to a wide range of animal species, including cattle, pigs, dogs, and other small animals. Engineered nucleases cut the genome at specific target positions, triggering the cell's mechanisms to repair the damage and introduce a mutation to a specific genomic site. This review discusses novel genome-based CRISPR/Cas9 editing tools, methods developed to improve efficiency and specificity, the use of gene-editing on animal models and translational medicine, and the main challenges and limitations of CRISPR-based gene-editing approaches.
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Affiliation(s)
- Clésio Gomes Mariano
- Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo – USP, Pirassununga, SP, Brasil
| | - Vanessa Cristina de Oliveira
- Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo – USP, Pirassununga, SP, Brasil
| | - Carlos Eduardo Ambrósio
- Departamento de Medicina Veterinária, Faculdade de Zootecnia e Engenharia de Alimentos, Universidade de São Paulo – USP, Pirassununga, SP, Brasil
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17
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Sun X, Hong J, Ding T, Wu Z, Lin D. Snail microbiota and snail-schistosome interactions: axenic and gnotobiotic technologies. Trends Parasitol 2024; 40:241-256. [PMID: 38278688 DOI: 10.1016/j.pt.2024.01.002] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/04/2023] [Revised: 01/04/2024] [Accepted: 01/04/2024] [Indexed: 01/28/2024]
Abstract
The microbiota in the intermediate snail hosts of human schistosomes can significantly affect host biology. For decades, researchers have developed axenic snails to manipulate the symbiotic microbiota. This review summarizes the characteristics of symbiotic microbes in intermediate snail hosts and describes their interactions with snails, affecting snail growth, development, and parasite transmission ability. We focus on advances in axenic and gnotobiotic technologies for studying snail-microbe interactions and exploring the role of microbiota in snail susceptibility to Schistosoma infection. We discuss the challenges related to axenic and gnotobiotic snails, possible solutions to address these challenges, and future research directions to deepen our understanding of snail-microbiota interactions, with the aim to develop microbiota-based strategies for controlling snail populations and reducing their competence in transmitting parasites.
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Affiliation(s)
- Xi Sun
- Department of Parasitology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China; Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-Sen University, Guangzhou, China; Provincial Engineering Technology Research Center for Diseases-vectors Control and Chinese Atomic Energy Agency Center of Excellence on Nuclear Technology Applications for Insect Control, Sun Yat-Sen University, Guangzhou, China
| | - Jinni Hong
- Department of Traditional Chinese Medicine, Guangdong Provincial People's Hospital, Guangdong Academy of Medical Sciences, Southern Medical University, Guangzhou, China
| | - Tao Ding
- Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-Sen University, Guangzhou, China; Provincial Engineering Technology Research Center for Diseases-vectors Control and Chinese Atomic Energy Agency Center of Excellence on Nuclear Technology Applications for Insect Control, Sun Yat-Sen University, Guangzhou, China
| | - Zhongdao Wu
- Department of Parasitology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China; Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-Sen University, Guangzhou, China; Provincial Engineering Technology Research Center for Diseases-vectors Control and Chinese Atomic Energy Agency Center of Excellence on Nuclear Technology Applications for Insect Control, Sun Yat-Sen University, Guangzhou, China.
| | - Datao Lin
- Department of Parasitology, Zhongshan School of Medicine, Sun Yat-Sen University, Guangzhou, China; Key Laboratory of Tropical Disease Control, Ministry of Education, Sun Yat-Sen University, Guangzhou, China; Provincial Engineering Technology Research Center for Diseases-vectors Control and Chinese Atomic Energy Agency Center of Excellence on Nuclear Technology Applications for Insect Control, Sun Yat-Sen University, Guangzhou, China.
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18
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Dziubańska-Kusibab PJ, Nevedomskaya E, Haendler B. Preclinical Anticipation of On- and Off-Target Resistance Mechanisms to Anti-Cancer Drugs: A Systematic Review. Int J Mol Sci 2024; 25:705. [PMID: 38255778 PMCID: PMC10815614 DOI: 10.3390/ijms25020705] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/14/2023] [Revised: 12/22/2023] [Accepted: 12/28/2023] [Indexed: 01/24/2024] Open
Abstract
The advent of targeted therapies has led to tremendous improvements in treatment options and their outcomes in the field of oncology. Yet, many cancers outsmart precision drugs by developing on-target or off-target resistance mechanisms. Gaining the ability to resist treatment is the rule rather than the exception in tumors, and it remains a major healthcare challenge to achieve long-lasting remission in most cancer patients. Here, we discuss emerging strategies that take advantage of innovative high-throughput screening technologies to anticipate on- and off-target resistance mechanisms before they occur in treated cancer patients. We divide the methods into non-systematic approaches, such as random mutagenesis or long-term drug treatment, and systematic approaches, relying on the clustered regularly interspaced short palindromic repeats (CRISPR) system, saturated mutagenesis, or computational methods. All these new developments, especially genome-wide CRISPR-based screening platforms, have significantly accelerated the processes for identification of the mechanisms responsible for cancer drug resistance and opened up new avenues for future treatments.
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Affiliation(s)
| | | | - Bernard Haendler
- Research and Early Development Oncology, Pharmaceuticals, Bayer AG, Müllerstr. 178, 13353 Berlin, Germany; (P.J.D.-K.); (E.N.)
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19
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van den Berg van Saparoea ACH, van Loosen QC, Sarno F, Ntini E, Rots MG, Gjaltema RAF, Verschure PJ. Plasmid Delivery and Single-Cell Plasmid Expression Analysis for CRISPR/dCas9-Based Epigenetic Editing. Methods Mol Biol 2024; 2842:255-265. [PMID: 39012600 DOI: 10.1007/978-1-0716-4051-7_13] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 07/17/2024]
Abstract
To fully exploit the potentials of reprogramming the epigenome through CRISPR/dCas9 systems for epigenetic editing, there is a growing need for improved transfection methods. With the utilization of constructs often with large sizes and the wide array of cell types used to read out the effect of epigenetic editing in different biological applications, it is evident that ongoing optimalization of transfection protocols tailored to each specific experimental setup is essential. Whether the goal is the production of viral particles using human embryonic kidney (HEK) cells or the direct examination of epigenomic modifications in the target cell type, continuous refinement of transfection methods is crucial. In the hereafter outlined protocol, we focus on optimization of transfection protocols by comparing different reagents and methods, creating a streamlined setup for transfection efficiency optimization in cultured mammalian cells. Our protocol provides a comprehensive overview of flow cytometry analysis following transfection not just to improve transfection efficiency but also to assess the expression level of the utilized construct. We showcase our transfection protocol optimization using HEK293T Lenti-X™ and breast cancer MCF-7 cell lines, using a single-guide RNA-containing plasmid. Specifically, we incorporate heat shock treatment for increased transfection efficiency of the MCF-7 cell line. Our detailed optimization protocol for efficient plasmid delivery and measurement of single-cell plasmid expression provides a comprehensive instruction for assessing both transient and sustained effects of epigenetic reprogramming.
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Affiliation(s)
| | - Quint C van Loosen
- Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands
| | - Federica Sarno
- Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Evgenia Ntini
- Institute of Molecular Biology and Biotechnology, FORTH, Heraklion, Greece
| | - Marianne G Rots
- Department of Pathology and Medical Biology, University of Groningen, University Medical Center Groningen, Groningen, the Netherlands
| | - Rutger A F Gjaltema
- Molecular & Cellular Epigenetics, Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands
| | - Pernette J Verschure
- Swammerdam Institute for Life Sciences, University of Amsterdam, Amsterdam, The Netherlands.
- Department of Medical Biochemistry, Amsterdam University Medical Center, Amsterdam, The Netherlands.
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20
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Munir A, Ali M, Qari SH, Munawar N, Saleem MS, Ahmad A. CRISPR workflow solutions: Cargos and versatile delivery platforms in genome editing. CRISPRIZED HORTICULTURE CROPS 2024:67-90. [DOI: 10.1016/b978-0-443-13229-2.00014-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 01/04/2025]
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21
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Khoshandam M, Soltaninejad H, Mousazadeh M, Hamidieh AA, Hosseinkhani S. Clinical applications of the CRISPR/Cas9 genome-editing system: Delivery options and challenges in precision medicine. Genes Dis 2024; 11:268-282. [PMID: 37588217 PMCID: PMC10425811 DOI: 10.1016/j.gendis.2023.02.027] [Citation(s) in RCA: 2] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/20/2022] [Accepted: 02/08/2023] [Indexed: 03/29/2023] Open
Abstract
CRISPR/Cas9 is an effective gene editing tool with broad applications for the prevention or treatment of numerous diseases. It depends on CRISPR (clustered regularly interspaced short palindromic repeats) as a bacterial immune system and plays as a gene editing tool. Due to the higher specificity and efficiency of CRISPR/Cas9 compared to other editing approaches, it has been broadly investigated to treat numerous hereditary and acquired illnesses, including cancers, hemolytic diseases, immunodeficiency disorders, cardiovascular diseases, visual maladies, neurodegenerative conditions, and a few X-linked disorders. CRISPR/Cas9 system has been used to treat cancers through a variety of approaches, with stable gene editing techniques. Here, the applications and clinical trials of CRISPR/Cas9 in various illnesses are described. Due to its high precision and efficiency, CRISPR/Cas9 strategies may treat gene-related illnesses by deleting, inserting, modifying, or blocking the expression of specific genes. The most challenging barrier to the in vivo use of CRISPR/Cas9 like off-target effects will be discussed. The use of transfection vehicles for CRISPR/Cas9, including viral vectors (such as an Adeno-associated virus (AAV)), and the development of non-viral vectors is also considered.
