A highly sensitive selection method for directed evolution of homing endonucleases. Academic Article

abstract

• Homing endonucleases are enzymes that catalyze DNA sequence specific double-strand breaks and can significantly stimulate homologous recombination at these breaks. These enzymes have great potential for applications such as gene correction in gene therapy or gene alteration in systems biology and metabolic engineering. However, homing endonucleases have a limited natural repertoire of target sequences, which severely hamper their applications. Here we report the development of a highly sensitive selection method for the directed evolution of homing endonucleases that couples enzymatic DNA cleavage with the survival of host cells. Using I-SceI as a model homing endonuclease, we have demonstrated that cells with wild-type I-SceI showed a high cell survival rate of 80-100% in the presence of the original I-SceI recognition site, whereas cells without I-SceI showed a survival rate <0.003%. This system should also be readily applicable for directed evolution of other DNA cleavage enzymes.

authors

• Chen, Zhilei

published proceedings

• Nucleic Acids Res

altmetric score

• 3

author list (cited authors)

• Chen, Z., & Zhao, H.

citation count

• 146

complete list of authors

• Chen, Zhilei||Zhao, Huimin

publication date

• October 2005

published in

• n1362-4962ISSN  Journal

keywords

• Deoxyribonucleases, Type II Site-Specific
• Directed Molecular Evolution
• Endodeoxyribonucleases
• Escherichia Coli
• Plasmids
• Saccharomyces Cerevisiae Proteins
• Substrate Specificity
• Transformation, Bacterial

PubMed Central ID

• 16214805

Digital Object Identifier (DOI)

• 10.1093/nar/gni148

start page

• e154

volume

• 33

issue

• 18