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Sci Rep. 2018 Jan 11;8(1):474. doi: 10.1038/s41598-017-18826-5.

Electroporation of mice zygotes with dual guide RNA/Cas9 complexes for simple and efficient cloning-free genome editing.

Author information

1
SFR BioSciences, Plateau de Biologie Expérimentale de la Souris (AniRA-PBES), Ecole Normale Supérieure de Lyon, Université Lyon1, CNRS UMS3444, INSERM US8, 69007, Lyon, France.
2
CIRI, INSERM U1111, Université Claude Bernard Lyon 1, CNRS UMR 5308, École Normale Supérieure de Lyon, Université de Lyon, 69007, Lyon, France.
3
Université Grenoble Alpes, Grenoble Institut des Neurosciences, GIN, F-38000, Grenoble, France.
4
INSERM, U1216, F-38000, Grenoble, France.
5
Institut de Génomique Fonctionnelle de Lyon, INRA USC 1370, Université de Lyon, Université Lyon 1, CNRS UMR 5242, Ecole Normale Supérieure de Lyon, 46, allée d'Italie, 69007, Lyon, France.
6
Institut de Génomique Fonctionnelle de Lyon, INRA USC 1370, Université de Lyon, Université Lyon 1, CNRS UMR 5242, Ecole Normale Supérieure de Lyon, 46, allée d'Italie, 69007, Lyon, France. Suzy.Markossian@ens-lyon.fr.

Abstract

In this report, we present an improved protocol for CRISPR/Cas9 genome editing in mice. The procedure consists in the electroporation of intact mouse zygotes with ribonucleoprotein complexes prepared in vitro from recombinant Cas9 nuclease and synthetic dual guide RNA. This simple cloning-free method proves to be extremely efficient for the generation of indels and small deletions by non-homologous end joining, and for the generation of specific point mutations by homology-directed repair. The procedure, which avoids DNA construction, in vitro transcription and oocyte microinjection, greatly simplifies genome editing in mice.

PMID:
29323173
PMCID:
PMC5764989
DOI:
10.1038/s41598-017-18826-5
[Indexed for MEDLINE]
Free PMC Article

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