Award Abstract #0726267
2010: Targeted Mutagenesis in Arabidopsis Using Zinc Finger Nucleases

NSF Org:	MCB Division of Molecular and Cellular Biosciences
Initial Amendment Date:	July 30, 2008
Latest Amendment Date:	August 15, 2009
Award Number:	0726267
Award Instrument:	Continuing grant
Program Manager:	Karen C. Cone MCB Division of Molecular and Cellular Biosciences BIO Directorate for Biological Sciences
Start Date:	August 1, 2008
Expires:	July 31, 2010 (Estimated)
Awarded Amount to Date:	$867519
Investigator(s):	Daniel Voytas voytas@umn.edu (Principal Investigator) Thomas Peterson (Former Co-Principal Investigator) Erica Unger-Wallace (Former Co-Principal Investigator)
Sponsor:	University of Minnesota-Twin Cities 200 OAK ST SE MINNEAPOLIS, MN 55455 612/624-5599
NSF Program(s):	THE 2010 PROJECT, GENES AND GENOME SYSTEMS
Field Application(s):
Program Reference Code(s):	BIOT, 9183, 1684, 1154, 1116, 1112
Program Element Code(s):	1684, 1112

ABSTRACT

The next challenge in plant biology is to discern the function of the many genes revealed through the various genome sequencing projects. One important approach for understanding gene function is to study the consequence of removing or knocking out the function of a specific gene. In the model plant Arabidopsis, efficient methods are available to knock out many genes of interest. Functional analysis, however, is often frustrated by genetic redundancy or the presence of multiple copies of the same or closely related genes. Particularly prevalent in plants are tandem gene duplications, and as many as 15% of Arabidopsis genes are organized in tandem arrays. A targeted mutagenesis protocol will be developed to augment existing approaches for understanding Arabidopsis gene function, particularly for genes for which functional analysis has been confounded by genetic redundancy. The approach uses zinc finger nucleases (ZFNs) - chimeric proteins made up of a zinc finger array fused to the DNA cleavage domain of Fokl endonuclease. Zinc finger arrays of high affinity and specificity can be engineered to recognize virtually any target gene. Upon cleavage of the target DNA by the ZFN, the broken ends are repaired inefficiently, resulting in locus-specific mutations. In initial experiments, a model zinc finger nuclease and a reporter gene containing the cognate target site will be used to optimize methods for efficient recovery of heritable ZFN-induced mutations. Next, ZFNs will be engineered to recognized native Arabidopsis loci, including those that give observable phenotypes when mutated. ZFNs will also be engineered for a locus where mutatations do not result in an observable phenotype. To recover mutations at such a locus, robust DNA amplification and sequence-based detection methods will be employed. Lastly, a tandem array of duplicate genes will be targeted. Deletion of the array will be accomplished by engineering ZFNs to cleave within the outermost genes. The frequency of recovering deletions and individual mutations will be determined to assess the relative efficiency of this approach. In addition to Arabidopsis, this mutagenesis approach will also be valuable for plant species such as rice and maize, where genome sequencing projects are ongoing and tandem genes are abundant. Moreover, the ability to generate and regulate specific chromosomal double-strand breaks will facilitate studies of chromosome structure and DNA repair mechanisms in plants. The URL for the web site where protocols as well as results of gene knock-out studies can be accessed is http://www.beckmancenter.umn.edu/html/2010.html.

Understanding the function of plant genes is critical if plants are to be fully harnessed to meet the world's burgeoning need for fuel, feed and industrial raw materials. Although significant progress has been made in discerning plant gene function, existing approaches have inherent limitations, particularly with respect to ascribing function to redundant or duplicated genes. The goal of this project is to implement an efficient targeted mutagenesis approach to overcome these limitations. This project will train undergraduate interns and graduate students for work in plant molecular biology. Students from underrepresented groups will be recruited to the project through programs designed to enhance minority participation in science.

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