High-frequency modification of plant genes using engineered zinc-finger nucleases

Abstract

An efficient method for making directed DNA sequence modifications to plant genes (gene targeting) is at present lacking, thereby frustrating efforts to dissect plant gene function and engineer crop plants that better meet the world's burgeoning need for food, fibre and fuel. Zinc-finger nucleases (ZFNs)-enzymes engineered to create DNA double-strand breaks at specific loci-are potent stimulators of gene targeting; for example, they can be used to precisely modify engineered reporter genes in plants. Here we demonstrate high-frequency ZFN-stimulated gene targeting at endogenous plant genes, namely the tobacco acetolactate synthase genes (ALS SuRA and SuRB), for which specific mutations are known to confer resistance to imidazolinone and sulphonylurea herbicides. Herbicide-resistance mutations were introduced into SuR loci by ZFN-mediated gene targeting at frequencies exceeding 2% of transformed cells for mutations as far as 1.3 kilobases from the ZFN cleavage site. More than 40% of recombinant plants had modifications in multiple SuR alleles. The observed high frequency of gene targeting indicates that it is now possible to efficiently make targeted sequence changes in endogenous plant genes.

Figure 1

The tobacco SuRB locus. a) The diagram is drawn to scale and annotated with ZFN sites, amino substitutions that confer herbicide resistance, PCR primers used to characterize recombinants, and the region used as a donor template. b) ZFN target sites. Left and right denote bases recognized by each ZFA. Underlined bases are either SuRA sequences that differ from SuRB or mutated bases in the donor (bottom row) that prevent cleavage by the ZFN. c) Sequences at the sites of introduced mutations. The targeted amino acid is underlined, as are sequences in SuRA that differ from SuRB and silent nucleotide changes in the donor template that distinguish recombinants from spontaneous mutants.

Figure 2

Activity of engineered ZFAs and ZFNs. a) ZFAs as stimulators of recombination in yeast. Target sites for each ZFA are listed in vertical text below the chart; H, high-copy plasmid; L, low-copy plasmid. Error bars denote s.d.; n = 3. b) Engineered ZFNs as stimulators of mutagenesis by NHEJ in tobacco. ZFNs were expressed in protoplasts, and the SuRA and SuRB target sites were analyzed by pyrosequencing. The number of sequences with insertions/deletions (indels) was divided by the total number of reads for a given target and normalized to a Zif268 control. Values above the X axis indicate a higher proportion of sequences with indels than the control. Error bars denote 95% confidence intervals; n = 4.