Directed Evolution Of Homing Endonuclease Isce-I

Protein Engineering, Design & Selection vol. 22 no. 4 pp. 249– 256, 2009 Published online January 28, 2009 doi:10.1093/protein/gzp001

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
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Center for Biophysics and Computational Biology, 2Department of Chemical and Biomolecular Engineering,3Department of Biochemistry, 4 Department of Chemistry, 5Department of Bioengineering and 6Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA
7 8

These two authors contributed equally.

Present address: Chemical Engineering Department, Texas A&M University, College Station, TX 77843, USA. To whom correspondence should be addressed. E-mail:zhao5@illinois.edu

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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 homingendonucleases 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 ofselectivity 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 vivofor targeting the new cleavage sequence within genomic DNA. Keywords: directed evolution/DNA modifying enzymes/gene targeting/homing endonuclease/protein engineering

Introduction The process of homologous recombination (HR) enables in situ replacement of a disease gene with its therapeutic counterpart, so that the integrated gene can be transcribed from the natural promoter without requiringadditional regulatory elements. This process possesses great potential to facilitate treatment of genetic disorders via gene therapy. To date, the most efficient method for inducing HR involves the introduction of a DNA double-stranded break (DSB) in the target gene locus, which results in a 10- to 10 000-fold increased rate of HR (up to 3 – 5%) at this site (Rouet et al., 1994; Choulika et al., 1995;Smih et al., 1995; Porteus and Baltimore, 2003). It is believed that the introduction of a DSB stimulates the HR machinery of the cell, which then

repairs the break by gene exchange with any available homologous template, including an exogenously administered gene-targeting vector (Johnson and Jasin, 2001). Genomic DSBs can be introduced by either natural or artificial DNA cleavage enzymes. Oneof the natural endonucleases commonly used for gene targeting is the homing endonuclease I-SceI (Dujon, 1989). This enzyme recognizes an 18 bp asymmetric DNA sequence that is absent in most mammalian genomes (Colleaux et al., 1988; Jasin, 1996) and catalyzes a DSB at the sequence recognition site. However, this system cannot be used for endogenous gene targeting (Jasin, 1996; Cohen-Tannoudji etal., 1998; Donoho et al., 1998). In order to target an endogenous gene, the cleavage sequence of wild-type I-SceI needs to be modified at multiple sites to match the specific target gene. Towards this goal, researchers have attempted to develop in vivo selection systems for engineering homing endonucleases with altered specificity (Gruen et al., 2002; Seligman et al., 2002; Chen and Zhao, 2005a;...