Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair
- ETH Zurich, Switzerland
- University of Zurich, Switzerland
Abstract
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.
Data availability
The data that support the findings of this study are available within the paper and its Supplementary files. Source data files have been provided for Figure 4, Figure 5, Figure 6, Figure Supplement 1, Figure Supplement 2, and Figure Supplement 3.Scripts for mapping sequencing data, counting mutations and generating plots are available at https://github.com/HLindsay/Savic_CRISPR_HDR. Fastq files have been uploaded to ArrayExpress, and accession number is E-MTAB-6808.
Article and author information
Author details
Funding
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (PMPDP3_171388)
- Natasa Savic
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_160230)
- Gerald Schwank
Schweizerischer Nationalfonds zur Förderung der Wissenschaftlichen Forschung (31003A_149393)
- Martin Jinek
Vallee Foundation
- Martin Jinek
The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.
Reviewing Editor
- Bernard de Massy, Institute of Human Genetics, CNRS UPR 1142, France
Version history
- Received: November 23, 2017
- Accepted: May 26, 2018
- Accepted Manuscript published: May 29, 2018 (version 1)
- Version of Record published: June 28, 2018 (version 2)
- Version of Record updated: June 29, 2018 (version 3)
- Version of Record updated: July 4, 2018 (version 4)
- Version of Record updated: March 6, 2019 (version 5)
Copyright
© 2018, Savic et al.
This article is distributed under the terms of the Creative Commons Attribution License permitting unrestricted use and redistribution provided that the original author and source are credited.
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Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair
eLife 7:e33761.
https://doi.org/10.7554/eLife.33761
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