1. Macromolecular Crystallography Group,
2. NMR group, Structural Biology and Biocomputing Programme, Spanish National Cancer Research Centre (CNIO), c/Melchor Fdez. Almagro 3, 28029 Madrid, Spain
3. European Molecular Biology Laboratory (EMBL)-CRG Systems Biology Unit, Centre de Regulació Genòmica (CRG), Universitat Pompeu Fabra (UPF), Dr Aiguader 88, 08003 Barcelona, Spain
4. Institució Catalana de Recerca i Estudis Avançats (ICREA),
5. CELLECTIS S.A., 102 Avenue Gaston Roussel, 93235 Romainville, France
6. These authors contributed equally to this work.
7. Present address: CIC bioGUNE, Parque Tecnológico de Vizcaya, Edificio 800, 48160 Derio, Spain.

Correspondence to: Francisco J. Blanco^2,7Guillermo Montoya¹ Correspondence and requests for materials should be addressed to F.J.B. (Email: fblanco@cicbiogune.es) or G.M. (Email: gmontoya@cnio.es).

Abstract

Xeroderma pigmentosum is a monogenic disease characterized by hypersensitivity to ultraviolet light. The cells of xeroderma pigmentosum patients are defective in nucleotide excision repair, limiting their capacity to eliminate ultraviolet-induced DNA damage, and resulting in a strong predisposition to develop skin cancers¹. The use of rare cutting DNA endonucleases—such as homing endonucleases, also known as meganucleases—constitutes one possible strategy for repairing DNA lesions. Homing endonucleases have emerged as highly specific molecular scalpels that recognize and cleave DNA sites, promoting efficient homologous gene targeting through double-strand-break-induced homologous recombination. Here we describe two engineered heterodimeric derivatives of the homing endonuclease I-CreI, produced by a semi-rational approach. These two molecules—Amel3–Amel4 and Ini3–Ini4—cleave DNA from the human XPC gene (xeroderma pigmentosum group C), in vitro and in vivo. Crystal structures of the I-CreI variants complexed with intact and cleaved XPC target DNA suggest that the mechanism of DNA recognition and cleavage by the engineered homing endonucleases is similar to that of the wild-type I-CreI. Furthermore, these derivatives induced high levels of specific gene targeting in mammalian cells while displaying no obvious genotoxicity. Thus, homing endonucleases can be designed to recognize and cleave the DNA sequences of specific genes, opening up new possibilities for genome engineering and gene therapy in xeroderma pigmentosum patients whose illness can be treated ex vivo.

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