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Nat Commun. 2018 Mar 19;9(1):1133. doi: 10.1038/s41467-018-03475-7.

CtIP fusion to Cas9 enhances transgene integration by homology-dependent repair.

Author information

1
Museum National d'Histoire Naturelle, INSERM U1154, CNRS UMR 7196, Sorbonne Universités, 43 rue Cuvier, Paris, F-75231, France.
2
Translational Sciences, Sanofi, 13 Quai Jules Guesde, F-94400, Vitry-sur-Seine, France.
3
Centre de Recherche en Transplantation et Immunologie UMR1064, INSERM, Université de Nantes, CHU de Nantes, 30 Avenue Jean Monnet, F-44093, Nantes, France.
4
Equipe Labellisée Ligue Contre le Cancer, Institut de Cancérologie Gustave-Roussy, Université Paris-Saclay, CNRS UMR 8200, 114 rue Edouard Vaillant, Villejuif, F-94805, France.
5
Museum National d'Histoire Naturelle, INSERM U1154, CNRS UMR 7196, Sorbonne Universités, 43 rue Cuvier, Paris, F-75231, France. jean-paul.concordet@mnhn.fr.

Abstract

In genome editing with CRISPR-Cas9, transgene integration often remains challenging. Here, we present an approach for increasing the efficiency of transgene integration by homology-dependent repair (HDR). CtIP, a key protein in early steps of homologous recombination, is fused to Cas9 and stimulates transgene integration by HDR at the human AAVS1 safe harbor locus. A minimal N-terminal fragment of CtIP, designated HE for HDR enhancer, is sufficient to stimulate HDR and this depends on CDK phosphorylation sites and the multimerization domain essential for CtIP activity in homologous recombination. HDR stimulation by Cas9-HE, however, depends on the guide RNA used, a limitation that may be overcome by testing multiple guides to the locus of interest. The Cas9-HE fusion is simple to use and allows obtaining twofold or more efficient transgene integration than that with Cas9 in several experimental systems, including human cell lines, iPS cells, and rat zygotes.

PMID:
29556040
PMCID:
PMC5859065
DOI:
10.1038/s41467-018-03475-7
[Indexed for MEDLINE]
Free PMC Article

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