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Gähwiler EKN, Motta SE, Martin M, Nugraha B, Hoerstrup SP, Emmert MY. Human iPSCs and Genome Editing Technologies for Precision Cardiovascular Tissue Engineering. Front Cell Dev Biol 2021; 9:639699. [PMID: 34262897 PMCID: PMC8273765 DOI: 10.3389/fcell.2021.639699] [Citation(s) in RCA: 8] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 12/09/2020] [Accepted: 03/31/2021] [Indexed: 12/12/2022] Open
Abstract
Induced pluripotent stem cells (iPSCs) originate from the reprogramming of adult somatic cells using four Yamanaka transcription factors. Since their discovery, the stem cell (SC) field achieved significant milestones and opened several gateways in the area of disease modeling, drug discovery, and regenerative medicine. In parallel, the emergence of clustered regularly interspaced short palindromic repeats (CRISPR)-associated protein 9 (CRISPR-Cas9) revolutionized the field of genome engineering, allowing the generation of genetically modified cell lines and achieving a precise genome recombination or random insertions/deletions, usefully translated for wider applications. Cardiovascular diseases represent a constantly increasing societal concern, with limited understanding of the underlying cellular and molecular mechanisms. The ability of iPSCs to differentiate into multiple cell types combined with CRISPR-Cas9 technology could enable the systematic investigation of pathophysiological mechanisms or drug screening for potential therapeutics. Furthermore, these technologies can provide a cellular platform for cardiovascular tissue engineering (TE) approaches by modulating the expression or inhibition of targeted proteins, thereby creating the possibility to engineer new cell lines and/or fine-tune biomimetic scaffolds. This review will focus on the application of iPSCs, CRISPR-Cas9, and a combination thereof to the field of cardiovascular TE. In particular, the clinical translatability of such technologies will be discussed ranging from disease modeling to drug screening and TE applications.
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Affiliation(s)
- Eric K. N. Gähwiler
- Institute for Regenerative Medicine (IREM), University of Zurich, Zurich, Switzerland
| | - Sarah E. Motta
- Institute for Regenerative Medicine (IREM), University of Zurich, Zurich, Switzerland
- Wyss Zurich, University and ETH Zurich, Zurich, Switzerland
| | - Marcy Martin
- Division of Pediatric Cardiology, Department of Pediatrics, Stanford School of Medicine, Stanford, CA, United States
- Vera Moulton Wall Center for Pulmonary Vascular Disease, Stanford School of Medicine, Stanford, CA, United States
- Stanford Cardiovascular Institute, Stanford School of Medicine, Stanford, CA, United States
| | - Bramasta Nugraha
- Molecular Parasitology Lab, Institute of Parasitology, University of Zurich, Zurich, Switzerland
- Bioscience Cardiovascular, Research and Early Development, Cardiovascular, Renal and Metabolism, R&D BioPharmaceuticals, AstraZeneca, Gothenburg, Sweden
| | - Simon P. Hoerstrup
- Institute for Regenerative Medicine (IREM), University of Zurich, Zurich, Switzerland
- Wyss Zurich, University and ETH Zurich, Zurich, Switzerland
| | - Maximilian Y. Emmert
- Institute for Regenerative Medicine (IREM), University of Zurich, Zurich, Switzerland
- Wyss Zurich, University and ETH Zurich, Zurich, Switzerland
- Department of Cardiovascular Surgery, Charité Universitätsmedizin Berlin, Berlin, Germany
- Department of Cardiothoracic and Vascular Surgery, German Heart Center Berlin, Berlin, Germany
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Lujan H, Romer E, Salisbury R, Hussain S, Sayes C. Determining the Biological Mechanisms of Action for Environmental Exposures: Applying CRISPR/Cas9 to Toxicological Assessments. Toxicol Sci 2021; 175:5-18. [PMID: 32105327 DOI: 10.1093/toxsci/kfaa028] [Citation(s) in RCA: 6] [Impact Index Per Article: 1.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 12/15/2022] Open
Abstract
Toxicology is a constantly evolving field, especially in the area of developing alternatives to animal testing. Toxicological research must evolve and utilize adaptive technologies in an effort to improve public, environmental, and occupational health. The most commonly cited mechanisms of toxic action after exposure to a chemical or particle test substance is oxidative stress. However, because oxidative stress involves a plethora of genes and proteins, the exact mechanism(s) are not commonly defined. Exact mechanisms of toxicity can be revealed using an emerging laboratory technique referred to as CRISPR (clustered regularly interspaced short palindromic repeats). This article reviews the most common CRISPR techniques utilized today and how each may be applied in Toxicological Sciences. Specifically, the CRISPR/CRISPR-associated protein complex is used for single gene knock-outs, whereas CRISPR interference/activation is used for silencing or activating (respectively) ribonucleic acid. Finally, CRISPR libraries are used for knocking-out entire gene pathways. This review highlights the application of CRISPR in toxicology to elucidate the exact mechanism through which toxicants perturb normal cellular functions.
