One of the strategies used by scientists to introduce foreign genes into living cells involves the targeted insertion of the foreign gene into a pre-determined chromosomal position. This approach has been used to great effect in (for example) yeast and transgenic mice to disrupt genes (hence the term “knock-out mouse”), and thus study their functions. Except in chloroplasts, similar strategies have been hard to perfect in plants. However, new technologies may be bringing us closer to the day when targeted gene disruption is an accessible methodology in plant systems. A recent report from Joe Petolino’s group at Dow Agrosciences provides an example.
Briefly, this group is developing tools that capitalize on the ability to design tailor-made DNA binding proteins that recognize specific sequences. Over the past several years, much progress has been made in understanding and adapting the DNA binding properties of a specific class of proteins known as zinc-finger proteins. It turns out that the chemical rules for specificity in some classes of these proteins can be delineated, and that different sequence specificities can be engineered in a rational way by linking a desired sequence of bases with amino acid side chains predicted to facilitate interactions with the base. It also turns out that one can target double-stranded DNA cleavage to specific sequences by linking endonucleases with sequence-specific DNA-binding proteins. Putting these two things together – tailor-made DNA binding specificity and targeted double-stranded cleavage – brings us technology to introduce double-stranded breaks at pre-determined chromosomal positions. (These chimeric proteins are known as zinc finger nucleases.) Add to this the realization that transgene integration occurs at double-stranded breaks, and one gets to the point described in the recent paper from Dow Agrosciences.
The abstract from the paper is given beneath the fold. What the authors did was design zinc-finger proteins that would recognize sequences in any of a number of exogenous reporters of endogenous host genes and use a battery of physiological and PCR-based assays to evaluate the efficacy of their strategies. Thus, they showed that they could promote intra-genetic recombination so as to restore a split reporter gene (encoding green fluorescent protein, or GFP). They then showed that they could promote intergenic recombination between two DNA molecules introduced transiently into tobacco cells using Agrobacterium; in this instance, they assayed for the reconstitution of a transgene that confers resistance to a herbicide (Bialaphos). Finally, they demonstrated targeted insertion of foreign DNA into a tobacco chitinase gene. This demonstration involved the attempted insertion of a Bialaphos gene into the genome; when used in conjunction with the tailor-made zinc finger constructs, as many as 10% of the recovered Bialaphos-resistance cells has the desired insertion.
While 1 in 10 may not seem to be very efficient, it far exceeds random T-DNA insertion, and brings the possibility of targeted gene disruption within the realm of possibility in plants. Beyond the utility of this technology in the lab, it will also provide crop scientists with ways to genetically modify plants such that unanticipated molecular consequences (inadvertent inactivation of endogenous genes by random insertion events, disruption of genetic circuits due to triggering of RNAi, to name two) are avoided.
“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 CQ, Doyon Y, Ainley WM, Miller JC, Dekelver RC, Moehle EA, Rock JM, Lee YL, Garrison R, Schulenberg L, Blue R, Worden A, Baker L, Faraji F, Zhang L, Holmes MC, Rebar EJ, Collingwood TN, Rubin-Wilson B, Gregory PD, Urnov FD, Petolino JF. 2009. Targeted transgene integration in plant cells using designed zinc finger nucleases. Plant Mol Biol. 69(6):699-709.
(Ulterior motive – Joe Petolino worked in the Dept. of Agronomy at the University of Kentucky under a colleague of mine, Dr. Glenn Collins. This post is a small way of promoting our former colleagues.)