title: Trait stacking via targeted genome editing.
abstract: Modern agriculture demands crops carrying multiple traits. The current paradigm of randomly integrating and sorting independently segregating transgenes creates severe downstream breeding challenges. A versatile, generally applicable solution is hereby provided: the combination of high-efficiency targeted genome editing driven by engineered zinc finger nucleases (ZFNs) with modular 'trait landing pads' (TLPs) that allow 'mix-and-match', on-demand transgene integration and trait stacking in crop plants. We illustrate the utility of nuclease-driven TLP technology by applying it to the stacking of herbicide resistance traits. We first integrated into the maize genome an herbicide resistance gene, pat, flanked with a TLP (ZFN target sites and sequences homologous to incoming DNA) using WHISKERS™-mediated transformation of embryogenic suspension cultures. We established a method for targeted transgene integration based on microparticle bombardment of immature embryos and used it to deliver a second trait precisely into the TLP via cotransformation with a donor DNA containing a second herbicide resistance gene, aad1, flanked by sequences homologous to the integrated TLP along with a corresponding ZFN expression construct. Remarkably, up to 5% of the embryo-derived transgenic events integrated the aad1 transgene precisely at the TLP, that is, directly adjacent to the pat transgene. Importantly and consistent with the juxtaposition achieved via nuclease-driven TLP technology, both herbicide resistance traits cosegregated in subsequent generations, thereby demonstrating linkage of the two independently transformed transgenes. Because ZFN-mediated targeted transgene integration is becoming applicable across an increasing number of crop species, this work exemplifies a simple, facile and rapid approach to trait stacking.
authors: Dow AgroSciences LLC, Indianapolis, IN, USA.
authors: William M
authors: WM
authors: Ainley
authors: Lakshmi
authors: L
authors: Sastry-Dent
authors: Mary E
authors: ME
authors: Welter
authors: Michael G
authors: MG
authors: Murray
authors: Bryan
authors: B
authors: Zeitler
authors: Rainier
authors: R
authors: Amora
authors: David R
authors: DR
authors: Corbin
authors: Rebecca R
authors: RR
authors: Miles
authors: Nicole L
authors: NL
authors: Arnold
authors: Tonya L
authors: TL
authors: Strange
authors: Matthew A
authors: MA
authors: Simpson
authors: Zehui
authors: Z
authors: Cao
authors: Carley
authors: C
authors: Carroll
authors: Katherine S
authors: KS
authors: Pawelczak
authors: Ryan
authors: R
authors: Blue
authors: Kim
authors: K
authors: West
authors: Lynn M
authors: LM
authors: Rowland
authors: Douglas
authors: D
authors: Perkins
authors: Pon
authors: P
authors: Samuel
authors: Cristie M
authors: CM
authors: Dewes
authors: Liu
authors: L
authors: Shen
authors: Shreedharan
authors: S
authors: Sriram
authors: Steven L
authors: SL
authors: Evans
authors: Edward J
authors: EJ
authors: Rebar
authors: Lei
authors: L
authors: Zhang
authors: Phillip D
authors: PD
authors: Gregory
authors: Fyodor D
authors: FD
authors: Urnov
authors: Steven R
authors: SR
authors: Webb
authors: Joseph F
authors: JF
authors: Petolino
mesh_terms: D018556:Crops, Agricultural; D004720:Endonucleases / Q000235:genetics* / Q000378:metabolism; D018390:Gene Targeting / Q000379:methods*; D008040:Genetic Linkage; D018745:Genome, Plant / Q000235:genetics*; D053209:Herbicide Resistance; D006540:Herbicides / Q000494:pharmacology*; D010641:Phenotype; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D010940:Plant Proteins / Q000235:genetics / Q000378:metabolism; D030821:Plants, Genetically Modified; D019076:Transgenes; D003313:Zea mays / Q000235:genetics*; D016335:Zinc Fingers
chemical_list: D006540:Herbicides; D010940:Plant Proteins; D004720:Endonucleases
keywords: designed zinc finger nucleases; gene targeting; transgene stacking