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Elife. 2018 May 29;7. pii: e33761. doi: 10.7554/eLife.33761.

Covalent linkage of the DNA repair template to the CRISPR-Cas9 nuclease enhances homology-directed repair.

Author information

1
The Institute of Molecular Health Sciences, ETH Zurich, Zurich, Switzerland.
2
The Institute of Molecular Life Sciences, University of Zurich, Zurich, Switzerland.
3
SIB Swiss Institute of Bioinformatics, Zurich, Switzerland.
4
Institute for Pharmaceutical Sciences, ETH Zurich, Zurich, Switzerland.
5
Department of Biochemistry, University of Zurich, Zurich, Switzerland.
#
Contributed equally

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.

KEYWORDS:

CRISPR/Cas9; chromosomes; gene editing; gene expression; homology-directed repair; human; human biology; medicine; mouse

PMID:
29809142
PMCID:
PMC6023611
DOI:
10.7554/eLife.33761
[Indexed for MEDLINE]
Free PMC Article

Conflict of interest statement

NS, FR, HL, CB, KB, YL, DN, MR, CC, JH, MJ, GS No competing interests declared

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