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Nucleic Acids Research Advance Access originally published online on December 2, 2008
Nucleic Acids Research 2009 37(1):e9; doi:10.1093/nar/gkn943
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Nucleic Acids Research, 2009, Vol. 37, No. 1 e9
© 2008 The Author(s)
This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/by-nc/2.0/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited.

Methods Online

Mutagenic inverted repeat assisted genome engineering (MIRAGE)

Nikhil U. Nair1 and Huimin Zhao1,2,*

1Department of Chemical and Biomolecular Engineering and 2Departments of Chemistry, Biochemistry and Bioengineering, Institute for Genomic Biology, University of Illinois at Urbana-Champaign, Urbana, IL 61801, USA

*To whom correspondence should be addressed. Tel: +1 217 333 2631; Fax: +1 217 333 5052; Email: zhao5{at}uiuc.edu

Received June 3, 2008. Revised November 6, 2008. Accepted November 7, 2008.

Here we describe a one-step method to create precise modifications in the genome of Saccharomyces cerevisiae as a tool for synthetic biology, metabolic engineering, systems biology and genetic studies. Through homologous recombination, a mutagenesis cassette containing an inverted repeat of selection marker(s) is integrated into the genome. Due to its inherent instability in genomic DNA, the inverted repeat catalyzes spontaneous self-excision, resulting in precise genome modification. Since this excision occurs at very high frequencies, selection for the integration event can be followed immediately by counterselection, without the need for growth in permissive conditions. This is the first time a truly one-step method has been described for genome modification in any organism.


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