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Affiliation(s)
- Mohadeseh Khoshandam
- Department of Reproductive Biology, Academic Center for Education, Culture, and Research (ACECR), Qom Branch, Qom 3716986466, Iran
- National Institute of Genetic Engineering and Biotechnology (NIGEB), Tehran 14155-6463, Iran
| | - Hossein Soltaninejad
- Faculty of Interdisciplinary Science and Technology, Tarbiat Modares University, Tehran 14117-13116, Iran
- Pediatric Cell and Gene Therapy Research Center, Gene, Cell & Tissue Research Institute, Tehran University of Medical Sciences, Tehran 14155-6559, Iran
| | - Marziyeh Mousazadeh
- Department of Nanobiotechnology, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14117-13116, Iran
| | - Amir Ali Hamidieh
- Pediatric Cell and Gene Therapy Research Center, Gene, Cell & Tissue Research Institute, Tehran University of Medical Sciences, Tehran 14155-6559, Iran
| | - Saman Hosseinkhani
- Department of Biochemistry, Faculty of Biological Sciences, Tarbiat Modares University, Tehran 14117-13116, Iran
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22
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Kola NS, Patel D, Thakur A. RNA-Based Vaccines and Therapeutics Against Intracellular Pathogens. Methods Mol Biol 2024; 2813:321-370. [PMID: 38888787 DOI: 10.1007/978-1-0716-3890-3_21] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 06/20/2024]
Abstract
RNA-based vaccines have sparked a paradigm shift in the treatment and prevention of diseases by nucleic acid medicines. There has been a notable surge in the development of nucleic acid therapeutics and vaccines following the global approval of the two messenger RNA-based COVID-19 vaccines. This growth is fueled by the exploration of numerous RNA products in preclinical stages, offering several advantages over conventional methods, i.e., safety, efficacy, scalability, and cost-effectiveness. In this chapter, we provide an overview of various types of RNA and their mechanisms of action for stimulating immune responses and inducing therapeutic effects. Furthermore, this chapter delves into the varying delivery systems, particularly emphasizing the use of nanoparticles to deliver RNA. The choice of delivery system is an intricate process involved in developing nucleic acid medicines that significantly enhances their stability, biocompatibility, and site-specificity. Additionally, this chapter sheds light on the current landscape of clinical trials of RNA therapeutics and vaccines against intracellular pathogens.
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Affiliation(s)
- Naga Suresh Kola
- Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK, Canada
| | - Dhruv Patel
- Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK, Canada
| | - Aneesh Thakur
- Vaccine and Infectious Disease Organization, University of Saskatchewan, Saskatoon, SK, Canada.
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23
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Wal P, Aziz N, Prajapati H, Soni S, Wal A. Current Landscape of Various Techniques and Methods of Gene Therapy through CRISPR Cas9 along with its Pharmacological and Interventional Therapies in the Treatment of Type 2 Diabetes Mellitus. Curr Diabetes Rev 2024; 20:e201023222414. [PMID: 37867274 DOI: 10.2174/0115733998263079231011073803] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 05/19/2023] [Revised: 08/11/2023] [Accepted: 08/25/2023] [Indexed: 10/24/2023]
Abstract
BACKGROUND Type 2 diabetes mellitus (T2DM) is frequently referred to as a "lifestyle illness". In 2000, India (31.7 million) had the greatest global prevalence of diabetes mellitus, followed by China (20.8 million), the United States (17.7 million), and other countries. In recent years, the treatment of gene therapy (T2DM) has attracted intensive interest. OBJECTIVE We aimed to critically review the literature on the various techniques and methods, which may be a possible novel approach through the gene therapy CRISPR Cas9 and some other gene editing techniques for T2DM. Interventional and pharmacological approaches for the treatment of T2DM were also included to identify novel therapies for its treatment. METHOD An extensive literature survey was done on databases like PubMed, Elsevier, Science Direct and Springer. CONCLUSION It can be concluded from the study that recent advancements in gene-editing technologies, such as CRISPR Cas9, have opened new avenues for the development of novel therapeutic approaches for T2DM. CRISPR Cas9 is a powerful tool that enables precise and targeted modifications of the genome.
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Affiliation(s)
- Pranay Wal
- PSIT-Pranveer Singh Institute of Technology (Pharmacy), Bhauti, Kanpur, UP, 209305, India
| | - Namra Aziz
- PSIT-Pranveer Singh Institute of Technology (Pharmacy), Bhauti, Kanpur, UP, 209305, India
| | - Harshit Prajapati
- PSIT-Pranveer Singh Institute of Technology (Pharmacy), Bhauti, Kanpur, UP, 209305, India
| | - Shashank Soni
- Department of Pharmaceutics, Amity Institute of Pharmacy, Lucknow, Amity University, Uttar Pradesh, Sector 125, Noida, 201313, India
| | - Ankita Wal
- PSIT-Pranveer Singh Institute of Technology (Pharmacy), Bhauti, Kanpur, UP, 209305, India
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24
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Sharma A, Choudhary P, Chakdar H, Shukla P. Molecular insights and omics-based understanding of plant-microbe interactions under drought stress. World J Microbiol Biotechnol 2023; 40:42. [PMID: 38105277 DOI: 10.1007/s11274-023-03837-4] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/29/2023] [Accepted: 11/11/2023] [Indexed: 12/19/2023]
Abstract
The detrimental effects of adverse environmental conditions are always challenging and remain a major concern for plant development and production worldwide. Plants deal with such constraints by physiological, biochemical, and morphological adaptations as well as acquiring mutual support of beneficial microorganisms. As many stress-responsive traits of plants are influenced by microbial activities, plants have developed a sophisticated interaction with microbes to cope with adverse environmental conditions. The production of numerous bioactive metabolites by rhizospheric, endo-, or epiphytic microorganisms can directly or indirectly alter the root system architecture, foliage production, and defense responses. Although plant-microbe interactions have been shown to improve nutrient uptake and stress resilience in plants, the underlying mechanisms are not fully understood. "Multi-omics" application supported by genomics, transcriptomics, and metabolomics has been quite useful to investigate and understand the biochemical, physiological, and molecular aspects of plant-microbe interactions under drought stress conditions. The present review explores various microbe-mediated mechanisms for drought stress resilience in plants. In addition, plant adaptation to drought stress is discussed, and insights into the latest molecular techniques and approaches available to improve drought-stress resilience are provided.
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Affiliation(s)
- Aditya Sharma
- Enzyme Technology and Protein Bioinformatics Laboratory, School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, 221005, India
| | - Prassan Choudhary
- Microbial Technology Unit II, ICAR-National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, Uttar Pradesh, 275103, India
| | - Hillol Chakdar
- Microbial Technology Unit II, ICAR-National Bureau of Agriculturally Important Microorganisms (NBAIM), Mau, Uttar Pradesh, 275103, India
| | - Pratyoosh Shukla
- Enzyme Technology and Protein Bioinformatics Laboratory, School of Biotechnology, Institute of Science, Banaras Hindu University, Varanasi, Uttar Pradesh, 221005, India.
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25
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Ning L, Xi J, Zi Y, Chen M, Zou Q, Zhou X, Tang C. Prospects and challenges of CRISPR/Cas9 gene-editing technology in cancer research. Clin Genet 2023; 104:613-624. [PMID: 37706265 DOI: 10.1111/cge.14424] [Citation(s) in RCA: 3] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/09/2023] [Revised: 08/28/2023] [Accepted: 09/02/2023] [Indexed: 09/15/2023]
Abstract
Cancer, one of the leading causes of death, usually commences and progresses as a result of a series of gene mutations and dysregulation of expression. With the development of clustered regularly interspaced palindromic repeat (CRISPR)/Cas9 gene-editing technology, it is possible to edit and then decode the functions of cancer-related gene mutations, markedly advance the research of biological mechanisms and treatment of cancer. This review summarizes the mechanism and development of CRISPR/Cas9 gene-editing technology in recent years and describes its potential application in cancer-related research, such as the establishment of human tumor disease models, gene therapy and immunotherapy. The challenges and future development directions are highlighted to provide a reference for exploring pathological mechanisms and potential treatment protocols of cancer.
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Affiliation(s)
- Li Ning
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, South China Institute of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, China
- International Healthcare Innovation Institute (Jiangmen), Jiangmen, China
| | - Jiahui Xi
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, South China Institute of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, China
- International Healthcare Innovation Institute (Jiangmen), Jiangmen, China
| | - Yin Zi
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, South China Institute of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, China
- International Healthcare Innovation Institute (Jiangmen), Jiangmen, China
| | - Min Chen
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, South China Institute of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, China
- International Healthcare Innovation Institute (Jiangmen), Jiangmen, China
| | - Qingjian Zou
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, South China Institute of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, China
- International Healthcare Innovation Institute (Jiangmen), Jiangmen, China
| | - Xiaoqing Zhou
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, South China Institute of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, China
- International Healthcare Innovation Institute (Jiangmen), Jiangmen, China
| | - Chengcheng Tang
- Guangdong Provincial Key Laboratory of Large Animal Models for Biomedicine, South China Institute of Large Animal Models for Biomedicine, School of Biotechnology and Health Science, Wuyi University, Jiangmen, China
- International Healthcare Innovation Institute (Jiangmen), Jiangmen, China
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26
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Chowdhry R, Lu SZ, Lee S, Godhulayyagari S, Ebrahimi SB, Samanta D. Enhancing CRISPR/Cas systems with nanotechnology. Trends Biotechnol 2023; 41:1549-1564. [PMID: 37451945 DOI: 10.1016/j.tibtech.2023.06.005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/18/2023] [Revised: 06/13/2023] [Accepted: 06/14/2023] [Indexed: 07/18/2023]
Abstract
CRISPR/Cas systems have revolutionized biology and medicine, and have led to new paradigms in disease diagnostics and therapeutics. However, these complexes suffer from key limitations regarding barriers to cellular entry, stability in biological environments, and off-target effects. Integrating nanotechnology with CRISPR/Cas systems has emerged as a promising strategy to overcome these challenges and has further unlocked structures that accumulate preferentially in tissues of interest, have tunable pharmacological properties, and are activated in response to desired stimuli. Nanomaterials can also enhance CRISPR/Cas-mediated detection platforms by enabling faster, more sensitive, and convenient readouts. We highlight recent advances in this rapidly growing field. We also outline areas that need further development to fully realize the potential of CRISPR technologies.
