Targeted transgene integration in plant cells using designed zinc finger nucleases.
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.
Cai
Charles Q
CQ
Dow AgroSciences, LLC, 9330 Zionsville Road, Indianapolis, IN 46268, USA.
Doyon
Yannick
Y
Ainley
W Michael
WM
Miller
Jeffrey C
JC
Dekelver
Russell C
RC
Moehle
Erica A
EA
Rock
Jeremy M
JM
Lee
Ya-Li
YL
Garrison
Robbi
R
Schulenberg
Lisa
L
Blue
Ryan
R
Worden
Andrew
A
Baker
Lisa
L
Faraji
Farhoud
F
Zhang
Lei
L
Holmes
Michael C
MC
Rebar
Edward J
EJ
Collingwood
Trevor N
TN
Rubin-Wilson
Beth
B
Gregory
Philip D
PD
Urnov
Fyodor D
FD
Petolino
Joseph F
JF
D000595:Amino Acid Sequence; D001483:Base Sequence; D001665:Binding Sites; D002478:Cells, Cultured; D002688:Chitinases / Q000235:genetics; D004720:Endonucleases / Q000235:genetics / Q000378:metabolism*; D005966:Glucuronidase / Q000235:genetics / Q000378:metabolism; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism; D011995:Recombination, Genetic; D014026:Nicotiana / Q000166:cytology / Q000235:genetics / Q000378:metabolism; D014162:Transfection / Q000379:methods; D019076:Transgenes / Q000235:genetics*; D016335:Zinc Fingers / Q000235:genetics*
D011993:Recombinant Fusion Proteins; D049452:Green Fluorescent Proteins; D004720:Endonucleases; D002688:Chitinases; D005966:Glucuronidase