title: Directed evolution of homing endonuclease I-SceI with altered sequence specificity.
abstract: Homing endonucleases recognize specific long DNA sequences and catalyze double-stranded breaks that significantly stimulate homologous recombination, representing an attractive tool for genome targeting and editing. We previously described a two-plasmid selection system that couples enzymatic DNA cleavage with the survival of host cells, and enables directed evolution of homing endonucleases with altered cleavage sequence specificity. Using this selection system, we successfully evolved mutant I-SceI homing endonucleases with greatly increased cleavage activity towards a new target DNA sequence that differs from the wild-type cleavage sequence by 4 bp. The most highly evolved mutant showed a survival rate approximately 100-fold higher than that of wild-type I-SceI enzyme. The degree of selectivity displayed by a mutant isolated from one round of saturation mutagenesis for the new target sequence is comparable to that of wild-type I-SceI for the natural sequence. These results highlight the ability and efficiency of our selection system for engineering homing endonucleases with novel DNA cleavage specificities. The mutant identified from this study can potentially be used in vivo for targeting the new cleavage sequence within genomic DNA.
authors.affiliation: Center for Biophysics and Computational Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA.
authors.forename: Zhilei
authors.initials: Z
authors.lastname: Chen
authors.forename: Fei
authors.initials: F
authors.lastname: Wen
authors.forename: Ning
authors.initials: N
authors.lastname: Sun
authors.forename: Huimin
authors.initials: H
authors.lastname: Zhao
mesh_terms: D001483:Base Sequence; D003198:Computer Simulation; D004247:DNA / Q000378:metabolism; D053837:DNA Cleavage; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D019020:Directed Molecular Evolution; D004926:Escherichia coli / Q000235:genetics; D008958:Models, Molecular; D015202:Protein Engineering; D029701:Saccharomyces cerevisiae Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism; D016415:Sequence Alignment; D013379:Substrate Specificity
chemical_list: D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific