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    High-efficiency plant genome engineering via CRISPR/Cas9 system

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    Name:
    Ayman Eid_Dissertation.pdf
    Size:
    17.39Mb
    Format:
    PDF
    Description:
    PhD Dissertation
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    Type
    Dissertation
    Authors
    Eid, Ayman cc
    Advisors
    Mahfouz, Magdy M. cc
    Committee members
    Blilou, Ikram cc
    Saikaly, Pascal cc
    Benhamed, Moussa cc
    Program
    Bioscience
    KAUST Department
    Biological and Environmental Sciences and Engineering (BESE) Division
    Date
    2019-04
    Permanent link to this record
    http://hdl.handle.net/10754/632513
    
    Metadata
    Show full item record
    Abstract
    Precise engineering of genomes holds great promise to advance our understanding of gene function and biotechnological applications. DNA double strand breaks are repaired via imprecise non-homologous end joining repair or via precise homology-directed repair processes. Therefore, we could harness the DSBs to engineer the genomes with a variety of genetic outcomes and with singlebase- level precision. The major barrier for genome engineering was the generation of site-specific DNA DSBs. Programmable DNA enzymes capable of making a complete and site-specific cut in the genome do not exist in nature. However, these enzymes can be made in in vitro as chimeric fusions of two modules, a DNA binding module and a DNA cleaving module. The DNA cleaving module can be programmed to bind to any user-defined sequence and the DNA cleaving module would generate DSBs in the target sequence. These enzymes called molecular scissors include zinc finger nucleases (ZFNs) and transcriptional activator like effector nucleases (TALENs). The programmability of these enzymes depends on protein engineering for DNA binding specificity which may be complicated, recourse intensive and suffer from reproducibility issues. Recently, clustered regularly interspaced palindromic repeats (CRISPR)/ CRISPR associated endonuclease 9 (Cas9) an adaptive immune system of bacterial and archaeal species has been developed for genome engineering applications. CRISPR/Cas9 is an RNA-guided DNA endonuclease and can be reprogrammed through the engineering of single guide RNA molecule (sgRNA). CRISPR/Cas9 activity has been shown across eukaryotic species including plants. Although the engineering of CRISPR/Cas9 is quite predictable and reproducible, there are many technological challenges and improvements that need to be made to achieve robust, specific, and efficient plant genome engineering. Here in this thesis, I developed a number of technologies to improve specificity, delivery and expression and heritability of CRISRP/Cas9-modification in planta. Moreover, I used these technologies to answer basic questions to understand the molecular underpinning of the interplay between splicing and abiotic stress. To improve Cas9 specificity, I designed and constructed a chimeric fusion between catalytically dead Cas9 (dCas9) and FOKI catalytic DNA cleaving domain (dCas9.FoKI). This synthetic chimeric fusion enzyme improved Cas9 specificity which enable precision genome engineering. Delivery of genome engineering reagents into plant cells is quite challenging, I developed a virus-based system to deliver sgRNAs into plants which facilitates plant genome engineering and could bypass the need for tissue culture in engineering plant genomes. To improve the expression of the CRISPR/Cas9 machinery in plant species, I developed a meiotically-driven expression of CRISPR/Cas9 which improved genome editing and heritability of editing in seed progeny, thereby facilitating robust genome engineering applications. To understand the molecular basis of the interplay between splicing stress and abiotic stress, I used the CRISPR/Cas9 machinery to engineer components of the U2snRNP complex coupled which chemical genomics to understand the splicing stress regulation in response to abiotic stress conditions. Finally, I harnessed the technological improvements and developments I have achieved with CRISPR/Cas9 system to develop a directed evolution platform for targeted trait engineering which expands and accelerates trait discovery and engineering of plant species resilient to climate change conditions.
    Citation
    Eid, A. (2019). High-efficiency plant genome engineering via CRISPR/Cas9 system. KAUST Research Repository. https://doi.org/10.25781/KAUST-89046
    DOI
    10.25781/KAUST-89046
    ae974a485f413a2113503eed53cd6c53
    10.25781/KAUST-89046
    Scopus Count
    Collections
    Biological and Environmental Sciences and Engineering (BESE) Division; Bioscience Program; Dissertations

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