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- Cre I homing endonuclease. A single-chain endonuclease, comprising a pair of I- Cre I 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 T₀ plants were screened for mutations introduced at the liguleless1 locus. We found mutations at the target locus in 3% of the T₀ 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.