TitleAuthorsAbstractMethods SummaryReferencesMethodsZFN design and proxy system validationPlasmid constructionNucleic acid analysesPlant cell cultureInorganic phosphate and phytate analysisMethods ReferencesFigure 1 ZFNs designed to target Z. mays IPK1 induce DSBs at the endogenous locus.Figure 2 Targeted gene addition to IPK1 in maize.Figure 3 ZFN-mediated gene disruption of Z. mays IPK1 is stable and heritable.Table 1 Frequency of targeted integration of PAT into Z. mays IPK1LETTERSPrecise genome modification in the crop species Zeamays using zinc-finger nucleasesVipula K. Shukla1, Yannick Doyon2, Jeffrey C. Miller2, Russell C. DeKelver2, Erica A. Moehle2, Sarah E. Worden1,Jon C. Mitchell1, Nicole L. Arnold1, Sunita Gopalan2, Xiangdong Meng2, Vivian M. Choi2, Jeremy M. Rock2,Ying-Ying Wu2, George E. Katibah2, Gao Zhifang1, David McCaskill1, Matthew A. Simpson1, Beth Blakeslee1,Scott A. Greenwalt1, Holly J. Butler1, Sarah J. Hinkley2, Lei Zhang2, Edward J. Rebar2, Philip D. Gregory2& Fyodor D. Urnov2Agricultural biotechnology is limited by the inefficiencies of con-ventional random mutagenesis and transgenesis. Because targetedgenome modification in plants has been intractable1, plant traitengineering remains a laborious, time-consuming and unpredict-able undertaking. Here we report a broadly applicable, versatilesolution to this problem: the use of designed zinc-finger nucleases(ZFNs) that induce a double-stranded break at their target locus2.We describe the use of ZFNs to modify endogenous loci in plantsof the crop species Zea mays. We show that simultaneous expres-sion of ZFNs and delivery of a simple heterologous donor moleculeleads to precise targeted addition of an herbicide-tolerance gene atthe intended locus in a significant number of isolated events. ZFN-modified maize plants faithfully transmit these genetic changes tothe next generation. Insertional disruption of one target locus,IPK1, results in both herbicide tolerance and the expected altera-tion of the inositol phosphate profile in developing seeds. ZFNscan be used in any plant species amenable to DNA delivery; ourresults therefore establish a new strategy for plant genetic manipu-lation in basic science and agricultural applications.Current approaches to the challenge of improving agriculturalproductivity and global food production (for example, enhancingyield or engineering pest resistance) rely on conventional biotech-nology approaches such as mutation breeding or transformation ofnovel genes into crop genomes. Both processes are inherently non-specific and relatively inefficient. Targeted genome modification inplant systems, which has been a long-standing but elusive goal, canovercome many of these logistical challenges. Previous efforts todrive targeted gene addition at endogenous loci in rice andArabidopsis have relied on analysis of large numbers of transformantsin order to recover extremely rare desired events3.Zinc finger nucleases (ZFNs) are a fusion of zinc-finger-basedDNA recognition modules to an endonuclease domain4. ZFNs actby invoking the recombinogenic repair potential of a double-stranded break (DSB) in the DNA of living cells5–7. ZFN-inducedDSBs enhance gene targeting at engineered loci in human cells8and in model plants9,10. Because the zinc-finger domain11,12can beengineered to recognize novel DNA sequences13,14, ZFNs have beenwidely exploited for genome engineering at endogenous loci ineukaryotic systems (reviewed in ref. 2).In this study, we assessed whether ZFN-driven gene addition couldbe used for trait engineering at an endogenous locus in maize. Wetargeted the IPK1 gene, which encodes inositol-1,3,4,5,6-penta-kisphosphate 2-kinase, an enzyme that catalyses the final step inphytate biosynthesis in maize seeds15. IPK1 represents an attractivechoice for targeting because phytate reduction is agriculturallyimportant: phytate accounts for ,75% of total seed phosphorus16,is an anti-nutritional component of feed grains and contributes toenvironmental pollution through the waste stream. Efforts to mani-pulate phytate accumulation via genetic modification have focused onreducing/eliminating the activities of enzymes that catalyse the con-version of inositol phosphate intermediates17,18.Two Z. mays IPK geneparalogues, herereferred to as IPK1 and IPK2,exist in the maize genome and share 98% sequence identity in thecoding regions15; IPK1 was selected for targeting based on its expres-sion pattern. Using an archive of pre-validated 2-finger modules19,wegenerated a panel of 66 ZFNs against 5 distinct positions of IPK1,focusing on the first two-thirds of the coding region and selectingsequences containing inter-paralogous single nucleotide polymor-phisms (SNPs; Fig. 1a and Supplementary Fig. 1; see also Sup-plementary Table 6 for ZFN engineering and testing statistics). Toovercome the low rates of DNA delivery to plant embryos or culturedcells20,21, the ZFNs were initially assessed for efficacy using a mam-malian reporter assay system8(Supplementary Fig. 2), followed by ayeast-based proxy system22(Fig. 1b). On the basis of this analysis, fourZFN pairs targeting the DNA sequences of Ile 71 and His 100 in exon 2were selected for the specific editing of IPK1 (Fig. 1a andSupplementary Table 1).Repair of ZFN-induced DSBs by non-homologous end-joining(NHEJ) generates small deletions and insertions at the ZFN cleavagesite23,24, which provide a rapid indicator of ZFN activity at endoge-nous loci. In cultured maize cells that were transiently expressingZFN 12 (but not controls), analyses of 6.5 3 104chromatids revealed28 deletions and 2 insertions aligning to the ZFN target site (Fig. 1c).This result illustrates that the human and yeast proxy system (Fig. 1b)identify ZFNs that induce a DSB at their intended target in plant cells.Having established ZFN cleavage activity in planta, we used aselection-based scheme to disrupt IPK1 by insertional gene addition.Two different donor constructs were generated, each containingshort homology arms19,25–27: one carried an autonomous herbicide-tolerance gene expression cassette (PAT), whereas the second carrieda non-autonomous donor that relied on precise trapping of theendogenous IPK1 promoter for expression of the marker (Fig. 2a).Four ZFN pairs designed to cleave IPK1 at two positions in exon 2were independently del ivered to maize cells, along with either autono-mous or non-autonomous donor plasmids. Transformed, herbicide-tolerant calli were genotyped at the IPK1
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