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Affiliation(s)
- Rupali Chowdhry
- Department of Public Health, The University of Texas at Austin, Austin, TX 78712, USA
| | - Steven Z Lu
- Department of Biology, The University of Texas at Austin, Austin, TX 78712, USA
| | - Seungheon Lee
- Department of Chemistry, The University of Texas at Austin, Austin, TX 78712, USA
| | | | - Sasha B Ebrahimi
- Drug Product Development - Steriles, GlaxoSmithKline, Collegeville, PA 19426, USA
| | - Devleena Samanta
- Department of Chemistry, The University of Texas at Austin, Austin, TX 78712, USA.
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27
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Liu L, Gray JL, Tate EW, Yang A. Bacterial enzymes: powerful tools for protein labeling, cell signaling, and therapeutic discovery. Trends Biotechnol 2023; 41:1385-1399. [PMID: 37328400 DOI: 10.1016/j.tibtech.2023.05.004] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/09/2023] [Revised: 05/01/2023] [Accepted: 05/16/2023] [Indexed: 06/18/2023]
Abstract
Bacteria have evolved a diverse set of enzymes that enable them to subvert host defense mechanisms as well as to form part of the prokaryotic immune system. Due to their unique and varied biochemical activities, these bacterial enzymes have emerged as key tools for understanding and investigating biological systems. In this review, we summarize and discuss some of the most prominent bacterial enzymes used for the site-specific modification of proteins, in vivo protein labeling, proximity labeling, interactome mapping, signaling pathway manipulation, and therapeutic discovery. Finally, we provide a perspective on the complementary advantages and limitations of using bacterial enzymes compared with chemical probes for exploring biological systems.
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Affiliation(s)
- Lu Liu
- School of Life Sciences, Chongqing University, Chongqing 401331, China
| | - Janine L Gray
- Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, London W12 0BZ, UK
| | - Edward W Tate
- Department of Chemistry, Molecular Sciences Research Hub, Imperial College London, London W12 0BZ, UK.
| | - Aimin Yang
- School of Life Sciences, Chongqing University, Chongqing 401331, China.
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28
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Preta G. Development of New Genome Editing Tools for the Treatment of Hyperlipidemia. Cells 2023; 12:2466. [PMID: 37887310 PMCID: PMC10605581 DOI: 10.3390/cells12202466] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/12/2023] [Revised: 10/10/2023] [Accepted: 10/12/2023] [Indexed: 10/28/2023] Open
Abstract
Hyperlipidemia is a medical condition characterized by high levels of lipids in the blood. It is often associated with an increased risk of cardiovascular diseases such as heart attacks and strokes. Traditional treatment approaches for hyperlipidemia involve lifestyle modifications, dietary changes, and the use of medications like statins. Recent advancements in genome editing technologies, including CRISPR-Cas9, have opened up new possibilities for the treatment of this condition. This review provides a general overview of the main target genes involved in lipid metabolism and highlights the progress made during recent years towards the development of new treatments for dyslipidemia.
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Affiliation(s)
- Giulio Preta
- VU LSC-EMBL Partnership Institute for Genome Editing Technologies, Life Sciences Center, Vilnius University, LT-10257 Vilnius, Lithuania;
- Institute of Biochemistry, Life Science Center, Vilnius University, LT-10257 Vilnius, Lithuania
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29
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Feng S, Xie X, Liu J, Li A, Wang Q, Guo D, Li S, Li Y, Wang Z, Guo T, Zhou J, Tang DYY, Show PL. A potential paradigm in CRISPR/Cas systems delivery: at the crossroad of microalgal gene editing and algal-mediated nanoparticles. J Nanobiotechnology 2023; 21:370. [PMID: 37817254 PMCID: PMC10563294 DOI: 10.1186/s12951-023-02139-z] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/12/2023] [Accepted: 10/03/2023] [Indexed: 10/12/2023] Open
Abstract
Microalgae as the photosynthetic organisms offer enormous promise in a variety of industries, such as the generation of high-value byproducts, biofuels, pharmaceuticals, environmental remediation, and others. With the rapid advancement of gene editing technology, CRISPR/Cas system has evolved into an effective tool that revolutionised the genetic engineering of microalgae due to its robustness, high target specificity, and programmability. However, due to the lack of robust delivery system, the efficacy of gene editing is significantly impaired, limiting its application in microalgae. Nanomaterials have become a potential delivery platform for CRISPR/Cas systems due to their advantages of precise targeting, high stability, safety, and improved immune system. Notably, algal-mediated nanoparticles (AMNPs), especially the microalgae-derived nanoparticles, are appealing as a sustainable delivery platform because of their biocompatibility and low toxicity in a homologous relationship. In addition, living microalgae demonstrated effective and regulated distribution into specified areas as the biohybrid microrobots. This review extensively summarised the uses of CRISPR/Cas systems in microalgae and the recent developments of nanoparticle-based CRISPR/Cas delivery systems. A systematic description of the properties and uses of AMNPs, microalgae-derived nanoparticles, and microalgae microrobots has also been discussed. Finally, this review highlights the challenges and future research directions for the development of gene-edited microalgae.
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Affiliation(s)
- Shuying Feng
- Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China.
| | - Xin Xie
- Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China
| | - Junjie Liu
- Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China
| | - Aifang Li
- Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China
| | - Qianqian Wang
- Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China
| | - Dandan Guo
- Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China
| | - Shuxuan Li
- Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China
| | - Yalan Li
- Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China
| | - Zilong Wang
- Medical College, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China
| | - Tao Guo
- Department of Pharmacy, Henan University of Chinese Medicine, Zhengzhou, 450046, Henan, China.
| | - Jin Zhou
- Institute for Ocean Engineering, Shenzhen International Graduate School, Tsinghua University, Shenzhen, 518055, Guangdong, China.
| | - Doris Ying Ying Tang
- Department of Chemical and Environmental Engineering, Faculty of Science and Engineering, University of Nottingham Malaysia, 43500, Semenyih, Malaysia
| | - Pau Loke Show
- Department of Chemical Engineering, Khalifa University, P.O. Box 127788, Abu Dhabi, United Arab Emirates.
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Ahmad A, Jamil A, Munawar N. GMOs or non-GMOs? The CRISPR Conundrum. FRONTIERS IN PLANT SCIENCE 2023; 14:1232938. [PMID: 37877083 PMCID: PMC10591184 DOI: 10.3389/fpls.2023.1232938] [Citation(s) in RCA: 5] [Impact Index Per Article: 2.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 06/01/2023] [Accepted: 09/15/2023] [Indexed: 10/26/2023]
Abstract
CRISPR-Cas9, the "genetic scissors", is being presaged as a revolutionary technology, having tremendous potential to create designer crops by introducing precise and targeted modifications in the genome to achieve global food security in the face of climate change and increasing population. Traditional genetic engineering relies on random and unpredictable insertion of isolated genes or foreign DNA elements into the plant genome. However, CRISPR-Cas based gene editing does not necessarily involve inserting a foreign DNA element into the plant genome from different species but introducing new traits by precisely altering the existing genes. CRISPR edited crops are touching markets, however, the world community is divided over whether these crops should be considered genetically modified (GM) or non-GM. Classification of CRISPR edited crops, especially transgene free crops as traditional GM crops, will significantly affect their future and public acceptance in some regions. Therefore, the future of the CRISPR edited crops is depending upon their regulation as GM or non-GMs, and their public perception. Here we briefly discuss how CRISPR edited crops are different from traditional genetically modified crops. In addition, we discuss different CRISPR reagents and their delivery tools to produce transgene-free CRISPR edited crops. Moreover, we also summarize the regulatory classification of CRISPR modifications and how different countries are regulating CRISPR edited crops. We summarize that the controversy of CRISPR-edited plants as GM or non-GM will continue until a universal, transparent, and scalable regulatory framework for CRISPR-edited plants will be introduced worldwide, with increased public awareness by involving all stakeholders.
