CRISPR-Cas9 mediated mutagenesis of the arsenic methyl-3- transferase gene in zebrafish

Wang, Guangning (2021) CRISPR-Cas9 mediated mutagenesis of the arsenic methyl-3- transferase gene in zebrafish. Masters thesis, Northern Arizona University.

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Abstract

Arsenic pollution is a global problem. People exposed to arsenic suffer from health associated diseases such as skin lesions, liver damage, cardiovascular failure, and cancer. In the Southwest United States, geologically sourced inorganic arsenic can be detected in surface and groundwater, and in some cases contaminated drinking water supplies exceed safety limits set by regulatory agencies. Many studies have shown serious adverse health impacts from inorganic arsenic exposure in human and animals; however, studies have also identified a putative arsenic chemical defense mechanism, which is the essential activity of an arsenic methyltransferase enzyme, arsenic 3-methylene transferase (AS3MT). Previously known as CYT19, AS3MT orthologues have been identified in all kingdoms of life, and AS3MT is presumed to be a key enzyme for arsenic detoxification in vivo, by conversion of inorganic arsenic into the less toxic forms monomethylarsonous acid (MMAIII) and dimethylarsinous acid (DMAIII). Despite the worldwide problem of arsenic contamination and identification of AS3MT as a key arsenic defense enzyme, the mechanisms that drive AS3MT as a protective enzyme are not fully understood. Herein, bioinformatics tools and molecular experiments were used to predict critical amino acid motifs and to disrupt those motifs, respectively. The zebrafish AS3MT protein structure model was constructed by I-TASSER service and the amino acid network was analyzed by Residue Centrality Analysis (RCA). We utilize the CRISPR-Cas9 gene editing system to create two separate as3mt loss-of-function zebrafish lines, by targeting domains upstream (i.e., 5’ to the critical residues). We describe the design, gene editing, and genotype diagnostics that yielded the engineered zebrafish. Thus, our research activities represent the important steps toward testing the hypothesis that AS3MT is necessary for zebrafish survival and/or health in an arsenic rich environment.

Item Type:	Thesis (Masters)
Publisher’s Statement:	© Copyright is held by the author. Digital access to this material is made possible by the Cline Library, Northern Arizona University. Further transmission, reproduction or presentation of protected items is prohibited except with permission of the author.
Keywords:	AS3MT; CRISPR/Cas9; Gene knockout; Zebrafish; Methyltransferase enzyme; Arsenic; Drinking water
Subjects:	T Technology > TD Environmental technology. Sanitary engineering
MeSH Subjects:	D Chemicals and Drugs > D01 Inorganic Chemicals
NAU Depositing Author Academic Status:	Student
Department/Unit:	Graduate College > Theses and Dissertations College of the Environment, Forestry, and Natural Sciences > Biological Sciences
Date Deposited:	15 Feb 2022 17:44
Last Modified:	15 Feb 2022 17:44
URI:	https://openknowledge.nau.edu/id/eprint/5704

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