title: Mining endonuclease cleavage determinants in genomic sequence data.
abstract: Homing endonucleases have great potential as tools for targeted gene therapy and gene correction, but identifying variants of these enzymes capable of cleaving specific DNA targets of interest is necessary before the widespread use of such technologies is possible. We identified homologues of the LAGLIDADG homing endonuclease I-AniI and their putative target insertion sites by BLAST searches followed by examination of the sequences of the flanking genomic regions. Amino acid substitutions in these homologues that were located close to the target site DNA, and thus potentially conferring differences in target specificity, were grafted onto the I-AniI scaffold. Many of these grafts exhibited novel and unexpected specificities. These findings show that the information present in genomic data can be exploited for endonuclease specificity redesign.
authors.affiliation: Department of Biochemistry, Howard Hughes Medical Institute, University of Washington, Seattle, Washington 98195, USA.
authors.forename: Mindy D
authors.initials: MD
authors.lastname: Szeto
authors.forename: Sandrine J S
authors.initials: SJ
authors.lastname: Boissel
authors.forename: David
authors.initials: D
authors.lastname: Baker
authors.forename: Summer B
authors.initials: SB
authors.lastname: Thyme
mesh_terms: D004247:DNA / Q000235:genetics*; D004706:Endodeoxyribonucleases; D016678:Genome / Q000235:genetics*; D015183:Restriction Mapping
chemical_list: D004247:DNA; D004706:Endodeoxyribonucleases