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Affiliation(s)
- Aftab Ahmad
- Center for Advanced Studies in Agriculture and Food Security (CASAFS), University of Agriculture Faisalabad, Faisalabad, Pakistan
- Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan
| | - Amer Jamil
- Department of Biochemistry, University of Agriculture, Faisalabad, Pakistan
| | - Nayla Munawar
- Department of Chemistry, College of Science, United Arab Emirates University, Al-Ain, United Arab Emirates
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31
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Lin YQ, Feng KK, Lu JY, Le JQ, Li WL, Zhang BC, Li CL, Song XH, Tong LW, Shao JW. CRISPR/Cas9-based application for cancer therapy: Challenges and solutions for non-viral delivery. J Control Release 2023; 361:727-749. [PMID: 37591461 DOI: 10.1016/j.jconrel.2023.08.028] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/21/2023] [Revised: 07/04/2023] [Accepted: 08/06/2023] [Indexed: 08/19/2023]
Abstract
CRISPR/Cas9 genome editing is a promising therapeutic technique, which makes precise and rapid gene editing technology possible on account of its high sensitivity and efficiency. CRISPR/Cas9 system has been proved to able to effectively disrupt and modify genes, which shows great potential for cancer treatment. Current researches proves that virus vectors are capable of effectively delivering the CRISPR/Cas9 system, but immunogenicity and carcinogenicity caused by virus transmission still trigger serious consequences. Therefore, the greatest challenge of CRISPR/Cas9 for cancer therapy lies on how to deliver it to the target tumor site safely and effectively. Non-viral delivery systems with specific targeting, high loading capacity, and low immune toxicity are more suitable than viral vectors, which limited by uncontrollable side effects. Their medical advances and applications have been widely concerned. Herein, we present the molecule mechanism and different construction strategies of CRISPR/Cas9 system for editing genes at the beginning of this research. Subsequently, several common CRISPR/Cas9 non-viral deliveries for cancer treatment are introduced. Lastly, based on the main factors limiting the delivery efficiency of non-viral vectors proposed in the existing researches and literature, we summarize and discuss the main methods to solve these limitations in the existing tumor treatment system, aiming to introduce further optimization and innovation of the CRISPR/Cas9 non-viral delivery system suitable for cancer treatment.
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Affiliation(s)
- Ying-Qi Lin
- Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Ke-Ke Feng
- Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Jie-Ying Lu
- Faculty of Foreign Studies, Guangdong Baiyun University, Guangzhou 510450, China
| | - Jing-Qing Le
- Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Wu-Lin Li
- Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Bing-Chen Zhang
- Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Cheng-Lei Li
- Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Xun-Huan Song
- Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Ling-Wu Tong
- Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China
| | - Jing-Wei Shao
- Fujian Provincial Key Laboratory of Cancer Metastasis Chemoprevention and Chemotherapy, College of Chemistry, Fuzhou University, Fuzhou 350108, China.
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Zhang CP, Qiu HY, Zhang CX, Zhang YM, Zhang YZ, Yin H, Zhang KQ, Zhang Y. Efficient non-viral delivery of macromolecules in human primary hematopoietic stem cells and lymphocytes. J Mol Cell Biol 2023; 15:mjad018. [PMID: 36945107 PMCID: PMC10481097 DOI: 10.1093/jmcb/mjad018] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2022] [Revised: 02/08/2023] [Accepted: 03/20/2023] [Indexed: 03/23/2023] Open
Affiliation(s)
- Chuan-Ping Zhang
- State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, School of Life Sciences, Yunnan University, Kunming 650091, China
- Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Hou-Yuan Qiu
- Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Cai-Xiang Zhang
- Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Yu-Ming Zhang
- Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Yi-Zhou Zhang
- Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
| | - Hao Yin
- Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
- State Key Laboratory of Virology, Wuhan University, Wuhan 430071, China
| | - Ke-Qin Zhang
- State Key Laboratory for Conservation and Utilization of Bio-Resources in Yunnan, School of Life Sciences, Yunnan University, Kunming 650091, China
| | - Ying Zhang
- Department of Rheumatology and Immunology, Medical Research Institute, Frontier Science Center for Immunology and Metabolism, Zhongnan Hospital of Wuhan University, Wuhan University, Wuhan 430071, China
- State Key Laboratory of Virology, Wuhan University, Wuhan 430071, China
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Shirani-Bidabadi S, Mirian M, Varshosaz J, Tavazohi N, Sadeghi HMM, Shariati L. Gene network analysis of oxaliplatin-resistant colorectal cancer to target a crucial gene using chitosan/hyaluronic acid/protamine polyplexes containing CRISPR-Cas9. Biochim Biophys Acta Gen Subj 2023; 1867:130385. [PMID: 37230419 DOI: 10.1016/j.bbagen.2023.130385] [Citation(s) in RCA: 2] [Impact Index Per Article: 1.0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/18/2023] [Revised: 05/18/2023] [Accepted: 05/19/2023] [Indexed: 05/27/2023]
Abstract
Colorectal cancer (CRC) treatment is dramatically hampered by resistance to oxaliplatin alone or in the combination of irinotecan or 5-fluorouracil and leucovorin. This study aims to design and assess Chitosan/Hyaluronic Acid/Protamine sulfate (CS/HA/PS) polyplexes loaded with CRISPR plasmid for targeting a key gene in cancer drug resistance. Here, recent findings were considered to validate oxaliplatin-resistant CRC-related genes and systems biology approaches employed to detect the critical gene. The polyplexes were characterized according to particle size, zeta potential, and stability. Moreover, carrier toxicity and transfection efficiency were assessed on oxaliplatin-resistant HT-29 cells. The post-transfection evaluations were performed to confirm gene disruption-mediated CRISPR. Eventually, excision cross complementation group 1(ERCC1), a crucial member of the nucleotide excision repair pathway, was selected to be targeted using CRISPR/Cas9 to reverse oxaliplatin resistance in HT-29 cells. CS/HA/PS polyplexes containing CRISPR/Cas9 plasmid exhibited negligible toxicity and comparable transfection efficiency with Lipofectamine™. Following the efficient gene delivery, sequences in CRISPR/Cas9 target sites were altered, ERCC1 was downregulated, and drug sensitivity was successfully restored in oxaliplatin-resistant cells. Findings indicate that CS/HA/PS/CRISPR polyplexes provide a potential strategy for delivering cargo and targeting oxaliplatin resistance-related gene to manipulate drug resistance as a rising concern in cancer therapeutic approaches.
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Affiliation(s)
- Shiva Shirani-Bidabadi
- Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan 8174673461, Iran
| | - Mina Mirian
- Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan 8174673461, Iran.
| | - Jaleh Varshosaz
- Department of Pharmaceutics, Faculty of Pharmacy and Novel Drug Delivery Systems Research Centre, Isfahan University of Medical Sciences, Isfahan 8174673461, Iran.
| | - Nazita Tavazohi
- Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan 8174673461, Iran
| | - Hamid Mir Mohammad Sadeghi
- Department of Pharmaceutical Biotechnology, Faculty of Pharmacy, Isfahan University of Medical Sciences, Isfahan 8174673461, Iran.
| | - Laleh Shariati
- Department of Biomaterials, Nanotechnology and Tissue Engineering, School of Advanced Technologies in Medicine, Isfahan University of Medical Sciences, Isfahan 8174673461, Iran.
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Hu Y, Lu B, Deng Z, Xing F, Hsu W. Virus-like particle-based delivery of Cas9/guide RNA ribonucleoprotein efficiently edits the brachyury gene and inhibits chordoma growth in vivo. Discov Oncol 2023; 14:70. [PMID: 37198417 DOI: 10.1007/s12672-023-00680-9] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 01/31/2023] [Accepted: 05/05/2023] [Indexed: 05/19/2023] Open
Abstract
PURPOSE Chordoma is a rare and aggressive bone cancer driven by the developmental transcription factor brachyury. Efforts to target brachyury are hampered by the absence of ligand-accessible small-molecule binding pockets. Genome editing with CRISPR systems provides an unprecedented opportunity to modulate undruggable transcription factor targets. However, delivery of CRISPR remains a bottleneck for in vivo therapy development. The aim was to investigate the in vivo therapeutic efficiency of Cas9/guide RNA (gRNA) ribonucleoprotein (RNP) delivery through a novel virus-like particle (VLP) by fusing an aptamer-binding protein to the lentiviral nucleocapsid protein. METHODS The p24 based ELISA and transmission electron microscopy were used to determine the characterization of engineered VLP-packaged Cas9/gRNA RNP. The deletion efficiency of brachyury gene in chordoma cells and tissues was measured by genome cleavage detection assay. RT-PCR, Western blot, immunofluorescence staining, and IHC were employed to test the function of brachyury deletion. Cell growth and tumor volume were measured to evaluate the therapeutic efficiency of brachyury deletion by VLP-packaged Cas9/gRNA RNP. RESULTS Our "all-in-one" VLP-based Cas9/gRNA RNP system allows for transient expression of Cas9 in chordoma cells, but maintains efficient editing capacity leading to approximately 85% knockdown of brachyury with subsequent inhibition of chordoma cell proliferation and tumor progression. In addition, this VLP-packaged brachyury-targeting Cas9 RNP avoids systemic toxicities in vivo. CONCLUSION Our preclinical studies demonstrate the potential of VLP-based Cas9/gRNA RNP gene therapy for the treatment of brachyury-dependent chordoma.
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Affiliation(s)
- Yunping Hu
- Department of Neurological Surgery, Medical Center Boulevard, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA.
| | - Baisong Lu
- Medical Center Boulevard, Wake Forest University Institute for Regenerative Medicine, Winston-Salem, NC, 27157, USA
| | - Zhiyong Deng
- Department of Physiology and Pharmacology, Medical Center Boulevard, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA
| | - Fei Xing
- Department of Cancer Biology, Medical Center Boulevard, Wake Forest University School of Medicine, Winston- Salem, NC, 27157, USA
| | - Wesley Hsu
- Department of Neurological Surgery, Medical Center Boulevard, Wake Forest University School of Medicine, Winston-Salem, NC, 27157, USA.
