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Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template
The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome, where the Cas9 nuclease then induces a double-stranded break (DSB). The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directe...
Autores principales: | , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Nature Publishing Group UK
2018
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123678/ https://www.ncbi.nlm.nih.gov/pubmed/30271937 http://dx.doi.org/10.1038/s42003-018-0054-2 |
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author | Aird, Eric J. Lovendahl, Klaus N. St. Martin, Amber Harris, Reuben S. Gordon, Wendy R. |
author_facet | Aird, Eric J. Lovendahl, Klaus N. St. Martin, Amber Harris, Reuben S. Gordon, Wendy R. |
author_sort | Aird, Eric J. |
collection | PubMed |
description | The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome, where the Cas9 nuclease then induces a double-stranded break (DSB). The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded oligodeoxynucleotide (ssODN) to the Cas9-guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to a 30-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci and is more pronounced at low RNP concentrations. |
format | Online Article Text |
id | pubmed-6123678 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2018 |
publisher | Nature Publishing Group UK |
record_format | MEDLINE/PubMed |
spelling | pubmed-61236782018-09-28 Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template Aird, Eric J. Lovendahl, Klaus N. St. Martin, Amber Harris, Reuben S. Gordon, Wendy R. Commun Biol Article The CRISPR-Cas9 system is a powerful genome-editing tool in which a guide RNA targets Cas9 to a site in the genome, where the Cas9 nuclease then induces a double-stranded break (DSB). The potential of CRISPR-Cas9 to deliver precise genome editing is hindered by the low efficiency of homology-directed repair (HDR), which is required to incorporate a donor DNA template encoding desired genome edits near the DSB. We present a strategy to enhance HDR efficiency by covalently tethering a single-stranded oligodeoxynucleotide (ssODN) to the Cas9-guide RNA ribonucleoprotein (RNP) complex via a fused HUH endonuclease, thus spatially and temporally co-localizing the DSB machinery and donor DNA. We demonstrate up to a 30-fold enhancement of HDR using several editing assays, including repair of a frameshift and in-frame insertions of protein tags. The improved HDR efficiency is observed in multiple cell types and target loci and is more pronounced at low RNP concentrations. Nature Publishing Group UK 2018-05-31 /pmc/articles/PMC6123678/ /pubmed/30271937 http://dx.doi.org/10.1038/s42003-018-0054-2 Text en © The Author(s) 2018 Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons license, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons license and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this license, visit http://creativecommons.org/licenses/by/4.0/. |
spellingShingle | Article Aird, Eric J. Lovendahl, Klaus N. St. Martin, Amber Harris, Reuben S. Gordon, Wendy R. Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template |
title | Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template |
title_full | Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template |
title_fullStr | Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template |
title_full_unstemmed | Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template |
title_short | Increasing Cas9-mediated homology-directed repair efficiency through covalent tethering of DNA repair template |
title_sort | increasing cas9-mediated homology-directed repair efficiency through covalent tethering of dna repair template |
topic | Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6123678/ https://www.ncbi.nlm.nih.gov/pubmed/30271937 http://dx.doi.org/10.1038/s42003-018-0054-2 |
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