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Affiliation(s)
- Henry Lujan
- Department of Environmental Science, Baylor University, Waco, Texas 76706
| | - Eric Romer
- Molecular Bioeffects Branch, Bioeffects Division, 711th Human Performance Wing, Air Force Research Laboratory, Dayton, Ohio 45433
| | - Richard Salisbury
- Molecular Bioeffects Branch, Bioeffects Division, 711th Human Performance Wing, Air Force Research Laboratory, Dayton, Ohio 45433
| | - Saber Hussain
- Molecular Bioeffects Branch, Bioeffects Division, 711th Human Performance Wing, Air Force Research Laboratory, Dayton, Ohio 45433
| | - Christie Sayes
- Department of Environmental Science, Baylor University, Waco, Texas 76706
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Vermersch E, Jouve C, Hulot JS. CRISPR/Cas9 gene-editing strategies in cardiovascular cells. Cardiovasc Res 2020; 116:894-907. [PMID: 31584620 DOI: 10.1093/cvr/cvz250] [Citation(s) in RCA: 51] [Impact Index Per Article: 10.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 12/12/2018] [Revised: 05/05/2019] [Accepted: 09/26/2019] [Indexed: 01/05/2024] Open
Abstract
Cardiovascular diseases are among the main causes of morbidity and mortality in Western countries and considered as a leading public health issue. Therefore, there is a strong need for new disease models to support the development of novel therapeutics approaches. The successive improvement of genome editing tools with zinc finger nucleases (ZFNs), transcription activator-like effector nucleases (TALENs), and more recently with clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated 9 (Cas9) has enabled the generation of genetically modified cells and organisms with much greater efficiency and precision than before. The simplicity of CRISPR/Cas9 technology made it especially suited for different studies, both in vitro and in vivo, and has been used in multiple studies evaluating gene functions, disease modelling, transcriptional regulation, and testing of novel therapeutic approaches. Notably, with the parallel development of human induced pluripotent stem cells (hiPSCs), the generation of knock-out and knock-in human cell lines significantly increased our understanding of mutation impacts and physiopathological mechanisms within the cardiovascular domain. Here, we review the recent development of CRISPR-Cas9 genome editing, the alternative tools, the available strategies to conduct genome editing in cardiovascular cells with a focus on its use for correcting mutations in vitro and in vivo both in germ and somatic cells. We will also highlight that, despite its potential, CRISPR/Cas9 technology comes with important technical and ethical limitations. The development of CRISPR/Cas9 genome editing for cardiovascular diseases indeed requires to develop a specific strategy in order to optimize the design of the genome editing tools, the manipulation of DNA repair mechanisms, the packaging and delivery of the tools to the studied organism, and the assessment of their efficiency and safety.
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Affiliation(s)
- Eva Vermersch
- Paris Cardiovascular Research Center PARCC, Université de Paris, INSERM, 56 Rue Leblanc, 75015 Paris, France
| | - Charlène Jouve
- Paris Cardiovascular Research Center PARCC, Université de Paris, INSERM, 56 Rue Leblanc, 75015 Paris, France
| | - Jean-Sébastien Hulot
- Paris Cardiovascular Research Center PARCC, Université de Paris, INSERM, 56 Rue Leblanc, 75015 Paris, France
- Centre d'Investigations Cliniques CIC1418, AP-HP, Hôpital Européen Georges Pompidou, 75015 Paris, France
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Schmidt C, Schindele P, Puchta H. From gene editing to genome engineering: restructuring plant chromosomes via CRISPR/Cas. ABIOTECH 2020; 1:21-31. [PMID: 36305002 PMCID: PMC9584095 DOI: 10.1007/s42994-019-00002-0] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Subscribe] [Scholar Register] [Received: 05/10/2019] [Accepted: 07/03/2019] [Indexed: 01/16/2023]
Abstract
In the last years, tremendous progress has been achieved in the field of gene editing in plants. By the induction of single site-specific double-strand breaks (DSBs), the knockout of genes by non-homologous end joining has become routine in many plant species. Recently, the efficiency of inducing pre-planned mutations by homologous recombination has also been improved considerably. However, very little effort has been undertaken until now to achieve more complex changes in plant genomes by the simultaneous induction of several DSBs. Several reports have been published on the efficient induction of deletions. However, the induction of intrachromosomal inversions and interchromosomal recombination by the use of CRISPR/Cas has only recently been reported. In this review, we want to sum up these results and put them into context with regards to what is known about natural chromosome rearrangements in plants. Moreover, we review the recent progress in CRISPR/Cas-based mammalian chromosomal rearrangements, which might be inspiring for plant biologists. In the long run, the controlled restructuring of plant genomes should enable us to link or break linkage of traits at will, thus defining a new area of plant breeding.