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Karrer LG, Mathew EN, Nava-Chavez J, Bhatti A, Delong RK. Evidence of Copper Nanoparticles and Poly I:C Modulating Cas9 Interaction and Cleavage of COR (Conserved Omicron RNA). Bioengineering (Basel) 2023; 10:bioengineering10050512. [PMID: 37237582 DOI: 10.3390/bioengineering10050512] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/24/2023] [Revised: 04/10/2023] [Accepted: 04/18/2023] [Indexed: 05/28/2023] Open
Abstract
Conserved omicron RNA (COR) is a 40 base long 99.9% conserved sequence in SARS-CoV-2 Omicron variant, predicted to form a stable stem loop, the targeted cleavage of which can be an ideal next step in controlling the spread of variants. The Cas9 enzyme has been traditionally utilized for gene editing and DNA cleavage. Previously Cas9 has been shown to be capable of RNA editing under certain conditions. Here we investigated the ability of Cas9 to bind to single-stranded conserved omicron RNA (COR) and examined the effect of copper nanoparticles (Cu NPs) and/or polyinosinic-polycytidilic acid (poly I:C) on the RNA cleavage ability of Cas9. The interaction of the Cas9 enzyme and COR with Cu NPs was shown by dynamic light scattering (DLS) and zeta potential measurements and was confirmed by two-dimensional fluorescence difference spectroscopy (2-D FDS). The interaction with and enhanced cleavage of COR by Cas9 in the presence of Cu NPs and poly I:C was shown by agarose gel electrophoresis. These data suggest that Cas9-mediated RNA cleavage may be potentiated at the nanoscale level in the presence of nanoparticles and a secondary RNA component. Further explorations in vitro and in vivo may contribute to the development of a better cellular delivery platform for Cas9.
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Affiliation(s)
- Lindy G Karrer
- Division of Biology, Kansas State University, Manhattan, KS 66506, USA
| | - Elza Neelima Mathew
- Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA
| | - Juliet Nava-Chavez
- Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA
| | - Abeera Bhatti
- Department of Anatomy and Physiology, Kansas State University, Manhattan, KS 66506, USA
| | - Robert K Delong
- Landmark Bio, Innovation Development Laboratory, Watertown, MA 02472, USA
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Rodgers TM, Muzzio N, Valero A, Ahmad I, Lüdtke TU, Moya SE, Romero G. Poly (β-amino Ester) Nanoparticles Modified with a Rabies Virus-derived peptide for the Delivery of ASCL1 Across a 3D In Vitro Model of the Blood Brain Barrier. ACS APPLIED NANO MATERIALS 2023; 6:6299-6311. [PMID: 37274933 PMCID: PMC10234607 DOI: 10.1021/acsanm.3c00651] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 06/07/2023]
Abstract
Gene editing has emerged as a therapeutic approach to manipulate the genome for killing cancer cells, protecting healthy tissues, and improving immune response to a tumor. The gene editing tool achaete-scute family bHLH transcription factor 1 CRISPR guide RNA (ASCL1-gRNA) is known to restore neuronal lineage potential, promote terminal differentiation, and attenuate tumorigenicity in glioblastoma tumors. Here, we fabricated a polymeric nonviral carrier to encapsulate ASCL1-gRNA by electrostatic interactions and deliver it into glioblastoma cells across a 3D in vitro model of the blood-brain barrier (BBB). To mimic rabies virus (RV) neurotropism, gene-loaded poly (β-amino ester) nanoparticles are surface functionalized with a peptide derivative of rabies virus glycoprotein (RVG29). The capability of the obtained NPs, hereinafter referred to as RV-like NPs, to travel across the BBB, internalize into glioblastoma cells and deliver ASCL1-gRNA are investigated in a 3D BBB in vitro model through flow cytometry and CLSM microscopy. The formation of nicotinic acetylcholine receptors in the 3D BBB in vitro model is confirmed by immunochemistry. These receptors are known to bind to RVG29. Unlike Lipofectamine that primarily internalizes and transfects endothelial cells, RV-like NPs are capable to travel across the BBB, preferentially internalize glioblastoma cells and deliver ASCL1-gRNA at an efficiency of 10 % causing non-cytotoxic effects.
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Affiliation(s)
- Tina M Rodgers
- Department of Biomedical Engineering and Chemical Engineering, The University of Texas at San Antonio, San Antonio, Texas, 78249, USA
| | - Nicolas Muzzio
- Department of Biomedical Engineering and Chemical Engineering, The University of Texas at San Antonio, San Antonio, Texas, 78249, USA
| | - Andrea Valero
- Department of Biomedical Engineering and Chemical Engineering, The University of Texas at San Antonio, San Antonio, Texas, 78249, USA
| | - Ikram Ahmad
- Department of Biomedical Engineering and Chemical Engineering, The University of Texas at San Antonio, San Antonio, Texas, 78249, USA
| | - Tanja Ursula Lüdtke
- Soft Matter Nanotechnology, CIC biomaGUNE, Basque Research and Technology Alliance (BRTA), Paseo Miramon 182, Donostia/San Sebastian, Gipuzkoa, 20014 Spain
| | - Sergio E Moya
- Soft Matter Nanotechnology, CIC biomaGUNE, Basque Research and Technology Alliance (BRTA), Paseo Miramon 182, Donostia/San Sebastian, Gipuzkoa, 20014 Spain
| | - Gabriela Romero
- Department of Biomedical Engineering and Chemical Engineering, The University of Texas at San Antonio, San Antonio, Texas, 78249, USA
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Allemailem KS, Almatroodi SA, Almatroudi A, Alrumaihi F, Al Abdulmonem W, Al-Megrin WAI, Aljamaan AN, Rahmani AH, Khan AA. Recent Advances in Genome-Editing Technology with CRISPR/Cas9 Variants and Stimuli-Responsive Targeting Approaches within Tumor Cells: A Future Perspective of Cancer Management. Int J Mol Sci 2023; 24:7052. [PMID: 37108214 PMCID: PMC10139162 DOI: 10.3390/ijms24087052] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 03/06/2023] [Revised: 04/06/2023] [Accepted: 04/09/2023] [Indexed: 04/29/2023] Open
Abstract
The innovative advances in transforming clustered regularly interspaced short palindromic repeats-associated protein 9 (CRISPR/Cas9) into different variants have taken the art of genome-editing specificity to new heights. Allosteric modulation of Cas9-targeting specificity by sgRNA sequence alterations and protospacer adjacent motif (PAM) modifications have been a good lesson to learn about specificity and activity scores in different Cas9 variants. Some of the high-fidelity Cas9 variants have been ranked as Sniper-Cas9, eSpCas9 (1.1), SpCas9-HF1, HypaCas9, xCas9, and evoCas9. However, the selection of an ideal Cas9 variant for a given target sequence remains a challenging task. A safe and efficient delivery system for the CRISPR/Cas9 complex at tumor target sites faces considerable challenges, and nanotechnology-based stimuli-responsive delivery approaches have significantly contributed to cancer management. Recent innovations in nanoformulation design, such as pH, glutathione (GSH), photo, thermal, and magnetic responsive systems, have modernized the art of CRISPR/Cas9 delivery approaches. These nanoformulations possess enhanced cellular internalization, endosomal membrane disruption/bypass, and controlled release. In this review, we aim to elaborate on different CRISPR/Cas9 variants and advances in stimuli-responsive nanoformulations for the specific delivery of this endonuclease system. Furthermore, the critical constraints of this endonuclease system on clinical translations towards the management of cancer and prospects are described.
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Affiliation(s)
- Khaled S. Allemailem
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
| | - Saleh A. Almatroodi
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
| | - Ahmad Almatroudi
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
| | - Faris Alrumaihi
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
| | - Waleed Al Abdulmonem
- Department of Pathology, College of Medicine, Qassim University, Buraydah 51452, Saudi Arabia
| | - Wafa Abdullah I. Al-Megrin
- Department of Biology, College of Science, Princess Nourah bint Abdulrahman University, P.O. Box 84428, Riyadh 11671, Saudi Arabia
| | | | - Arshad Husain Rahmani
- Department of Medical Laboratories, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
| | - Amjad Ali Khan
- Department of Basic Health Sciences, College of Applied Medical Sciences, Qassim University, Buraydah 51452, Saudi Arabia
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Sinclair F, Begum AA, Dai CC, Toth I, Moyle PM. Recent advances in the delivery and applications of nonviral CRISPR/Cas9 gene editing. Drug Deliv Transl Res 2023; 13:1500-1519. [PMID: 36988873 PMCID: PMC10052255 DOI: 10.1007/s13346-023-01320-z] [Citation(s) in RCA: 29] [Impact Index Per Article: 14.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 02/21/2023] [Indexed: 03/30/2023]
Abstract
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.
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Affiliation(s)
- Frazer Sinclair
- School of Pharmacy, The University of Queensland, Woolloongabba, QLD, 4102, Australia
| | - Anjuman A Begum
- School of Pharmacy, The University of Queensland, Woolloongabba, QLD, 4102, Australia.
| | - Charles C Dai
- School of Pharmacy, The University of Queensland, Woolloongabba, QLD, 4102, Australia
| | - Istvan Toth
- School of Pharmacy, The University of Queensland, Woolloongabba, QLD, 4102, Australia
- School of Chemistry and Molecular Biosciences, The University of Queensland, St Lucia, QLD, 4072, Australia
- Institute for Molecular Bioscience, The University of Queensland, St Lucia, QLD, 4072, Australia
| | - Peter M Moyle
- School of Pharmacy, The University of Queensland, Woolloongabba, QLD, 4102, Australia.
