Engineered I-Crel derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells

Meganucleases are sequence-specific endonucleases which recognize large (> 12 bp) target sites in living cells and can stimulate homologous gene targeting by a 1000-fold factor at the cleaved locus. We have recently described a combinatorial approach to redesign the I-Crel meganuclease DNA-binding interface, in order to target chosen sequences. However, engineering was limited to the protein regions shown to directly interact with DNA in a base-specific manner. Here, we take advantage of I-Crel natural degeneracy, and of additional refinement steps to extend the number of sequences that can be efficiently cleaved. We searched the sequence of the human XPC gene, involved in the disease Xeroderma Pigmentosum (XP), for potential targets, and chose three sequences that differed from the I-Crel cleavage site over their entire length, including the central four base-pairs, whose role in the DNA/protein recognition and cleavage steps remains very elusive. Two out of these targets could be cleaved by engineered I-Crel derivatives, and we could improve the activity of weak novel meganucleases, to eventually match the activity of the parental I-Crel scaffold. The novel proteins maintain a narrow cleavage pattern for cognate targets, showing that the extensive redesign of the I-Crel protein was not made at the expense of its specificity. Finally, we used a chromosomal reporter system in CHO-K1 cells to compare the gene targeting frequencies induced by natural and engineered meganucleases. Tailored I-Crel derivatives cleaving sequences from the XPC gene were found to induce high levels of gene targeting, similar to the I-Crel scaffold or the I-SceI gold standard. This is the first time an engineered homing endonuclease has been used to modify a chromosomal locus. (c) 2007 Elsevier Ltd. All rights reserved.