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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...

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Autores principales: Savic, Natasa, Ringnalda, Femke CAS, Lindsay, Helen, Berk, Christian, Bargsten, Katja, Li, Yizhou, Neri, Dario, Robinson, Mark D, Ciaudo, Constance, Hall, Jonathan, Jinek, Martin, Schwank, Gerald
Formato: Online Artículo Texto
Lenguaje:English
Publicado: eLife Sciences Publications, Ltd 2018
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023611/
https://www.ncbi.nlm.nih.gov/pubmed/29809142
http://dx.doi.org/10.7554/eLife.33761
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author Savic, Natasa
Ringnalda, Femke CAS
Lindsay, Helen
Berk, Christian
Bargsten, Katja
Li, Yizhou
Neri, Dario
Robinson, Mark D
Ciaudo, Constance
Hall, Jonathan
Jinek, Martin
Schwank, Gerald
author_facet Savic, Natasa
Ringnalda, Femke CAS
Lindsay, Helen
Berk, Christian
Bargsten, Katja
Li, Yizhou
Neri, Dario
Robinson, Mark D
Ciaudo, Constance
Hall, Jonathan
Jinek, Martin
Schwank, Gerald
author_sort Savic, Natasa
collection PubMed
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.
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spelling pubmed-60236112018-07-06 Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair Savic, Natasa Ringnalda, Femke CAS Lindsay, Helen Berk, Christian Bargsten, Katja Li, Yizhou Neri, Dario Robinson, Mark D Ciaudo, Constance Hall, Jonathan Jinek, Martin Schwank, Gerald eLife Chromosomes and Gene Expression 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. eLife Sciences Publications, Ltd 2018-05-29 /pmc/articles/PMC6023611/ /pubmed/29809142 http://dx.doi.org/10.7554/eLife.33761 Text en © 2018, Savic et al http://creativecommons.org/licenses/by/4.0/ http://creativecommons.org/licenses/by/4.0/This article is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use and redistribution provided that the original author and source are credited.
spellingShingle Chromosomes and Gene Expression
Savic, Natasa
Ringnalda, Femke CAS
Lindsay, Helen
Berk, Christian
Bargsten, Katja
Li, Yizhou
Neri, Dario
Robinson, Mark D
Ciaudo, Constance
Hall, Jonathan
Jinek, Martin
Schwank, Gerald
Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances 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 Chromosomes and Gene Expression
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6023611/
https://www.ncbi.nlm.nih.gov/pubmed/29809142
http://dx.doi.org/10.7554/eLife.33761
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