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Bhatia S, Pooja, Yadav SK. CRISPR-Cas for genome editing: Classification, mechanism, designing and applications. Int J Biol Macromol 2023; 238:124054. [PMID: 36933595 DOI: 10.1016/j.ijbiomac.2023.124054] [Citation(s) in RCA: 33] [Impact Index Per Article: 16.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/20/2023] [Revised: 02/24/2023] [Accepted: 03/12/2023] [Indexed: 03/18/2023]
Abstract
Clustered regularly interspersed short pallindromic repeats (CRISPR) and CRISPR associated proteins (Cas) system (CRISPR-Cas) came into light as prokaryotic defence mechanism for adaptive immune response. CRISPR-Cas works by integrating short sequences of the target genome (spacers) into the CRISPR locus. The locus containing spacers interspersed repeats is further expressed into small guide CRISPR RNA (crRNA) which is then deployed by the Cas proteins to evade the target genome. Based on the Cas proteins CRISPR-Cas is classified according to polythetic system of classification. The characteristic of the CRISPR-Cas9 system to target DNA sequences using programmable RNAs has opened new arenas due to which today CRISPR-Cas has evolved as cutting end technique in the field of genome editing. Here, we discuss about the evolution of CRISPR, its classification and various Cas systems including the designing and molecular mechanism of CRISPR-Cas. Applications of CRISPR-Cas as a genome editing tools are also highlighted in the areas such as agriculture, and anticancer therapy. Briefly discuss the role of CRISPR and its Cas systems in the diagnosis of COVID-19 and its possible preventive measures. The challenges in existing CRISP-Cas technologies and their potential solutions are also discussed briefly.
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Affiliation(s)
- Simran Bhatia
- Center of Innovative and applied Bioprocessing, Sector-81, Knowledge City, Mohali, India; Regional Center for Biotechnology, Faridabad, India
| | - Pooja
- Center of Innovative and applied Bioprocessing, Sector-81, Knowledge City, Mohali, India
| | - Sudesh Kumar Yadav
- Center of Innovative and applied Bioprocessing, Sector-81, Knowledge City, Mohali, India; Regional Center for Biotechnology, Faridabad, India.
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Papaioannou NY, Patsali P, Naiisseh B, Papasavva PL, Koniali L, Kurita R, Nakamura Y, Christou S, Sitarou M, Mussolino C, Cathomen T, Kleanthous M, Lederer CW. High-efficiency editing in hematopoietic stem cells and the HUDEP-2 cell line based on in vitro mRNA synthesis. Front Genome Ed 2023; 5:1141618. [PMID: 36969374 PMCID: PMC10030607 DOI: 10.3389/fgeed.2023.1141618] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Accepted: 02/17/2023] [Indexed: 03/11/2023] Open
Abstract
Introduction: Genome editing tools, such as CRISPR/Cas, TALE nucleases and, more recently, double-strand-break-independent editors, have been successfully used for gene therapy and reverse genetics. Among various challenges in the field, tolerable and efficient delivery of editors to target cells and sites, as well as independence from commercially available tools for flexibility and fast adoption of new editing technology are the most pressing. For many hematopoietic research applications, primary CD34+ cells and the human umbilical cord-derived progenitor erythroid 2 (HUDEP-2) cell line are highly informative substrates and readily accessible for in vitro manipulation. Moreover, ex vivo editing of CD34+ cells has immediate therapeutic relevance. Both cell types are sensitive to standard transfection procedures and reagents, such as lipofection with plasmid DNA, calling for more suitable methodology in order to achieve high efficiency and tolerability of editing with editors of choice. These challenges can be addressed by RNA delivery, either as a mixture of guide RNA and mRNA for CRISRP/Cas-based systems or as a mixture of mRNAs for TALENs. Compared to ribonucleoproteins or proteins, RNA as vector creates flexibility by removing dependence on commercial availability or laborious in-house preparations of novel editor proteins. Compared to DNA, RNA is less toxic and by obviating nuclear transcription and export of mRNA offers faster kinetics and higher editing efficiencies. Methods: Here, we detail an in vitro transcription protocol based on plasmid DNA templates with the addition of Anti-Reverse Cap Analog (ARCA) using T7 RNA polymerase, and poly (A) tailing using poly (A) polymerase, combined with nucleofection of HUDEP-2 and patient-derived CD34+ cells. Our protocol for RNA-based delivery employs widely available reagents and equipment and can easily be adopted for universal in vitro delivery of genome editing tools. Results and Discussion: Drawing on a common use case, we employ the protocol to target a β-globin mutation and to reactivate γ-globin expression as two potential therapies for β-hemoglobinopathies, followed by erythroid differentiation and functional analyses. Our protocol allows high editing efficiencies and unimpaired cell viability and differentiation, with scalability, suitability for functional assessment of editing outcomes and high flexibility in the application to different editors.
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Affiliation(s)
- Nikoletta Y. Papaioannou
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Petros Patsali
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Basma Naiisseh
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Panayiota L. Papasavva
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Lola Koniali
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Ryo Kurita
- Research and Development Department, Central Blood Institute, Blood Service Headquarters Japanese Red Cross Society, Tokyo, Japan
| | - Yukio Nakamura
- Cell Engineering Division, RIKEN BioResource Research Center, Tsukuba, Japan
| | - Soteroula Christou
- Thalassaemia Centre, State Health Services Organisation of Cyprus, Nicosia, Cyprus
| | - Maria Sitarou
- Thalassaemia Centre, State Health Services Organisation of Cyprus, Larnaca, Cyprus
| | - Claudio Mussolino
- Institute for Transfusion Medicine and Gene Therapy, Medical Center—University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Toni Cathomen
- Institute for Transfusion Medicine and Gene Therapy, Medical Center—University of Freiburg, Freiburg, Germany
- Center for Chronic Immunodeficiency (CCI), Faculty of Medicine, University of Freiburg, Freiburg, Germany
| | - Marina Kleanthous
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
| | - Carsten W. Lederer
- Department of Molecular Genetics Thalassemia, The Cyprus Institute of Neurology and Genetics, Nicosia, Cyprus
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41
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Padmaswari MH, Agrawal S, Jia MS, Ivy A, Maxenberger DA, Burcham LA, Nelson CE. Delivery challenges for CRISPR-Cas9 genome editing for Duchenne muscular dystrophy. BIOPHYSICS REVIEWS 2023; 4:011307. [PMID: 36864908 PMCID: PMC9969352 DOI: 10.1063/5.0131452] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Subscribe] [Scholar Register] [Received: 10/20/2022] [Accepted: 01/19/2023] [Indexed: 06/18/2023]
Abstract
Duchene muscular dystrophy (DMD) is an X-linked neuromuscular disorder that affects about one in every 5000 live male births. DMD is caused by mutations in the gene that codes for dystrophin, which is required for muscle membrane stabilization. The loss of functional dystrophin causes muscle degradation that leads to weakness, loss of ambulation, cardiac and respiratory complications, and eventually, premature death. Therapies to treat DMD have advanced in the past decade, with treatments in clinical trials and four exon-skipping drugs receiving conditional Food and Drug Administration approval. However, to date, no treatment has provided long-term correction. Gene editing has emerged as a promising approach to treating DMD. There is a wide range of tools, including meganucleases, zinc finger nucleases, transcription activator-like effector nucleases, and, most notably, RNA-guided enzymes from the bacterial adaptive immune system clustered regularly interspaced short palindromic repeats (CRISPR). Although challenges in using CRISPR for gene therapy in humans still abound, including safety and efficiency of delivery, the future for CRISPR gene editing for DMD is promising. This review will summarize the progress in CRISPR gene editing for DMD including key summaries of current approaches, delivery methodologies, and the challenges that gene editing still faces as well as prospective solutions.
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Affiliation(s)
| | - Shilpi Agrawal
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
| | - Mary S. Jia
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
| | - Allie Ivy
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
| | - Daniel A. Maxenberger
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
| | - Landon A. Burcham
- Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas 72701, USA
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42
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Toward the Development of Epigenome Editing-Based Therapeutics: Potentials and Challenges. Int J Mol Sci 2023; 24:ijms24054778. [PMID: 36902207 PMCID: PMC10003136 DOI: 10.3390/ijms24054778] [Citation(s) in RCA: 18] [Impact Index Per Article: 9.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/10/2023] [Revised: 02/27/2023] [Accepted: 02/28/2023] [Indexed: 03/06/2023] Open
Abstract
The advancement in epigenetics research over the past several decades has led to the potential application of epigenome-editing technologies for the treatment of various diseases. In particular, epigenome editing is potentially useful in the treatment of genetic and other related diseases, including rare imprinted diseases, as it can regulate the expression of the epigenome of the target region, and thereby the causative gene, with minimal or no modification of the genomic DNA. Various efforts are underway to successfully apply epigenome editing in vivo, such as improving target specificity, enzymatic activity, and drug delivery for the development of reliable therapeutics. In this review, we introduce the latest findings, summarize the current limitations and future challenges in the practical application of epigenome editing for disease therapy, and introduce important factors to consider, such as chromatin plasticity, for a more effective epigenome editing-based therapy.
