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Efficient single copy integration via homology-directed repair (scHDR) by 5’modification of large DNA donor fragments in mice

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CRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as conca...

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Main Authors: Medert, Rebekka (Author) , Thumberger, Thomas (Author) , Tavhelidse-Suck, Tinatini (Author) , Hub, Tobias (Author) , Kellner, Tanja (Author) , Oguchi, Yoko (Author) , Dlugosz, Sascha (Author) , Zimmermann, Frank (Author) , Wittbrodt, Joachim (Author) , Freichel, Marc (Author)
Format: Article (Journal) Chapter/Article
Language:English
Published: September 30, 2021
In: bioRxiv beta
Year: 2021, Pages: 1-37
DOI:10.1101/2021.09.30.462539
Online Access:Verlag, lizenzpflichtig, Volltext: https://doi.org/10.1101/2021.09.30.462539
Verlag, lizenzpflichtig, Volltext: https://www.biorxiv.org/content/10.1101/2021.09.30.462539v1
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Author Notes:Rebekka Medert, Thomas Thumberger, Tinatini Tavhelidse, Tobias Hub, Tanja Kellner, Yoko Oguchi, Sascha Dlugosz, Frank Zimmermann, Joachim Wittbrodt, Marc Freichel
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Summary:CRISPR/Cas approaches have largely replaced conventional gene targeting strategies. However, homology-directed repair (HDR) in the mouse genome is not very efficient, and precisely inserting longer sequences using HDR remains challenging, given that donor constructs preferentially integrate as concatemers. Here, we show that injecting 5’biotinylated donor DNA in mouse embryos at the two-cell stage leads to efficient single-copy HDR (scHDR) alleles. Our dedicated genotyping strategy showed that these alleles occurred with a frequency of 19%, 20%, and 26%, respectively, in three independent gene loci, indicating that scHDR is dramatically boosted by 5’biotinylation. Thus, we suggest that a combination of a 5’biotinylated donor and diligent analysis of concatemer integration are prerequisites for efficiently and reliably generating conditional alleles or other large fragment knock-ins into the mouse genome.
Item Description:Gesehen am 26.02.2024
Physical Description:Online Resource
DOI:10.1101/2021.09.30.462539

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