Vol 459 21 May 2009 doi 10 1038 nature07992 LETTERS Precise genome modification in the crop species Zea mays using zinc finger nucleases Vipula 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 Urnov2 Agricultural biotechnology is limited by the inefficiencies of conventional random mutagenesis and transgenesis Because targeted genome modification in plants has been intractable1 plant trait engineering remains a laborious time consuming and unpredictable undertaking Here we report a broadly applicable versatile solution 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 plants of the crop species Zea mays We show that simultaneous expression of ZFNs and delivery of a simple heterologous donor molecule leads to precise targeted addition of an herbicide tolerance gene at the intended locus in a significant number of isolated events ZFNmodified maize plants faithfully transmit these genetic changes to the next generation Insertional disruption of one target locus IPK1 results in both herbicide tolerance and the expected alteration of the inositol phosphate profile in developing seeds ZFNs can be used in any plant species amenable to DNA delivery our results therefore establish a new strategy for plant genetic manipulation in basic science and agricultural applications Current approaches to the challenge of improving agricultural productivity and global food production for example enhancing yield or engineering pest resistance rely on conventional biotechnology approaches such as mutation breeding or transformation of novel genes into crop genomes Both processes are inherently nonspecific and relatively inefficient Targeted genome modification in plant systems which has been a long standing but elusive goal can overcome many of these logistical challenges Previous efforts to drive targeted gene addition at endogenous loci in rice and Arabidopsis have relied on analysis of large numbers of transformants in order to recover extremely rare desired events3 Zinc finger nucleases ZFNs are a fusion of zinc finger based DNA recognition modules to an endonuclease domain4 ZFNs act by invoking the recombinogenic repair potential of a doublestranded break DSB in the DNA of living cells5 7 ZFN induced DSBs enhance gene targeting at engineered loci in human cells8 and in model plants9 10 Because the zinc finger domain11 12 can be engineered to recognize novel DNA sequences13 14 ZFNs have been widely exploited for genome engineering at endogenous loci in eukaryotic systems reviewed in ref 2 In this study we assessed whether ZFN driven gene addition could be used for trait engineering at an endogenous locus in maize We targeted the IPK1 gene which encodes inositol 1 3 4 5 6 pentakisphosphate 2 kinase an enzyme that catalyses the final step in phytate biosynthesis in maize seeds15 IPK1 represents an attractive choice for targeting because phytate reduction is agriculturally important phytate accounts for 75 of total seed phosphorus16 is an anti nutritional component of feed grains and contributes to environmental pollution through the waste stream Efforts to manipulate phytate accumulation via genetic modification have focused on reducing eliminating the activities of enzymes that catalyse the conversion of inositol phosphate intermediates17 18 Two Z mays IPK gene paralogues here referred to as IPK1 and IPK2 exist in the maize genome and share 98 sequence identity in the coding regions15 IPK1 was selected for targeting based on its expression pattern Using an archive of pre validated 2 finger modules19 we generated a panel of 66 ZFNs against 5 distinct positions of IPK1 focusing on the first two thirds of the coding region and selecting sequences containing inter paralogous single nucleotide polymorphisms SNPs Fig 1a and Supplementary Fig 1 see also Supplementary Table 6 for ZFN engineering and testing statistics To overcome the low rates of DNA delivery to plant embryos or cultured cells20 21 the ZFNs were initially assessed for efficacy using a mammalian reporter assay system8 Supplementary Fig 2 followed by a yeast based proxy system22 Fig 1b On the basis of this analysis four ZFN pairs targeting the DNA sequences of Ile 71 and His 100 in exon 2 were selected for the specific editing of IPK1 Fig 1a and Supplementary Table 1 Repair of ZFN induced DSBs by non homologous end joining NHEJ generates small deletions and insertions at the ZFN cleavage site23 24 which provide a rapid indicator of ZFN activity at endogenous loci In cultured maize cells that were transiently expressing ZFN 12 but not controls analyses of 6 5 3 104 chromatids revealed 28 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 a selection based scheme to disrupt IPK1 by insertional gene addition Two different donor constructs were generated each containing short homology arms19 25 27 one carried an autonomous herbicidetolerance gene expression cassette PAT whereas the second carried a non autonomous donor that relied on precise trapping of the endogenous IPK1 promoter for expression of the marker Fig 2a Four ZFN pairs designed to cleave IPK1 at two positions in exon 2 were independently delivered to maize cells along with either autonomous or non autonomous donor plasmids Transformed herbicidetolerant calli were genotyped at the IPK1 locus Fig 2b and Table 1 our 1 Dow AgroSciences 9330 Zionsville Road Indianapolis Indiana 46268 USA 2Sangamo BioSciences Point Richmond Tech Center 501 Canal Boulevard Suite A100 Richmond California 94804 USA 437 2009 Macmillan Publishers Limited All rights reserved LETTERS a NATURE Vol 459 21 May 2009 Zea mays IPK1 1 2 3 4 5 Exon 2 1 ZFN 8 12 Activity mU b 60 50 40 30 20 10 0 ZFN 12 exon 2 1 7 Exon 2 2 ZFN 15 16 IPK1 Control 6 IPK2 ZFN 15 exon 2 2 Control ZFN 12 exon 2 1 ZFN 15 exon 2 2 60 50 40 30 20 10 0 0h 2h 4h 6h c Wild
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