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CRISPR-Cas9 mediated gene editing of promoter cis-elements in core Arabidopsis clock genes

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The circadian clock in plants coordinates internal biological processes to exhibit daily oscillations that are in synchrony with environmental rhythms. The roles of core clock genes CCA1, LHY, and TOC1 that regulate the molecular mechanism have already been well established in Arabidopsis thaliana and the mechanism by which TOC1 regulates CCA1 and LHY appears to be mediated by transcription factors of the TCP family. However, the role of cis-elements in the promoters of these clock genes has not been fully addressed. In this thesis, we utilized CRISPR/Cas9 technology to create mutations within the CCA1, LHY and TOC1 promoter regions. Through this, we hope to better understand the role of cis-elements in the promoters of core clock genes and how they influence the clock feedback regulatory mechanism. We propose a procedure to efficiently identify genome editing when Cas9 is expressed in early gametogenesis stages. Using this procedure, we generated indel mutations in the CCA1 and LHY promoters and found that one of them resulted in a short hypocotyl phenotype. However, the visual observation of mutant plants suggested that off-target mutations may be present, thus backcrossing is required before further analysis. As such, our results suggest that cis-elements in the promoters of core clock genes may play an important role in modulating the clock function and therefore, clock-controlled processes.

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This item is under embargo until June 22, 2023.