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A_Gunadi_Dissertation 2019Final.pdf (5.13 MB)

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Advancing CRISPR Applications Using Soybean [Glycine max (L.) Merr.] Promoters

Gunadi, Andika
http://rave.ohiolink.edu/etdc/view?acc_num=osu1566169449003179

Abstract Details

2019, Doctor of Philosophy, Ohio State University, Horticulture and Crop Science.
The advent of clustered regularly interspaced short palindromic repeats (CRISPR) technology has enabled targeted genome modifications in plants through genome editing and gene transcriptional modifications. For high value crops such as soybean [Glycine max (L.) Merr.], CRISPR has the potential to induce targeted genome modifications for crop improvements faster and with higher precision than conventional breeding and transgenic strategies. In its basic form, CRISPR functions through the engineered use of compatible guide RNA and Cas nuclease, which are both simpler and more accessible than presently available alternative targeted genome modification tools such as meganuclease, zinc finger nuclease (ZFN) and transcription activator-like effector nuclease (TALEN). Nevertheless, all of these genome modification tools need to be customized and calibrated for optimal function in plants. A critical customization for CRISPR in soybean is the use of appropriate promoters for introducing the DNA precursors of guide RNA and Cas nuclease. In addition, CRISPR can also be directed to target soybean promoters, leading to precise promoter modifications and modulation of native gene expression. CHAPTER 1 of this dissertation reviews the important functions and applications of promoters in CRISPR-based plant genome modifications. Next, CHAPTER 2 describes the isolation and characterization of 40 unique promoters from soybean, which broaden the toolbox of genetic regulatory components for genome modifications. CHAPTER 3 describes the adaptation of CRISPR interference (CRISPRi) for interrogating cis-elements within a soybean promoter, resulting in pertinent information for future applications of this transcriptional modification strategy. Lastly, CHAPTER 4 describes multiple applications of soybean promoters to both confirm and improve a CRISPR-based targeted DNA integration strategy in plants, termed homology-independent targeted integration (HITI). Customized, particle bombardment-based plant bioassays utilizing the green fluorescence protein marker were developed for testing CRISPRi and HITI efficacies, and have enabled rapid optimizations of both CRISPR approaches. Altogether, the information gleaned from the interconnections of promoters and CRISPR in these studies pave the way towards identifying fundamental mechanisms in plant biology and creative solutions for crop improvements.
John Finer (Advisor)
Jonathan Fresnedo Ramirez (Committee Member)
Feng Qu (Committee Member)
Eric Stockinger (Committee Member)
249 p.
Agriculture; Biology; Botany; Cellular Biology; Molecular Biology; Plant Biology; Plant Sciences; Technology
CRISPR; promoters; particle bombardment; GFP; plant biology; CRISPRi; HITI; genome editing; sgRNA; gene expression; plant biotechnology

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Citations

  • Gunadi, A. (2019). Advancing CRISPR Applications Using Soybean [Glycine max (L.) Merr.] Promoters [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1566169449003179

    APA Style (7th edition)

  • Gunadi, Andika. Advancing CRISPR Applications Using Soybean [Glycine max (L.) Merr.] Promoters. 2019. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1566169449003179.

    MLA Style (8th edition)

  • Gunadi, Andika. "Advancing CRISPR Applications Using Soybean [Glycine max (L.) Merr.] Promoters." Doctoral dissertation, Ohio State University, 2019. http://rave.ohiolink.edu/etdc/view?acc_num=osu1566169449003179

    Chicago Manual of Style (17th edition)

osu1566169449003179
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This open access ETD is published by The Ohio State University and OhioLINK.