Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair
The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, howe...
| Main Authors: | , , , , , , , , , , , |
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| Format: | Article |
| Language: | English |
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eLife Sciences Publications Ltd
2018-05-01
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| Series: | eLife |
| Subjects: | |
| Online Access: | https://elifesciences.org/articles/33761 |
| _version_ | 1811181122126610432 |
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| author | Natasa Savic Femke CAS Ringnalda Helen Lindsay Christian Berk Katja Bargsten Yizhou Li Dario Neri Mark D Robinson Constance Ciaudo Jonathan Hall Martin Jinek Gerald Schwank |
| author_facet | Natasa Savic Femke CAS Ringnalda Helen Lindsay Christian Berk Katja Bargsten Yizhou Li Dario Neri Mark D Robinson Constance Ciaudo Jonathan Hall Martin Jinek Gerald Schwank |
| author_sort | Natasa Savic |
| collection | DOAJ |
| description | The CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired. |
| first_indexed | 2024-04-11T09:14:19Z |
| format | Article |
| id | doaj.art-d383e78bf9ad4d79a251b869c68ad33c |
| institution | Directory Open Access Journal |
| issn | 2050-084X |
| language | English |
| last_indexed | 2024-04-11T09:14:19Z |
| publishDate | 2018-05-01 |
| publisher | eLife Sciences Publications Ltd |
| record_format | Article |
| series | eLife |
| spelling | doaj.art-d383e78bf9ad4d79a251b869c68ad33c2022-12-22T04:32:25ZengeLife Sciences Publications LtdeLife2050-084X2018-05-01710.7554/eLife.33761Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repairNatasa Savic0https://orcid.org/0000-0003-3110-5780Femke CAS Ringnalda1https://orcid.org/0000-0002-0684-4613Helen Lindsay2Christian Berk3Katja Bargsten4Yizhou Li5Dario Neri6Mark D Robinson7Constance Ciaudo8https://orcid.org/0000-0002-0857-4506Jonathan Hall9https://orcid.org/0000-0003-4160-7135Martin Jinek10https://orcid.org/0000-0002-7601-210XGerald Schwank11https://orcid.org/0000-0003-0767-2953The Institute of Molecular Health Sciences, ETH Zurich, Zurich, SwitzerlandThe Institute of Molecular Health Sciences, ETH Zurich, Zurich, SwitzerlandThe Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland; SIB Swiss Institute of Bioinformatics, Zurich, SwitzerlandInstitute for Pharmaceutical Sciences, ETH Zurich, Zurich, SwitzerlandDepartment of Biochemistry, University of Zurich, Zurich, SwitzerlandInstitute for Pharmaceutical Sciences, ETH Zurich, Zurich, SwitzerlandInstitute for Pharmaceutical Sciences, ETH Zurich, Zurich, SwitzerlandThe Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland; SIB Swiss Institute of Bioinformatics, Zurich, SwitzerlandThe Institute of Molecular Health Sciences, ETH Zurich, Zurich, SwitzerlandInstitute for Pharmaceutical Sciences, ETH Zurich, Zurich, SwitzerlandDepartment of Biochemistry, University of Zurich, Zurich, SwitzerlandThe Institute of Molecular Health Sciences, ETH Zurich, Zurich, SwitzerlandThe CRISPR-Cas9 targeted nuclease technology allows the insertion of genetic modifications with single base-pair precision. The preference of mammalian cells to repair Cas9-induced DNA double-strand breaks via error-prone end-joining pathways rather than via homology-directed repair mechanisms, however, leads to relatively low rates of precise editing from donor DNA. Here we show that spatial and temporal co-localization of the donor template and Cas9 via covalent linkage increases the correction rates up to 24-fold, and demonstrate that the effect is mainly caused by an increase of donor template concentration in the nucleus. Enhanced correction rates were observed in multiple cell types and on different genomic loci, suggesting that covalently linking the donor template to the Cas9 complex provides advantages for clinical applications where high-fidelity repair is desired.https://elifesciences.org/articles/33761CRISPR/Cas9gene editinghomology-directed repair |
| spellingShingle | Natasa Savic Femke CAS Ringnalda Helen Lindsay Christian Berk Katja Bargsten Yizhou Li Dario Neri Mark D Robinson Constance Ciaudo Jonathan Hall Martin Jinek Gerald Schwank Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair eLife CRISPR/Cas9 gene editing homology-directed repair |
| title | Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair |
| title_full | Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair |
| title_fullStr | Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair |
| title_full_unstemmed | Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair |
| title_short | Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair |
| title_sort | covalent linkage of the dna repair template to the crispr cas9 nuclease enhances homology directed repair |
| topic | CRISPR/Cas9 gene editing homology-directed repair |
| url | https://elifesciences.org/articles/33761 |
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