The ability to make precise changes to chromosomal DNA has been a long sought goal for geneticists. Targeted genome modification has a variety of applications - ranging from correcting genetic defects in human cells to creating novel, agronomically important traits in crops. The ability to make such targeted DNA modifications has been enabled by nucleases that bind to specific sequences within a gene and create double-strand breaks. Through the action of cellular repair pathways, these targeted breaks lead to localized mutagenesis via non-homologous end joining and to gene editing or insertion via homologous recombination. A primary focus in the field of genome engineering has been to develop tools and techniques that allow precise manipulation of the DNA of various organisms. Zinc finger nucleases and meganucleases are well established as DNA targeting reagents; however, both have limitations in terms of the ease in which they can be engineered to recognize new target sites and their targeting range.
Several years ago, a novel class of DNA binding domain known as Transcriptional Activator-like (TAL) effectors was described. TAL effectors proteins bind to specific sequences in plant genomes and turn on plant genes that promote bacterial infection. DNA target specificity of TAL effectors is conferred by a central array of typically 14-24 repeats, with each repeat recognizing one DNA nucleotide. The one-to-one correspondence of a TAL repeat to a single DNA base constitutes a simple code that can be used to design a TAL effector to target almost any sequence in a given genome.
Studies in this dissertation were directed at developing, engineering and applying a novel tool for genetic manipulation, called TAL effector Nucleases, or TALENs. The first efforts of this work demonstrate that fusions of TAL effector proteins to a non-specific nuclease created a targeted DNA break, the repair of which can resulted in site-specific modification of the target sequence. To advance TALEN technology and extend it to target novel DNA targets, we developed a method for rapid construction of engineered TALENs. Using our Golden Gate system, custom TALENs for target sites in essentially any gene of interest can be constructed in 5 days. Finally, We used this method to engineer TALENs targeting genes in Nicotiana tabacum (tobacco) and Arabidopsis thaliana and tested their ability to create mutations at endogenous loci in both protoplasts and whole plants. Our results indicate that TALENs indeed cleave their intended targets in plant protoplasts of Arabidopsis, and these mutations are heritable. Studies conducted in this dissertation were the first to develop the TALENs, a tool that promises to facilitate the manipulation of natural genomic loci in organisms to a much greater degree than previous targeting reagents.
University of Minnesota Ph.D. dissertation. September 2013. Major: Molecular, Cellular, Developmental Biology and Genetics. Advisor: Daniel F. Voytas. 1 computer file (PDF); xv, 227 pages, appendices A-B.
Christian, Michelle L..
The development, engineering and application of TAL effector nucleases for targeted genome modification.
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