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Affiliation(s)
- Carla Schmidt
- Botanical Institute, Karlsruhe Institute of Technology, POB 6980, 76133 Karlsruhe, Germany
| | - Patrick Schindele
- Botanical Institute, Karlsruhe Institute of Technology, POB 6980, 76133 Karlsruhe, Germany
| | - Holger Puchta
- Botanical Institute, Karlsruhe Institute of Technology, POB 6980, 76133 Karlsruhe, Germany
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Stephens CJ, Lauron EJ, Kashentseva E, Lu ZH, Yokoyama WM, Curiel DT. Long-term correction of hemophilia B using adenoviral delivery of CRISPR/Cas9. J Control Release 2019; 298:128-141. [PMID: 30771412 PMCID: PMC6636336 DOI: 10.1016/j.jconrel.2019.02.009] [Citation(s) in RCA: 68] [Impact Index Per Article: 11.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/09/2018] [Revised: 01/28/2019] [Accepted: 02/08/2019] [Indexed: 12/25/2022]
Abstract
Hemophilia B (HB) is a life-threatening inherited disease caused by mutations in the FIX gene, leading to reduced protein function and abnormal blood clotting. Due to its monogenic nature, HB is one of the primary targets for gene therapy. Indeed, successful correction of HB has been shown in clinical trials using gene therapy approaches. However, application of these strategies to non-adult patients is limited due to high cell turnover as young patients develop, resulting in vector dilution and subsequent loss of therapeutic expression. Gene editing can potentially overcome this issue by permanently inserting the corrective gene. Integration allows replication of the therapeutic transgene at every cell division and can avoid issues associated with vector dilution. In this study, we explored adenovirus as a platform for corrective CRISPR/Cas9-mediated gene knock-in. We determined as a proof-of-principle that adenoviral delivery of CRISPR/Cas9 is capable of corrective gene addition, leading to long-term augmentation of FIX activity and phenotypic correction in a murine model of juvenile HB. While we found on-target error-free integration in all examined samples, some mice also contained mutations at the integration target site. Additionally, we detected adaptive immune responses against the vector and Cas9 nuclease. Overall, our findings show that the adenovirus platform is suitable for gene insertion in juveniles with inherited disease, suggesting this approach may be applicable to other diseases.
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Affiliation(s)
- Calvin J Stephens
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8224, St. Louis, MO 63110, USA; Molecular Genetics and Genomics Program, Division of Biology and Biomedical Sciences, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8226, St. Louis, MO 63110, USA
| | - Elvin J Lauron
- Division of Rheumatology, Department of Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8045, St. Louis, MO 63110, USA
| | - Elena Kashentseva
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8224, St. Louis, MO 63110, USA
| | - Zhi Hong Lu
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8224, St. Louis, MO 63110, USA
| | - Wayne M Yokoyama
- Division of Rheumatology, Department of Medicine, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8045, St. Louis, MO 63110, USA
| | - David T Curiel
- Cancer Biology Division, Department of Radiation Oncology, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8224, St. Louis, MO 63110, USA; Department of Radiation Oncology, Biologic Therapeutics Center, Washington University School of Medicine, 660 South Euclid Avenue, Campus Box 8224, St. Louis, MO 63110, USA.
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Sakuma T, Yamamoto T. Acceleration of cancer science with genome editing and related technologies. Cancer Sci 2018; 109:3679-3685. [PMID: 30315615 PMCID: PMC6272086 DOI: 10.1111/cas.13832] [Citation(s) in RCA: 14] [Impact Index Per Article: 2.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 05/30/2018] [Revised: 08/30/2018] [Accepted: 10/09/2018] [Indexed: 12/26/2022] Open
Abstract
Genome editing includes various edits of the genome, such as short insertions and deletions, substitutions, and chromosomal rearrangements including inversions, duplications, and translocations. These variations are based on single or multiple DNA double-strand break (DSB)-triggered in cellulo repair machineries. In addition to these "conventional" genome editing strategies, tools enabling customized, site-specific recognition of particular nucleic acid sequences have been coming into wider use; for example, single base editing without DSB introduction, epigenome editing with recruitment of epigenetic modifiers, transcriptome engineering using RNA editing systems, and in vitro detection of specific DNA and RNA sequences. In this review, we provide a quick overview of the current state of genome editing and related technologies that multilaterally contribute to cancer science.
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Affiliation(s)
- Tetsushi Sakuma
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan
| | - Takashi Yamamoto
- Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Hiroshima, Japan
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