Heritable targeted mutagenesis in maize using a designed endonuclease

Abstract

SUMMARYThe 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.

Tapping natural reservoirs of homing endonucleases for targeted gene modification

“…While many engineered ZFNs have been used successfully to modify chromosomal target loci in a variety of multicellular eukaryotic organisms (5,39), to date only the homodimeric I-CreI enzyme has been engineered to modify endogenous chromosomal loci (11,12). However, the properties of monomeric LHEs argue for their continued development as genome-modifying enzymes: They are encoded by particularly short open reading frames that are less than 1,000 base pairs in length, and their cleavage specificities are often very high (18)(19)(20)(21)(22).…”

“…These proteins possess one or two LAGLID-ADG catalytic motifs per protein chain and function as homodimers or monomers, respectively. To date, four naturally occurring LHEs (I-SceI, I-CreI, I-MsoI, and I-AniI) and one chimeric enzyme (DmoCre) have been engineered to alter their sequence recognition specificity (3,4,(8)(9)(10), but only I-CreI has been used to modify endogenous chromosomal targets (11,12). Successful gene targeting required extensive alteration of its DNA contact points and specificity, at up to two-thirds of the base-pair positions in its target site.…”

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…In this study, we used a single, well-characterized QQR ZFN. Naturally, the application of our strategy for native sequences will require the design and assembly of two ZFNs, or some other type of artificially designed restriction enzyme, such as transcription activator-like effector-based nucleases (Cermak et al, 2011;Li et al, 2012), and designed endonucleases (Gao et al, 2010).…”

Dynamic maize responses to aphid feeding are revealed by a time series of transcriptomic and metabolomic assays

“…For the full primer list, see Supplemental Table S9. Genomic DNA preparation was described previously (Gao et al, 2010). All PCRs were performed in a 20-mL GoTaq PCR device (Promega) with either 4% or 5% dimethyl sulfoxide added.…”