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Ahmadi SE, Soleymani M, Shahriyary F, Amirzargar MR, Ofoghi M, Fattahi MD, Safa M. Viral vectors and extracellular vesicles: innate delivery systems utilized in CRISPR/Cas-mediated cancer therapy. Cancer Gene Ther 2023:10.1038/s41417-023-00597-z. [PMID: 36854897 PMCID: PMC9971689 DOI: 10.1038/s41417-023-00597-z] [Citation(s) in RCA: 25] [Impact Index Per Article: 12.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/09/2022] [Revised: 01/13/2023] [Accepted: 02/01/2023] [Indexed: 03/02/2023]
Abstract
Gene editing-based therapeutic strategies grant the power to override cell machinery and alter faulty genes contributing to disease development like cancer. Nowadays, the principal tool for gene editing is the clustered regularly interspaced short palindromic repeats-associated nuclease 9 (CRISPR/Cas9) system. In order to bring this gene-editing system from the bench to the bedside, a significant hurdle remains, and that is the delivery of CRISPR/Cas to various target cells in vivo and in vitro. The CRISPR-Cas system can be delivered into mammalian cells using various strategies; among all, we have reviewed recent research around two natural gene delivery systems that have been proven to be compatible with human cells. Herein, we have discussed the advantages and limitations of viral vectors, and extracellular vesicles (EVs) in delivering the CRISPR/Cas system for cancer therapy purposes.
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Affiliation(s)
- Seyed Esmaeil Ahmadi
- grid.411746.10000 0004 4911 7066Department of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Maral Soleymani
- grid.411230.50000 0000 9296 6873School of Medicine, Ahvaz Jundishapur University of Medical Sciences, Ahvaz, Iran
| | - Fahimeh Shahriyary
- grid.411746.10000 0004 4911 7066Department of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Mohammad Reza Amirzargar
- grid.411746.10000 0004 4911 7066Department of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran
| | - Mahya Ofoghi
- Division of Clinical Laboratory, Tehran Hospital of Petroleum Industry, Tehran, Iran ,grid.411600.2Department of Immunology, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Mohammad Davood Fattahi
- grid.411600.2Department of Clinical Biochemistry, School of Medicine, Shahid Beheshti University of Medical Sciences, Tehran, Iran
| | - Majid Safa
- Department of Hematology and Blood Banking, Faculty of Allied Medicine, Iran University of Medical Sciences, Tehran, Iran.
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Mohr M, Damas N, Gudmand-Høyer J, Zeeberg K, Jedrzejczyk D, Vlassis A, Morera-Gómez M, Pereira-Schoning S, Puš U, Oliver-Almirall A, Lyholm Jensen T, Baumgartner R, Tate Weinert B, Gill RT, Warnecke T. The CRISPR-Cas12a Platform for Accurate Genome Editing, Gene Disruption, and Efficient Transgene Integration in Human Immune Cells. ACS Synth Biol 2023; 12:375-389. [PMID: 36750230 PMCID: PMC9942205 DOI: 10.1021/acssynbio.2c00179] [Citation(s) in RCA: 7] [Impact Index Per Article: 3.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/08/2022] [Indexed: 02/09/2023]
Abstract
CRISPR-Cas12a nucleases have expanded the toolbox for targeted genome engineering in a broad range of organisms. Here, using a high-throughput engineering approach, we explored the potential of a novel CRISPR-MAD7 system for genome editing in human cells. We evaluated several thousand optimization conditions and demonstrated accurate genome reprogramming with modified MAD7. We identified crRNAs that allow for ≤95% non-homologous end joining (NHEJ) and 66% frameshift mutations in various genes and observed the high-cleavage fidelity of MAD7 resulting in undetectable off-target activity. We explored the dsDNA delivery efficiency of CRISPR-MAD7, and by using our optimized transfection protocol, we obtained ≤85% chimeric antigen receptor (CAR) insertions in primary T cells, thus exceeding the baseline integration efficiencies of therapeutically relevant transgenes using currently available virus-free technologies. Finally, we evaluated multiplex editing efficiency with CRISPR-MAD7 and demonstrated simultaneous ≤35% CAR transgene insertions and ≤80% gene disruption efficiencies. Both the platform and our transfection procedure are easily adaptable for further preclinical studies and could potentially be used for clinical manufacturing of CAR T cells.
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Affiliation(s)
- Marina Mohr
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Nkerorema Damas
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Johanne Gudmand-Høyer
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Katrine Zeeberg
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Dominika Jedrzejczyk
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Arsenios Vlassis
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Martí Morera-Gómez
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Sara Pereira-Schoning
- Artisan
Bio, 363 Centennial Parkway,
Suite 310, Louisville, Colorado 80027, United States
| | - Urška Puš
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Anna Oliver-Almirall
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Tanja Lyholm Jensen
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Roland Baumgartner
- Artisan
Bio, 363 Centennial Parkway,
Suite 310, Louisville, Colorado 80027, United States
| | - Brian Tate Weinert
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
| | - Ryan T. Gill
- Novo
Nordisk Foundation Center for Biosustainability, Technical University of Denmark, Kemitorvet 220, 2800 Kongens Lyngby, Denmark
- Artisan
Bio, 363 Centennial Parkway,
Suite 310, Louisville, Colorado 80027, United States
| | - Tanya Warnecke
- Artisan
Bio, 363 Centennial Parkway,
Suite 310, Louisville, Colorado 80027, United States
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45
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Yanagawa Y, Suenaga Y, Iijima Y, Endo M, Sanada N, Katoh E, Toki S, Okino A, Mitsuhara I. Genome editing by introduction of Cas9/sgRNA into plant cells using temperature-controlled atmospheric pressure plasma. PLoS One 2023; 18:e0281767. [PMID: 36795787 PMCID: PMC9934431 DOI: 10.1371/journal.pone.0281767] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/29/2022] [Accepted: 01/31/2023] [Indexed: 02/17/2023] Open
Abstract
Previously, we developed a technique to introduce a superfolder green fluorescent protein (sGFP) fusion protein directly into plant cells using atmospheric-pressure plasma. In this study, we attempted genome editing using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/CRISPR associated protein 9) system using this protein introduction technique. As an experimental system to evaluate genome editing, we utilized transgenic reporter plants carrying the reporter genes L-(I-SceI)-UC and sGFP-waxy-HPT. The L-(I-SceI)-UC system allowed the detection of successful genome editing by measuring the chemiluminescent signal observed upon re-functionalization of the luciferase (LUC) gene following genome editing. Similarly, the sGFP-waxy-HPT system conferred hygromycin resistance caused by hygromycin phosphotransferase (HPT) during genome editing. CRISPR/Cas9 ribonucleoproteins targeting these reporter genes were directly introduced into rice calli or tobacco leaf pieces after treatment with N2 and/or CO2 plasma. Cultivation of the treated rice calli on a suitable medium plate produced the luminescence signal, which was not observed in the negative control. Four types of genome-edited sequences were obtained upon sequencing the reporter genes of genome-edited candidate calli. sGFP-waxy-HPT-carrying tobacco cells exhibited hygromycin resistance during genome editing. After repeated cultivation of the treated tobacco leaf pieces on a regeneration medium plate, the calli were observed with leaf pieces. A green callus that was hygromycin-resistant was harvested, and a genome-edited sequence in the tobacco reporter gene was confirmed. As direct introduction of the Cas9/sgRNA (single guide RNA) complex using plasma enables genome editing in plants without any DNA introduction, this method is expected to be optimized for many plant species and may be widely applied for plant breeding in the future.
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Affiliation(s)
- Yuki Yanagawa
- Graduate School of Horticulture, Chiba University, Matsudo, Chiba, Japan
- Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki, Japan
- RIKEN Center for Sustainable Resource Science, Yokohama, Kanagawa, Japan
- * E-mail: , (YY); (IM)
| | - Yuma Suenaga
- Laboratory for Future Interdisciplinary Research of Science and Technology, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
| | - Yusuke Iijima
- Laboratory for Future Interdisciplinary Research of Science and Technology, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
| | - Masaki Endo
- Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki, Japan
- Graduate School of Nanobioscience, Yokohama City University, Yokohama, Kanagawa, Japan
- Kihara Institute for Biological Research, Yokohama City University, Yokohama, Kanagawa, Japan
| | - Naoko Sanada
- Advanced Analysis Center, NARO, Tsukuba, Ibaraki, Japan
| | - Etsuko Katoh
- Advanced Analysis Center, NARO, Tsukuba, Ibaraki, Japan
- Faculty of Food and Nutritional Science, Toyo University, Ora-gun, Gunma, Japan
| | - Seiichi Toki
- Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki, Japan
- Graduate School of Nanobioscience, Yokohama City University, Yokohama, Kanagawa, Japan
- Kihara Institute for Biological Research, Yokohama City University, Yokohama, Kanagawa, Japan
- Faculty of Agriculture, Ryukoku University, Otsu, Shiga, Japan
| | - Akitoshi Okino
- Laboratory for Future Interdisciplinary Research of Science and Technology, Institute of Innovative Research, Tokyo Institute of Technology, Yokohama, Kanagawa, Japan
| | - Ichiro Mitsuhara
- Institute of Agrobiological Sciences, NARO, Tsukuba, Ibaraki, Japan
- * E-mail: , (YY); (IM)
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46
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Verma V, Kumar A, Partap M, Thakur M, Bhargava B. CRISPR-Cas: A robust technology for enhancing consumer-preferred commercial traits in crops. FRONTIERS IN PLANT SCIENCE 2023; 14:1122940. [PMID: 36824195 PMCID: PMC9941649 DOI: 10.3389/fpls.2023.1122940] [Citation(s) in RCA: 15] [Impact Index Per Article: 7.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 12/13/2022] [Accepted: 01/16/2023] [Indexed: 06/18/2023]
Abstract
The acceptance of new crop varieties by consumers is contingent on the presence of consumer-preferred traits, which include sensory attributes, nutritional value, industrial products and bioactive compounds production. Recent developments in genome editing technologies provide novel insight to identify gene functions and improve the various qualitative and quantitative traits of commercial importance in plants. Various conventional as well as advanced gene-mutagenesis techniques such as physical and chemical mutagenesis, CRISPR-Cas9, Cas12 and base editors are used for the trait improvement in crops. To meet consumer demand, breakthrough biotechnologies, especially CRISPR-Cas have received a fair share of scientific and industrial interest, particularly in plant genome editing. CRISPR-Cas is a versatile tool that can be used to knock out, replace and knock-in the desired gene fragments at targeted locations in the genome, resulting in heritable mutations of interest. This review highlights the existing literature and recent developments in CRISPR-Cas technologies (base editing, prime editing, multiplex gene editing, epigenome editing, gene delivery methods) for reliable and precise gene editing in plants. This review also discusses the potential of gene editing exhibited in crops for the improvement of consumer-demanded traits such as higher nutritional value, colour, texture, aroma/flavour, and production of industrial products such as biofuel, fibre, rubber and pharmaceuticals. In addition, the bottlenecks and challenges associated with gene editing system, such as off targeting, ploidy level and the ability to edit organelle genome have also been discussed.
