Trait stacking via targeted genome editing

被引：170

作者：

Ainley, William M. ^{[1
]}

Sastry-Dent, Lakshmi ^{[1
]}

Welter, Mary E. ^{[1
]}

Murray, Michael G. ^{[1
]}

Zeitler, Bryan ^{[2
]}

Amora, Rainier ^{[2
]}

Corbin, David R. ^{[1
]}

Miles, Rebecca R. ^{[1
]}

Arnold, Nicole L. ^{[1
]}

Strange, Tonya L. ^{[1
]}

Simpson, Matthew A. ^{[1
]}

Cao, Zehui ^{[1
]}

Carroll, Carley ^{[1
]}

Pawelczak, Katherine S. ^{[1
]}

Blue, Ryan ^{[1
]}

West, Kim ^{[1
]}

Rowland, Lynn M. ^{[1
]}

Perkins, Douglas ^{[1
]}

Samuel, Pon ^{[1
]}

Dewes, Cristie M. ^{[1
]}

Shen, Liu ^{[1
]}

Sriram, Shreedharan ^{[1
]}

Evans, Steven L. ^{[1
]}

Rebar, Edward J. ^{[2
]}

Zhang, Lei ^{[2
]}

Gregory, Phillip D. ^{[2
]}

Urnov, Fyodor D. ^{[2
]}

Webb, Steven R. ^{[1
]}

Petolino, Joseph F. ^{[1
]}

机构：

[1] Dow AgroSci LLC, Indianapolis, IN USA

[2] Sangamo BioSci Inc, Richmond, CA USA

来源：

PLANT BIOTECHNOLOGY JOURNAL | 2013年 / 11卷 / 09期

关键词：

gene targeting; designed zinc finger nucleases; transgene stacking; SITE-SPECIFIC INTEGRATION; HOMOLOGOUS RECOMBINATION; TRANSGENE INTEGRATION; MULTIPLE TRANSGENES; FINGER; GENE; RESISTANCE; PLANTS; DNA; ARABIDOPSIS;

D O I：

10.1111/pbi.12107

中图分类号：

Q81 [生物工程学（生物技术）]; Q93 [微生物学];

学科分类号：

071005 ; 0836 ; 090102 ; 100705 ;

摘要：

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.

引用

页码：1126 / 1134

页数：9

共 40 条

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