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Macaulay, Tessa (Magnus)

University of British Columbia

Microtubules are structural components of the cytoskeleton involved in cell mobility, division, and cellular shaping of all eukaryotes. Microtubule-associated proteins, such as MICROTUBULE ORGANIZATION 1 (MOR1) are known to control microtubule polymerization in the plant Arabidopsis thaliana. Previous studies have observed MOR1 activity through conditional mutants, since loss-of-function knockouts of this essential gene are homozygous lethal. The mor1-1L174F conditional mutant, a single nucleotide mutation, shows microtubule disorganization and left-handed organ twisting when grown above 29°C, but not 21°C. Fluorescent reporters of mor1-1 allow live in vivo study of MOR1 effects on microtubule dynamics. However, MOR1 mutants tagged with fluorescent protein reporters show masking of the mutant phenotype from the endogenous MOR1 gene still expressing functional MOR1 protein untagged with fluorescent protein. In this thesis, I describe a novel technique for establishing a MOR1 endogenous null background for MOR1 mutant reporters to be expressed using recombineering and CRISPR/Cas9 technologies. Recombineering was first used to generate silent point mutations in protospacer-adjacent motif sites within a mor1-1 fluorescent reporter, rendering it unrecognizable by CRISPR/Cas9 designed to target MOR1, and therefore CRISPR-resistant. When expressed in A. thaliana plants, the CRISPR/Cas9-resistant mor1-1 fluorescent reporter remained untargeted by CRISPR/Cas9, while the endogenous MOR1 became null. Expression of the CRISPR-resistant transgene with the mor1-1L174F amino acid substitution generated the characteristic left-handed root twisting observed in endogenous mor1-1 homozygous mutants. This confirmed that the CRISPR construct had knocked out the function of the endogenous gene, thus demonstrating the feasibility of the method. This proposed technique paves the way for future studies into the function of essential microtubule-associated proteins and further insights into how microtubule polymerization is regulated in eukaryotes. This guide can be easily adapted to generate endogenous-null backgrounds for reporters of other genes of interest for in vivo studies.

Text

2024-09-03

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/89154

Botany

University of British Columbia

UBCV

http://creativecommons.org/licenses/by-nc-nd/4.0/