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Affiliation(s)
- Vipasha Verma
- Floriculture Laboratory, Agrotechnology Division, Council of Scientific and Industrial Research (CSIR) –Institute of Himalayan Bioresource Technology (IHBT), Palampur, India
| | - Akhil Kumar
- Floriculture Laboratory, Agrotechnology Division, Council of Scientific and Industrial Research (CSIR) –Institute of Himalayan Bioresource Technology (IHBT), Palampur, India
| | - Mahinder Partap
- Floriculture Laboratory, Agrotechnology Division, Council of Scientific and Industrial Research (CSIR) –Institute of Himalayan Bioresource Technology (IHBT), Palampur, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
| | - Meenakshi Thakur
- Floriculture Laboratory, Agrotechnology Division, Council of Scientific and Industrial Research (CSIR) –Institute of Himalayan Bioresource Technology (IHBT), Palampur, India
| | - Bhavya Bhargava
- Floriculture Laboratory, Agrotechnology Division, Council of Scientific and Industrial Research (CSIR) –Institute of Himalayan Bioresource Technology (IHBT), Palampur, India
- Academy of Scientific and Innovative Research (AcSIR), Ghaziabad, Uttar Pradesh, India
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Aziz A, Rehman U, Sheikh A, Abourehab MAS, Kesharwani P. Lipid-based nanocarrier mediated CRISPR/Cas9 delivery for cancer therapy. JOURNAL OF BIOMATERIALS SCIENCE. POLYMER EDITION 2023; 34:398-418. [PMID: 36083788 DOI: 10.1080/09205063.2022.2121592] [Citation(s) in RCA: 12] [Impact Index Per Article: 6.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Indexed: 02/04/2023]
Abstract
CRISPR/Cas mediated gene-editing has opened new avenues for therapies that show great potential for treating or curing cancers, genetic disorders, and microbial infections such as HIV. CRISPR/Cas9 tool is highly efficacious in revolutionizing the advent of genome editing; however, its efficient and safe delivery is a major hurdle due to its cellular impermeability and instability. Nano vectors could be explored to scale up the safe and effective delivery of CRISPR/Cas9. This review highlights the importance of CRISPR/Cas9 genome editing system in cancer treatment along with the effect of lipid-based nanoparticles in its safe delivery to cancer cells. The solid-lipid nanoparticles, nanostructured lipid carrier, lipid nanoparticles and niosomes have shown great effect in the delivery of CRISPR compounds to the cancer cells. The design and genome editing application in cancer therapy has been discussed along with the future concern and prospects of lipid nanoparticle based CRISPR/Cas9 has been focused toward the end.
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Affiliation(s)
- Aisha Aziz
- Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India
| | - Urushi Rehman
- Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India
| | - Afsana Sheikh
- Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India
| | - Mohammed A S Abourehab
- Department of Pharmaceutics, College of Pharmacy, Umm Al-Qura University, Makkah, Saudi Arabia.,Department of Pharmaceutics and Industrial Pharmacy, College of Pharmacy, Minia University, Minia, Egypt
| | - Prashant Kesharwani
- Department of Pharmaceutics, School of Pharmaceutical Education and Research, Jamia Hamdard, New Delhi, India.,University Institute of Pharma Sciences, Chandigarh University, Mohali, Punjab, India
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48
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Ureña-Bailén G, Block M, Grandi T, Aivazidou F, Quednau J, Krenz D, Daniel-Moreno A, Lamsfus-Calle A, Epting T, Handgretinger R, Wild S, Mezger M. Automated Good Manufacturing Practice-Compatible CRISPR-Cas9 Editing of Hematopoietic Stem and Progenitor Cells for Clinical Treatment of β-Hemoglobinopathies. CRISPR J 2023; 6:5-16. [PMID: 36662546 PMCID: PMC9986018 DOI: 10.1089/crispr.2022.0086] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 01/21/2023] Open
Abstract
Cellular therapies hold enormous potential for the cure of severe hematological and oncological disorders. The forefront of innovative gene therapy approaches including therapeutic gene editing and hematopoietic stem cell transplantation needs to be processed by good manufacturing practice to ensure safe application in patients. In the present study, an effective transfection protocol for automated clinical-scale production of genetically modified hematopoietic stem and progenitor cells (HSPCs) using the CliniMACS Prodigy® system including the CliniMACS Electroporator (Miltenyi Biotec) was established. As a proof-of-concept, the enhancer of the BCL11A gene, clustered regularly interspaced short palindromic repeat (CRISPR) target in ongoing clinical trials for β-thalassemia and sickle-cell disease treatment, was disrupted by the CRISPR-Cas9 system simulating a large-scale clinical scenario, yielding 100 million HSPCs with high editing efficiency. In vitro erythroid differentiation and high-performance liquid chromatography analyses corroborated fetal hemoglobin resurgence in edited samples, supporting the feasibility of running the complete process of HSPC gene editing in an automated closed system.
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Affiliation(s)
- Guillermo Ureña-Bailén
- Department of General Pediatrics, Oncology and Hematology, University Children's Hospital, Tübingen, Germany
| | - Milena Block
- Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
| | - Tommaso Grandi
- Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
| | | | - Jona Quednau
- Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
| | - Dariusz Krenz
- Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
| | - Alberto Daniel-Moreno
- Department of General Pediatrics, Oncology and Hematology, University Children's Hospital, Tübingen, Germany
| | - Andrés Lamsfus-Calle
- Department of General Pediatrics, Oncology and Hematology, University Children's Hospital, Tübingen, Germany
| | - Thomas Epting
- Institute for Clinical Chemistry and Laboratory Medicine, University Hospital, Freiburg, Germany
| | - Rupert Handgretinger
- Department of General Pediatrics, Oncology and Hematology, University Children's Hospital, Tübingen, Germany.,Abu Dhabi Stem Cells Center, Abu Dhabi, United Arab Emirates
| | - Stefan Wild
- Miltenyi Biotec B.V. & Co. KG, Bergisch Gladbach, Germany
| | - Markus Mezger
- Department of General Pediatrics, Oncology and Hematology, University Children's Hospital, Tübingen, Germany
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49
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CRISPR/Cas9 in the era of nanomedicine and synthetic biology. Drug Discov Today 2023; 28:103375. [PMID: 36174966 DOI: 10.1016/j.drudis.2022.103375] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/19/2022] [Revised: 08/15/2022] [Accepted: 09/22/2022] [Indexed: 02/02/2023]
Abstract
The CRISPR/Cas system was first discovered as a defense mechanism in bacteria and is now used as a tool for precise gene-editing applications. Rapidly evolving, it is increasingly applied in therapeutics. However, concerns about safety, specificity, and delivery still limit its potential. In this context, we introduce the concept of nanogenetics and speculate how the rational engineering of the CRISPR/Cas machinery could advance the biomedical field. In nanogenetics, the advantages of traditional approaches of synthetic biology could be expanded by nanotechnology approaches, enabling the design of a new generation of intrinsically safe and specific genome-editing platforms.
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50
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Zhou Q, Chen Y, Wang R, Jia F, He F, Yuan F. Advances of CRISPR-Cas13 system in COVID-19 diagnosis and treatment. Genes Dis 2022; 10:S2352-3042(22)00317-8. [PMID: 36591005 PMCID: PMC9793954 DOI: 10.1016/j.gendis.2022.11.016] [Citation(s) in RCA: 2] [Impact Index Per Article: 0.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/04/2022] [Revised: 11/15/2022] [Accepted: 11/17/2022] [Indexed: 12/28/2022] Open
Abstract
The ongoing global pandemic of coronavirus disease 2019 (COVID-19), caused by the severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), has resulted in over 570 million infections and 6 million deaths worldwide. Early detection and quarantine are essential to arrest the spread of the highly contagious COVID-19. High-risk groups, such as older adults and individuals with comorbidities, can present severe symptoms, including pyrexia, pertussis, and acute respiratory distress syndrome, on SARS-CoV-2 infection that can prove fatal, demonstrating a clear need for high-throughput and sensitive platforms to detect and eliminate SARS-CoV-2. CRISPR-Cas13, an emerging CRISPR system targeting RNA with high specificity and efficiency, has recently drawn much attention for COVID-19 diagnosis and treatment. Here, we summarized the current research progress on CRISPR-Cas13 in COVID-19 diagnosis and treatment and highlight the challenges and future research directions of CRISPR-Cas13 for effectively counteracting COVID-19.
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Affiliation(s)
| | | | - Ruolei Wang
- Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Fengjing Jia
- Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Feng He
- Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
| | - Fuwen Yuan
- Academy of Integrative Medicine, Shanghai University of Traditional Chinese Medicine, Shanghai 201203, China
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