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PEDS Advance Access originally published online on January 28, 2009
Protein Engineering Design and Selection 2009 22(4):249-256; doi:10.1093/protein/gzp001
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© The Author 2009. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oxfordjournals.org

Directed evolution of homing endonuclease I-SceI with altered sequence specificity

Zhilei Chen1,7,8, Fei Wen2,7, Ning Sun3 and Huimin Zhao1,2,3,4,5,6,9

1Center for Biophysics and Computational Biology 2Department of Chemical and Biomolecular Engineering 3Department of Biochemistry 4Department of Chemistry 5Department of Bioengineering 6Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

9 To whom correspondence should be addressed. E-mail: zhao5{at}illinois.edu

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 ~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.

Keywords: directed evolution/DNA modifying enzymes/gene targeting/homing endonuclease/protein engineering

Received November 24, 2008; revised December 30, 2008; accepted December 30, 2008.

7 These two authors contributed equally.

8 Present address: Chemical Engineering Department, Texas A&M University, College Station, TX 77843, USA.


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