Date of Award

2019

Document Type

Thesis

Degree Name

Bachelors

Department

Natural Sciences

First Advisor

Walstrom, Katherine

Area of Concentration

Biochemistry

Abstract

RNA helicase A (RHA) is a conserved protein in Caenorhabditis elegans which has been identified as playing a role in transcriptional silencing in germline development. To further study the function and expression of RHA-1 in C. elegans, a GFP tagged deletion mutant model needs to be developed. The objective of this thesis was to design and construct a single guide RNA plasmid (sgRNA) and a repair template for homology directed repair to allow for modification of the C. elegans genome using a CRISPR-Cas9 system, replacing the rha-1 gene with a GFP gene. The HiFi assembly method, using overlapping segments to create a plasmid construct, was used. The repair template showed evidence of a successful assembly, though attempts to transform the construct into competent E. coli cells were not successful. The linear assembly of the three insertion fragments of the repair template appeared successful when run on an agarose gel. A PCR reaction using the M13 reverse and 5’ homology arm reverse primers showed a band around 850 bp, suggesting a successful assembly of the complete plasmid. Sequencing of the sgRNA plasmid assembly showed that the plasmid did not anneal using the designed sgRNA overlaps, but instead reformed a circular plasmid by annealing to itself. Through this project, plans for design of sgRNA plasmids and repair plasmids have been developed but need further refinement.

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