Heritable targeted mutagenesis in maize using a designed endonuclease.
The liguleless locus (liguleless1) was chosen for demonstration of targeted mutagenesis in maize using an engineered endonuclease derived from the I-CreI homing endonuclease. A single-chain endonuclease, comprising a pair of I-CreI monomers fused into a single polypeptide, was designed to recognize a target sequence adjacent to the LIGULELESS1 (LG1) gene promoter. The endonuclease gene was delivered to maize cells by Agrobacterium-mediated transformation of immature embryos, and transgenic T(0) plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T(0) plants, each of which was regenerated from independently selected callus. Plants that were monoallelic, biallelic and chimeric for mutations at the liguleless1 locus were found. Relatively short deletions (shortest 2 bp, longest 220 bp) were most frequently identified at the expected cut site, although short insertions were also detected at this site. We show that rational re-design of an endonuclease can produce a functional enzyme capable of introducing double-strand breaks at selected chromosomal loci. In combination with DNA repair mechanisms, the system produces targeted mutations with sufficient frequency that dedicated selection for such mutations is not required. Re-designed homing endonucleases are a useful molecular tool for introducing targeted mutations in a living organism, specifically a maize plant.
Gao
Huirong
H
Pioneer Hi-Bred International, Research Center, 7300 NW 62nd Avenue, Johnston, IA 50131-1004, USA.
Smith
Jeff
J
Yang
Meizhu
M
Jones
Spencer
S
Djukanovic
Vesna
V
Nicholson
Michael G
MG
West
Ande
A
Bidney
Dennis
D
Falco
S Carl
SC
Jantz
Derek
D
Lyznik
L Alexander
LA
D004262:DNA Restriction Enzymes / Q000235:genetics / Q000378:metabolism*; D016296:Mutagenesis / Q000235:genetics*; D010940:Plant Proteins / Q000235:genetics; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic / Q000235:genetics; D003313:Zea mays / Q000235:genetics*
C105241:LG1 protein, Zea mays; D010940:Plant Proteins; D004262:DNA Restriction Enzymes; C048901:endodeoxyribonuclease CreI