Site-directed mutagenesis in Arabidopsis using custom-designed zinc finger nucleases.pdf

Site-directed mutagenesis in Arabidopsis using
custom-designed zinc finger nucleases
Keishi Osakabea, Yuriko Osakabeb, and Seiichi Tokia,c,1
aPlant ic Engineering Research Unit, Division of Plant Sciences, National Institute of Agrobiological Sciences, Tsukuba 305-8602, Japan; bGraduate School
of Agricultural and Life Sciences, The University of Tokyo, Tokyo 113-8657, Japan; and cKihara Institute of Biological Research, Yokohama City University,
Yokohama 244-0813, Japan
Edited by Joseph R. Ecker, Salk Institute, La Jolla, CA, and approved May 3, 2010 (received for review January 9, 2010)
Site-directed mutagenesis in higher plants remains a significant Indeed, expression of I-Sce I, a rare cutting restriction enzyme, has
technical challenge for basic research and molecular breeding. Here, been shown to introduce mutations at I-Sce I cleavage sites in
we demonstrate targeted-gene inactivation for an endogenous Arabidopsis and o (12). Nevertheless, the use of restriction
gene in Arabidopsis using zinc finger nucleases (ZFNs). Engineered enzymes is limited to rarely occurring natural recognition sites or
ZFNs for a stress-response regulator, the ABA-INSENSITIVE4 (ABI4) to artificial target sites. To e this problem, zinc finger
gene, cleaved their recognition sequences specifically in vitro, and nucleases (ZFNs) have been developed. ZFNs are chimeric pro-
ZFN genes driven by