Editing of HSPG2 Nucleic Acids using CRISPR/Cas Tools

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Copyright: Li, Bitong
Abstract
In the past decade, the discovery of the CRISPR/Cas system launched a new era of genome editing and has rapidly become a universal research tool. The specific recognition between guide RNA and target nucleic acids enables CRISPR/Cas systems to exhibit high specificity compared to gene editing systems such as zinc finger or transcription activator-like effector nucleases. In this thesis, the CRISPR/Cas12a and CRISPR/Cas13a systems were designed to modify DNA or RNA associated with the HSPG2 gene which encodes perlecan, the major extracellular heparan sulphate (HS) proteoglycan in basement membranes. Perlecan has essential roles in organ development and contributes angiogenesis in pathological processes including cancer. The Cas12a and Cas13a nucleases were expressed in transformed E. coli and purified via His-tag affinity followed by size exclusion chromatography. The purified Cas12a nuclease exhibited high activity and specificity in a collateral activity assay in which the reporter sequence was only cleaved in the presence of the Cas12a protein and target ssDNA. Similarly, Cas13a nuclease exhibited high activity and specificity in a collateral activity assay with target ssRNA. A CRISPR/Cas12a gRNA was designed to target exon 2 of HSPG2 and was able to cleave amplified genomic DNA extracted from human melanoma cell line, MM200. Additionally, the collateral activity assay revealed that Cas12a nuclease dose-dependently cleaved the reporter ssDNA when used with target HSPG2 DNA. Similarly, a CRISPR/Cas13a gRNA was designed to target exon 2 of HSPG2 RNA and was able to cleave the target RNA extracted from MM200 cells and was active in the collateral activity assay when used with target HSPG2 RNA. Modification of HSPG2 nucleic acids in both MM200 cells and human umbilical vein endothelial cells (HUVECs) was also established. The CRISPR/Cas12a system resulted in up to 39 and 24 % reduction in HSPG2 gene expression in MM200 and HUVECs, respectively. Moreover, the CRISPR/Cas13a system achieved up to 69 and 99% reduction in HSPG2 RNA in MM200 and HUVECs, respectively. The HSPG2 mRNA modification in both MM200 and HUVECs resulted in decreased expression of FGF2 and VEGF-A, genes involved in the perlecan signalling pathway networks and associated with angiogenesis. The established CRISPR/Cas12a and CRISPR/Cas13a systems provide novel and efficient nucleic acid editing tools to further study the functions of perlecan in vitro and potentially in vivo. In addition, the LbCas12a or LwCas13a-based collateral cleavage assay enabled efficient and specific detection of the HSPG2 genome or transcripts, suggestive of its potential in perlecan-related disease diagnoses, such as cancer or genetic disorders.
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Publication Year
2021
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Thesis
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Masters Thesis
UNSW Faculty
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