Journal Article

A highly sensitive selection method for directed evolution of homing endonucleases

Zhilei Chen and Huimin Zhao

in Nucleic Acids Research

Volume 33, issue 18, pages e154-e154
Published in print January 2005 | ISSN: 0305-1048
Published online January 2005 | e-ISSN: 1362-4962 | DOI: https://dx.doi.org/10.1093/nar/gni148

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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.

Journal Article.  5592 words.  Illustrated.

Subjects: Research Methods in Life Sciences

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