title: Engineered I-CreI derivatives cleaving sequences from the human XPC gene can induce highly efficient gene correction in mammalian cells.
abstract: 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-CreI 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-CreI 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-CreI 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-CreI derivatives, and we could improve the activity of weak novel meganucleases, to eventually match the activity of the parental I-CreI scaffold. The novel proteins maintain a narrow cleavage pattern for cognate targets, showing that the extensive redesign of the I-CreI 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-CreI derivatives cleaving sequences from the XPC gene were found to induce high levels of gene targeting, similar to the I-CreI scaffold or the I-SceI "gold standard". This is the first time an engineered homing endonuclease has been used to modify a chromosomal locus.
authors.affiliation: CELLECTIS S.A., 102 route de Noisy 93235 Romainville, France.
authors.forename: Sylvain
authors.initials: S
authors.lastname: Arnould
authors.forename: Christophe
authors.initials: C
authors.lastname: Perez
authors.forename: Jean-Pierre
authors.initials: JP
authors.lastname: Cabaniols
authors.forename: Julianne
authors.initials: J
authors.lastname: Smith
authors.forename: Agnès
authors.initials: A
authors.lastname: Gouble
authors.forename: Sylvestre
authors.initials: S
authors.lastname: Grizot
authors.forename: Jean-Charles
authors.initials: JC
authors.lastname: Epinat
authors.forename: Aymeric
authors.initials: A
authors.lastname: Duclert
authors.forename: Philippe
authors.initials: P
authors.lastname: Duchateau
authors.forename: Frédéric
authors.initials: F
authors.lastname: Pâques
mesh_terms: D000595:Amino Acid Sequence; D000818:Animals; D001483:Base Sequence; D016466:CHO Cells; D006224:Cricetinae; D003412:Cricetulus; D004262:DNA Restriction Enzymes / Q000737:chemistry / Q000235:genetics / Q000378:metabolism*; D004268:DNA-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019281:Dimerization; D018390:Gene Targeting; D017930:Genes, Reporter; D006801:Humans; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009154:Mutation; D015202:Protein Engineering; D017434:Protein Structure, Tertiary; D012441:Saccharomyces cerevisiae / Q000235:genetics / Q000378:metabolism
chemical_list: D004268:DNA-Binding Proteins; C086984:XPC protein, human; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI