Efficient single-copy HDR by 5' modified long dsDNA donors

Abstract
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Abstract

CRISPR/Cas9 efficiently induces targeted mutations via non-homologous-end-joining but for genome editing, precise, homology-directed repair (HDR) of endogenous DNA stretches is a prerequisite. To favor HDR, many approaches interfere with the repair machinery or manipulate Cas9 itself. Using Medaka we show that the modification of 5' ends of long dsDNA donors strongly enhances HDR, favors efficient single-copy integration by retaining a monomeric donor conformation thus facilitating successful gene replacement or tagging.

Data availability

All data generated or analyzed during this study are included in the manuscript and supporting files. Source data files have been provided for Figure 2-figure supplement 1.

Article and author information

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Jose Arturo Gutierrez-Triana

Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

Competing interests
The authors declare that no competing interests exist.
Tinatini Tavhelidse

Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-6103-9019
Thomas Thumberger

Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

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The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8485-457X
Isabelle Thomas

Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

Competing interests
The authors declare that no competing interests exist.
Beate Wittbrodt

Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

Competing interests
The authors declare that no competing interests exist.
Tanja Kellner

Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

Competing interests
The authors declare that no competing interests exist.
Kerim Anlas

Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

Competing interests
The authors declare that no competing interests exist.
Erika Tsingos

Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0002-7267-160X
Joachim Wittbrodt

Centre for Organismal Studies, Heidelberg University, Heidelberg, Germany

For correspondence
jochen.wittbrodt@cos.uni-heidelberg.de

Competing interests
The authors declare that no competing interests exist.

"This ORCID iD identifies the author of this article:" 0000-0001-8550-7377

Funding

Deutsche Forschungsgemeinschaft (CRC 873 project A3)

Joachim Wittbrodt

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Copyright

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.