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dc.contributor.advisor
dc.contributor.authorNeda Kianpour
dc.date.accessioned2024-01-04T17:10:29Z
dc.date.available2024-01-04T17:10:29Z
dc.date.issued2023
dc.identifierno.inn:inspera:180789498:65284356
dc.identifier.urihttps://hdl.handle.net/11250/3109914
dc.description.abstract
dc.description.abstractPlant genome editing, a transformative technology in agriculture and biotechnology, faces challenges in efficiently delivering CRISPR/Cas components. Current methods, including Agrobacterium-mediated delivery, particle bombardment, and PEG-mediated protoplast transformation, have limitations due to random plasmid DNA integration and unpredictable genetic expression. As a solution, there's a growing interest in developing gene-edited lines with reduced foreign genetic integration risk using DNA-free delivery methods like RNP complexes (pre-assembled ribonucleoprotein) or transient plasmid DNA expression. Hence, this study aimed to create tools for CRISPR/Cas9-based gene editing in Ipomoea batatas (sweet potato) by targeting the ALS1 gene as a reliable null-mutant phenotype. The focus was designing and evaluating optimal sgRNA candidates for CRISPR/Cas9 RNP-based genome editing. Additionally, the performance characteristics of an engineered recombinant Cas9 with those of a commercially available Cas9 were aimed to be compared. Firstly, the selection of target plasmids DNA, PCR_IbALS1_S1.1 and PCR_IbALS1_S4.1, represented the specific genomic region of interest (ALS1 gene) in Ipomoea batatas among four plasmids DNA. Then, five distinct sgRNAs were designed based on coding sequences and evaluated through in-vitro cleavage assays. The findings demonstrated effective cleavage by SpCas9 guided by a single sgRNA (FP_IVT_T7_Spa2_S1.1) in the complementary DNA sequence of plasmid DNA PCR_IbALS1_S1.1. Efforts were made to create in-vitro recombinant Cas9 protein through expression vector construction, primer designing, in-fusion cloning, and PCR. However, this endeavour yielded no product, primarily attributed to the substantial size of the insert fragment (CDS of Cas9 and NLS ~ 9849 bp). Thus, this study has laid the foundation and provided fundamental insights for future in-vitro ALS1 CRISPR/Cas9-based genome editing using RNP complexes in Ipomoea batatas, despite the challenges encountered and the outcome.
dc.languageeng
dc.publisherInland Norway University
dc.titleIdentification of optimal sgRNA candidates for mutagenesis of the ALS1 gene in Ipomoea batatas using in-vitro cleavage assay and CRISPR/Cas9 technology
dc.typeMaster thesis


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