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Browsing by Subject "CRISPR Cas9"

Now showing 1 - 2 of 2
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    The Mechanism of Precise Genome Engineering in Human Cells
    (2015-09) Kan, Yinan
    Genome engineering is the intentional alteration of the genetic information in living cells or organisms. Since Clustered Regularly Interspaced Short Palindromic Repeat/CRISPR-associated 9 (CRISPR/Cas9) was repurposed for genome engineering, the “CRISPR Craze” is quickly bridging the genotype and phenotype worlds and transforming the biological, biomedical and biotechnological research. Interestingly, CRISRP/Cas9 does not perform precise genome engineering (PGE) by itself, but it only induces a targeted genomic lesion and invites the HDR pathways to introduce the desired modifications. Although PGE has a wide application in genome modification and gene therapy, the identity, property and hierarchy of the HDR pathways leading to the formation of PGE products remain obscure. In my doctorial dissertation, I demonstrated that double-strand DNA (dsDNA) donors with a sizable central heterology preferentially utilize the double-strand break repair (DSBR) pathway in the absence and presence of chromosomal double-strand breaks (DSBs). This pathway generates long, bidirectional conversion tracts with linear distribution. In contrast, single-strand oligonucleotide (ODN) donors utilize the synthesis-dependent strand annealing (SDSA) and single-strand DNA incorporation (ssDI) pathways, respectively, depending on the strandedness of the genomic lesions and ODN donors. These pathways produce short, unidirectional and bidirectional conversion tracts with Gaussian distributions. The SDSA pathway is preferentially utilized in the presence of compound genomic lesions such as DSBs and paired nicks. In summary, this work systematically determined the identity, property and hierarchy of the HDR pathways underlying PGE with definitive molecular evidence, and provided practical guidelines for the improvement of PGE.
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    Viral vector-mediated CRISPR-Cas9 gene editing for fundamental and applied plant research
    (2023-06) Tibebu, Redeat
    Agricultural systems face significant challenges in achieving sustainable and productive growth in light of increasing global population and climate change. In the past decade, advances in genome editing and sequencing technologies have offered promising opportunities for improving various aspects of plant productivity and sustainability. However, while major crops such as maize and rice have been the primary focus of research in this area, regional staple crops also referred to as “underutilized”, “minor” or “orphan” crops have been largely neglected. This has resulted in significant yield gaps for these crops, which could be closed through the application of gene editing techniques. The main challenge in using gene editing technologies for plant improvement is the inefficient delivery of gene editing reagents into plant cells and the subsequent regeneration of an edited plant. This is especially challenging for orphan crops, which often lack reference genomes and efficient plant transformation methods.This thesis comprises three manuscripts and a concluding chapter that address challenges, opportunities, and potential solution in utilizing advanced plant biotechnology tools for fundamental and applied plant research, with a particular focus on orphan crops. The first manuscript reviews biotechnological approaches that have been attempted in Eragrostis tef, a cereal orphan crop, emphasizing the potential of gene editing for crop improvement. In the second manuscript, we demonstrated the use of plant viral vectors to deliver editing reagents into the model plant Nicotiana benthamiana, aiming to regulate stomatal development for functional characterization and enhanced drought tolerance. In the third manuscript, we extended this technique to the orphan fruit crop P. grisea (groundcherry), demonstrating the introduction of domestication trait focused on boosting fruit quality and production. The final chapter concludes the thesis covering the available plant transformation and delivery methods and their potential application in rapid improvement of orphan crops including monocots such as tef. Ultimately, this thesis demonstrates the viability of gene editing technologies to enhance orphan crops and prompts the need for further research aimed at improving the current bottleneck of delivering editing reagents to a wide array of crops thereby supporting global food security and resilient agricultural systems.