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Lo, Ivan Hok Yin

University of British Columbia

The yellow fever mosquito, Aedes aegypti, is a prevalent vector that spreads transmissible diseases in human populations. In the past few decades, increasing effort have been made to study these fascinating animals. The rising research in CRISPR/Cas9 technology has allowed the potential for gene editing to be done in Ae. aegypti. This thesis focuses on three novel applications of CRISPR/Cas9 in Ae. aegypti that focus on improving gene editing efficiency and expanding the mosquito genetic toolbox. In the first chapter, germline gene promoters (zpg and nanos) are used to promote the expression of Cas9 proteins. We hypothesized that germline gene promoters can bias Cas9 expression in space and time to favor Homology Directed Repair (HDR). We discovered that transient tail expression of fluorescence markers in injected mosquitoes successfully predicted integration of transgene into the germline. This transgene was able to be passed on to the next generation. The use of germline gene promoters can reduce efforts in creating transgenic mosquitoes, as it increases mosquito survival rate and reduce time needed to screen fluorescence. In the second chapter, a sgRNA and donor template cassette was inserted into the mosquito genome through piggyBac transposon integration to create a split CRISPR system. We hypothesized that the endogenous expression of CRISPR components in a split system can favor HDR. We found no offspring that had a stable integration of transgene through HDR, while most strains exhibited Mendelian inheritance of fluorescence genes. Our results show that split systems in gene editing should test multiple candidates to ensure Cas9 activity. In the third chapter, we aimed to create a novel balancer chromosome in Ae. aegypti through utilizing CRISPR/Cas9 to generate a large chromosomal inversion. We applied methods of CRISPR gene editing at two target sites simultaneously, which has the potential for HDR repair to invert the chromosome segment between these sites. Future work will validate the sequences of putative G0 founders. These are the first steps towards creating a novel balancer chromosome.

Text

2024-04-18

Attribution-NonCommercial-NoDerivatives 4.0 International

http://hdl.handle.net/2429/87868

Zoology

University of British Columbia

UBCV

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