Date of Graduation

12-2019

Document Type

Dissertation

Degree Name

Doctor of Philosophy in Cell & Molecular Biology (PhD)

Degree Level

Graduate

Department

Cell & Molecular Biology

Advisor/Mentor

Srivastava, Vibha

Committee Member

Pereira, Andy

Second Committee Member

Shi, Ainong

Third Committee Member

Thallapuranam, Suresh

Keywords

Crop biotechnology; Crop improvement; Gene stacking; Genome editing; Genome engineering; Targeted mutagenesis

Abstract

Crop improvement through biotechnology is an integrated effort, incorporating multiple approaches like integration of genes, editing of native genes, and removal of selection marker genes. Before streamlining the protocols, the efficiency and feasibility of the individual approach and their components must be tested. This study evaluated following approaches: 1) stacking an array of genes into a single locus by site-specific integration via Cre-lox recombination in rice, 2) determining the efficiency of I-SceI and the CCR5-ZFN in the targeted excisions of gene fragments in rice and Arabidopsis, and 3) determining the efficiency of CRISPR/Cas9 in generating targeted mutations for genome editing in rice. In gene stacking, >50% site-specific integration lines contained full-length integration of five genes. All genes were properly regulated by their promoters as indicated by the correlation of expression levels of the three constitutively expressed genes with their allelic number, and heat- or cold-induction levels of the two inducible genes. Analysis of I-SceI and CCR5-ZFN in rice and Arabidopsis found that these overexpressing constructs were refractory to plant transformation. The heat-inducible I-SceI expression in Arabidopsis was effective in creating somatic excisions but ineffective in generating heritable excisions. The inducible expression of CCR5-ZFN in rice, although transmitted stably to the progeny, appeared ineffective in creating detectable excisions. Finally, the application of CRISPR/Cas9 in rice was found to induce mutations at a high rate, but point-mutations occurred far more frequently than genomic deletions as determined in 114 rice lines including the primary transgenic lines and their progenies for 3 different genes. The heat-shock induced CRISPR/Cas9 was found to create heat-inducible targeted mutations that were inherited by the progeny. Additionally, mutations in the predicted off-target sites were undetectable or found at a lower rate in the heat-shock CRISPR/Cas9 lines as compared to their frequency in the constitutive‐overexpression CRISPR/Cas9 lines. In summary, while Cre-lox mediated site-specific integration and CRISPR/Cas9 mediated point-mutagenesis were highly effective in rice genome, application of I-SceI or CCR5-ZFN was problematic as tested in Arabidopsis and/or rice.

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