In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9
The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases...
| Main Authors: | , , , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
| Published: |
Elsevier
2020-12-01
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| Series: | Molecular Therapy: Methods & Clinical Development |
| Online Access: | http://www.sciencedirect.com/science/article/pii/S2329050120302114 |
| _version_ | 1818664364006178816 |
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| author | Sergio López-Manzaneda Isabel Ojeda-Pérez Nerea Zabaleta Aída García-Torralba Omaira Alberquilla Raúl Torres Rebeca Sánchez-Domínguez Laura Torella Emmanuel Olivier Joanne Mountford Juan C. Ramírez Juan A. Bueren Gloria González-Aseguinolaza Jose-Carlos Segovia |
| author_facet | Sergio López-Manzaneda Isabel Ojeda-Pérez Nerea Zabaleta Aída García-Torralba Omaira Alberquilla Raúl Torres Rebeca Sánchez-Domínguez Laura Torella Emmanuel Olivier Joanne Mountford Juan C. Ramírez Juan A. Bueren Gloria González-Aseguinolaza Jose-Carlos Segovia |
| author_sort | Sergio López-Manzaneda |
| collection | DOAJ |
| description | The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated. |
| first_indexed | 2024-12-17T05:31:34Z |
| format | Article |
| id | doaj.art-a89d25dc6deb4878ad9c49dd61bdf7ef |
| institution | Directory Open Access Journal |
| issn | 2329-0501 |
| language | English |
| last_indexed | 2024-12-17T05:31:34Z |
| publishDate | 2020-12-01 |
| publisher | Elsevier |
| record_format | Article |
| series | Molecular Therapy: Methods & Clinical Development |
| spelling | doaj.art-a89d25dc6deb4878ad9c49dd61bdf7ef2022-12-21T22:01:43ZengElsevierMolecular Therapy: Methods & Clinical Development2329-05012020-12-0119426437In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9Sergio López-Manzaneda0Isabel Ojeda-Pérez1Nerea Zabaleta2Aída García-Torralba3Omaira Alberquilla4Raúl Torres5Rebeca Sánchez-Domínguez6Laura Torella7Emmanuel Olivier8Joanne Mountford9Juan C. Ramírez10Juan A. Bueren11Gloria González-Aseguinolaza12Jose-Carlos Segovia13Cell Differentiation and Cytometry Unit. Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, SpainCell Differentiation and Cytometry Unit. Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, SpainCIMA, Pamplona, SpainCell Differentiation and Cytometry Unit. Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, SpainCell Differentiation and Cytometry Unit. Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, SpainMolecular Cytogenetics Group, Human Cancer Genetics Program, Spanish National Cancer Research Centre-CNIO, Madrid, SpainCell Differentiation and Cytometry Unit. Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, SpainCIMA, Pamplona, SpainScottish National Blood Transfusion Service and ICAMS, University of Glasgow, Glasgow, UKScottish National Blood Transfusion Service and ICAMS, University of Glasgow, Glasgow, UKVIVEbioTECH, San Sebastian, SpainUnidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain; Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, SpainCIMA, Pamplona, SpainCell Differentiation and Cytometry Unit. Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), Madrid, Spain; Unidad Mixta de Terapias Avanzadas. Instituto de Investigación Sanitaria Fundación Jiménez Díaz (IIS-FJD, UAM), Madrid, Spain; Corresponding author: Jose-Carlos Segovia, Cell Differentiation and Cytometry Unit. Hematopoietic Innovative Therapies Division, Centro de Investigaciones Energéticas, Medioambientales y Tecnológicas (CIEMAT) and Centro de Investigación Biomédica en Red de Enfermedades Raras (CIBERER), CIEMAT (Building 70), Avenida Complutense, 40, 28040 Madrid, Spain.The development of advanced gene and cell therapies for the treatment of genetic diseases requires reliable animal and cellular models to test their efficacy. Moreover, the availability of the target human primary cells of these therapies is reduced in many diseases. The development of endonucleases that can cut into specific sites of the cell genome, as well as the repair of the generated break by non-homologous end-joining, results in a variety of outcomes, insertions, deletions, and inversions that can induce the disruption of any specific gene. Among the many methods that have been developed for gene editing, CRISPR-Cas9 technology has become one of the most widely used endonuclease tools due to its easy design and its low cost. It has also been reported that the use of two guides, instead of just the one required, reduces the outcomes of non-homologous end joining mainly to the precise genomic sequences between the cutting sites of the guides used. We have explored this strategy to generate useful cellular and animal models. Different distances between the two guides have been tested (from 8 to 500 bp apart), and using the optimal range of 30–60 bp we have obtained a human primary cellular model of a genetic disease, pyruvate kinase deficiency, where the availability of the target cells is limited. We have also generated an in vivo model of glycolate oxidase (GO) deficiency, which is an enzyme involved in the glyoxylate metabolism following the same strategy. We demonstrate that the use of two-guide CRISPR-Cas9-induced non-homologous end joining is a feasible and useful tool for disease modeling, and it is most relevant to those diseases in which it is difficult to get the cells that will be genetically manipulated.http://www.sciencedirect.com/science/article/pii/S2329050120302114 |
| spellingShingle | Sergio López-Manzaneda Isabel Ojeda-Pérez Nerea Zabaleta Aída García-Torralba Omaira Alberquilla Raúl Torres Rebeca Sánchez-Domínguez Laura Torella Emmanuel Olivier Joanne Mountford Juan C. Ramírez Juan A. Bueren Gloria González-Aseguinolaza Jose-Carlos Segovia In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9 Molecular Therapy: Methods & Clinical Development |
| title | In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9 |
| title_full | In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9 |
| title_fullStr | In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9 |
| title_full_unstemmed | In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9 |
| title_short | In Vitro and In Vivo Genetic Disease Modeling via NHEJ-Precise Deletions Using CRISPR-Cas9 |
| title_sort | in vitro and in vivo genetic disease modeling via nhej precise deletions using crispr cas9 |
| url | http://www.sciencedirect.com/science/article/pii/S2329050120302114 |
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