Browsing by Subject "genome editing"

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    Efficient and Precise Genome Editing in Shewanella with Recombineering and CRISPR/Cas9-mediated Counter-selection
    (2019-06) Domenech Corts, Anna
    Shewanella are invaluable hosts for the discovery and engineering of pathways important for bioremediation of toxic and radioactive metals, to create microbial fuel cells and for understanding extracellular electron transfer. However, studies on this species have suffered from a lack of effective genetic tools for precise and high throughput genome manipulation. Previously, the only reliable method used for introducing DNA into Shewanella spp. at high efficiency was bacterial conjugation, enabling transposon mutagenesis and targeted knockouts using suicide vectors for gene disruptions. In this dissertation, I describe development of simple and efficient genome editing tools for precise and site-directed mutagenesis of Shewanella. First, In Chapter I, I review recent advances in synthetic biology that accelerate the study and engineering of bacterial phenotypes. In chapter II, I show the development of a robust and simple electroporation method in S. oneidensis that allows an efficiency of up to ~108 transformants/µg DNA and which is adaptable to other strains. Using this method for DNA transfer, in chapter III, I characterize a new phage recombinase, W3 Beta from Shewanella sp. W3-18-1 and show its utility for in vivo genome engineering (recombineering) using linear single-stranded DNA oligonucleotides. In my experiments the W3 Beta recombinase gives an efficiency of ~5% recombinants among total viable cells. In addition, I show the functionality of this new system in S. amazonensis, a strain with few genetic studies but of interest given its higher temperature range for growth and wide range of carbon sources utilized. In chapter IV, I demonstrate use of the CRISPR/Cas9 system as a counter-selection to isolate recombinants. When coupled to recombineering, this counter-selection results in an extremely high efficiency of >90% among total surviving cells, regardless of the gene or strain modified. This efficiency allows isolation of several different types of mutations made with recombineering, and even allows identification of rare recombinants that form independently of W3 Beta expression. This is the first effective and simple strategy for recombination with markerless mutations in Shewanella. With synthesized single-stranded DNA as substrates for homologous recombination and CRISPR/Cas9 as a counter-selection, this new system provides a rapid, scalable, versatile and scarless tool that will accelerate progress in Shewanella genomic engineering. Finally, I conclude in Chapter V with an overview of the challenges and future directions of the technologies demonstrated here, discussing possible advancements that could further enhance the study of Shewanella.
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    Genome editing in alfalfa (Medicago sativa) to hyper-accumulate phosphate
    (2019) Samac, Deborah A; Miller, Susan S; Dornbusch, Melinda R; Curtin, Shaun J
    Rock phosphate, the main source of phosphate (P) for crop fertilizers, is a finite resource that is predicted to be depleted in 50-100 years. P is a critical nutrient in agriculture and its application can dramatically improve plant productivity. However, many soils have excess amounts of P from application of animal manures and runoff of phosphate from agricultural lands is the major source of nonpoint water pollution in the Midwestern US. The goal of this project is to create mutations by gene editing in the ubiquitin E2 conjugating enzyme PHO2, involved in P signaling and P homeostasis in alfalfa so that plants hyper-accumulate phosphate. Such plants could be used to reduce soil P levels and reclaim P for use as a fertilizer. From a draft diploid Medicago sativa genome scaffold sequence and the alfalfa transcriptome database (AGED), three PHO2 genes were identified. The genes, two of which are >99% homologous (a/b), each have seven exons interspersed by six introns. The open reading frames are 912 amino acids except when an alternate splice site is used in a/b gene transcript resulting in a 902 amino acid sequence. Alfalfa plants grown under P limiting conditions expressed low levels of the a/b transcripts with higher levels seen for PHO2c, while application of higher P induced increased expression mainly of the a/b transcripts. Under high P conditions, roots and shoots accumulated 4.1x and 2.5x more P than in low P conditions, respectively. An initial CRISPR/Cas9/Cys4 reagent targeting all three genes was generated and used to transform alfalfa cv. RegenSY. A total of 67 verified transgenic plants were screened by acrylamide gel shift assays, cloning, and sequencing to identify plants with mutations. Mutations ranging from a 1 bp insertion to a 25 bp deletion were identified in a total of 10 plants and some plants had multiple targets hit. Recently, a second attempt at CRISPR/Cas9 mutation utilized a cassette vector system with either the tRNA or Cys4 splicing system and exonuclease components. Initial screening results indicate that the tRNA splicing system may have yielded greater numbers of mutations. TaqMan probes were designed to identify plants with changes in the target sites and were verified by restriction digestions, cloning, and sequencing. Data on inheritance of mutations and phosphate accumulation in edited plants will be presented. The results of these experiments demonstrate that editing of multiple targets can be accomplished in alfalfa, although the tetraploid inheritance of genes complicates analysis.