Targeted transgene integration in plant cells using designed zinc finger nucleases |
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Authors: | Charles Q Cai Yannick Doyon W Michael Ainley Jeffrey C Miller Russell C DeKelver Erica A Moehle Jeremy M Rock Ya-Li Lee Robbi Garrison Lisa Schulenberg Ryan Blue Andrew Worden Lisa Baker Farhoud Faraji Lei Zhang Michael C Holmes Edward J Rebar Trevor N Collingwood Beth Rubin-Wilson Philip D Gregory Fyodor D Urnov Joseph F Petolino |
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Institution: | (1) Dow AgroSciences, LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA;(2) Sangamo BioSciences, 501 Canal Blvd., Suite A 100, Richmond, CA 94804, USA |
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Abstract: | Targeted transgene integration in plants remains a significant technical challenge for both basic and applied research. Here
it is reported that designed zinc finger nucleases (ZFNs) can drive site-directed DNA integration into transgenic and native
gene loci. A dimer of designed 4-finger ZFNs enabled intra-chromosomal reconstitution of a disabled gfp reporter gene and site-specific transgene integration into chromosomal reporter loci following co-transformation of tobacco
cell cultures with a donor construct comprised of sequences necessary to complement a non-functional pat herbicide resistance gene. In addition, a yeast-based assay was used to identify ZFNs capable of cleaving a native endochitinase
gene. Agrobacterium delivery of a Ti plasmid harboring both the ZFNs and a donor DNA construct comprising a pat herbicide resistance gene cassette flanked by short stretches of homology to the endochitinase locus yielded up to 10% targeted,
homology-directed transgene integration precisely into the ZFN cleavage site. Given that ZFNs can be designed to recognize
a wide range of target sequences, these data point toward a novel approach for targeted gene addition, replacement and trait
stacking in plants. |
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Keywords: | Double strand DNA breaks Homology-directed repair Site-directed transgene integration Zinc finger nucleases |
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