Browsing by Subject "Genome engineering"

Now showing 1 - 4 of 4
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    The development, engineering and application of TAL effector nucleases for targeted genome modification
    (2013-09) Christian, Michelle L.
    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.
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    The Mechanism of Precise Genome Engineering in Human Cells
    (2015-09) Kan, Yinan
    Genome engineering is the intentional alteration of the genetic information in living cells or organisms. Since Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated 9 (CRISPR/Cas9) was repurposed for genome engineering, the “CRISPR Craze” is quickly bridging the genotype and phenotype worlds and transforming the biological, biomedical and biotechnological research. Interestingly, CRISRP/Cas9 does not perform precise genome engineering (PGE) by itself, but it only induces a targeted genomic lesion and invites the HDR pathways to introduce the desired modifications. Although PGE has a wide application in genome modification and gene therapy, the identity, property and hierarchy of the HDR pathways leading to the formation of PGE products remain obscure. In my doctorial dissertation, I demonstrated that double-strand DNA (dsDNA) donors with a sizable central heterology preferentially utilize the double-strand break repair (DSBR) pathway in the absence and presence of chromosomal double-strand breaks (DSBs). This pathway generates long, bidirectional conversion tracts with linear distribution. In contrast, single-strand oligonucleotide (ODN) donors utilize the synthesis-dependent strand annealing (SDSA) and single-strand DNA incorporation (ssDI) pathways, respectively, depending on the strandedness of the genomic lesions and ODN donors. These pathways produce short, unidirectional and bidirectional conversion tracts with Gaussian distributions. The SDSA pathway is preferentially utilized in the presence of compound genomic lesions such as DSBs and paired nicks. In summary, this work systematically determined the identity, property and hierarchy of the HDR pathways underlying PGE with definitive molecular evidence, and provided practical guidelines for the improvement of PGE.
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    Plant Genome Engineering With Sequence-Specific Nucleases: Methods For Editing DNA In Whole Plants
    (2014-08) Baltes, Nicholas
    The development and function of all living organisms, from bacteria to humans, is encoded within a universal blueprint-deoxyribonucleic acid (DNA). The ability to re-write this code of life promises great benefits, ranging from a better understanding of gene function to correcting genetic diseases. Therefore, there is high value for tools and techniques that enable genome editing in living cells. In the last 20 years, multiple classes of enzymes have been developed that can be `rewired' to recognize and break a DNA sequence of interest. These enzymes (sequence-specific nucleases) have proven to be powerful reagents for editing DNA in higher-eukaryotic cells. However, the ability to modify DNA, particularly in plant cells, does not solely depend on the activity of the sequence specific nuclease. Instead, it also depends on the efficiency with which the genome engineering reagents are delivered, the cells they are delivered to, and the effectiveness of selecting (or screening) for cells with the desired modification. Studies within this dissertation seek to develop novel methods for delivering genome engineering reagents to whole plants. First, we focused our attention on geminiviruses--a large family of plant DNA viruses. Prior to these studies, geminiviruses were primarily used as vectors for virus-induced gene silencing or for protein expression; however, their circular DNA genomes, and their ability to replicate extrachromosomally, makes them an attractive vector for delivering genome engineering reagents. Here, we describe proof-of-principle experiments showing that, in Nicotiana tabacum, replicons based on the bean yellow dwarf virus can indeed deliver genome engineering reagents to leaf cells, and that these modified cells could grow into calli and seedlings. Interestingly, we also observed an enhancement in homologous recombination in leaf cells, relative to our non-viral controls. This enhancement appeared to be due to replication of donor molecules and by pleiotropic activity of the virus replication proteins. In addition to DNA viruses, we also explored the use of RNA viruses for the delivery of sequence-specific nucleases in Arabidopsis. And, finally, we expanded the utility of stable integration into plant genomes by applying this approach to additional plants, additional target genes, and additional genome modifications.
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    Production of induced regulatory T-cells through CRISPR/Cas9-based gene editing
    (2018-12) Tschann, Madison
    Regulatory T-cells (Tregs) are a subset of T-cells essential for maintaining immune tolerance and their dysregulation has been found to have a central role in the progression of various autoimmune diseases. The transplantations of Treg as a form of immune therapy has and continues to be an attractive method for the treatment of such disease based on their immuno-modulatory properties. Despite its potential, Treg adoptive cell transfer therapy is hampered by limited isolation efficiency due to low frequencies in human peripheral blood and poor in vitro expansion of a pure population. Herein, a novel CRISPR/Cas9 based technique is described utilizing AAV incorporation of strong transcriptional elements into the promoter region of the Treg master transcription factor, FOXP3, to upregulate expression in isolated primary T-cells and drive them toward a Treg phenotype.