Directed evolution and substrate specificity profile of homing endonuclease I-SceI.
The laboratory evolution of enzymes with tailor-made DNA cleavage specificities would represent new tools for manipulating genomes and may enhance our understanding of sequence-specific DNA recognition by nucleases. Below we describe the development and successful application of an efficient in vivo positive and negative selection system that applies evolutionary pressure either to favor the cleavage of a desired target sequence or to disfavor the cleavage of nontarget sequences. We also applied a previously described in vitro selection method to reveal the comprehensive substrate specificity profile of the wild-type I-SceI homing endonuclease. Together these tools were used to successfully evolve mutant I-SceI homing endonucleases with altered DNA cleavage specificities. The most highly evolved enzyme cleaves the target mutant DNA sequence with a selectivity that is comparable to wild-type I-SceI's preference for its cognate substrate.
Doyon
Jeffrey B
JB
Howard Hughes Medical Institute and the Department of Chemistry and Chemical Biology, Harvard University, Cambridge, MA 02138, USA.
Pattanayak
Vikram
V
Meyer
Carissa B
CB
Liu
David R
DR
D004247:DNA / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D015252:Deoxyribonucleases, Type II Site-Specific / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*; D019143:Evolution, Molecular; D008958:Models, Molecular; D009154:Mutation; D010957:Plasmids / Q000235:genetics; D029701:Saccharomyces cerevisiae Proteins; D013379:Substrate Specificity
D029701:Saccharomyces cerevisiae Proteins; D004247:DNA; C037725:SCEI protein, S cerevisiae; D015252:Deoxyribonucleases, Type II Site-Specific