Date of Submission

12-21-2021

Document Type

Thesis

Department

Biology and Environmental Sciences

Advisor

Carter Takacs, Ph.D.

Keywords

CRISPR-Cas9, KCNN2, Zebrafish

MeSH

CRISPR-Associated Protein 9, Ion Channels, Genes, Zebrafish, Embryonic Structures

LCSH

CRISPR-associated protein 9, Ion channels, Genes, Zebra danio, Fishes--Embryos

Abstract

Introduction: Mutations in the KCNN2 gene have been linked to phenotypes of movement disorders and autism. The goal of this experiment was to use the CIUSPR-Cas9 system to create a deletion within the KCNN2 gene and to observe the effect on the expression of movement in zebra fish embryos.

Methods: Linearized Cas9 DNA and guide DNA underwent in vitm transcription (IVT) reactions to produce RNA to be injected into zebrafish embryos at the one cell stage. KCNN guide RNAs were produced for the KCNN2 and KCNN3 genes (to prevent KCNN3 protein function from compensating for loss ofKCNN2's protein function), and the SLC45A2 gene, which was used as a positive control to indicate success of reagent preparation and Cas9 function. Genorypic and phenotypic analyses were performed 2 days post-fertilization (dpf).

Results: No notable difference was seen with genotypic analysis, but KCNN-injected embryos moved less in unprovoked and provoked phenotypic movement tests than wild type (WT) embryos.

Discussion: Microdeletions may have been the cause of the observed phenotypic difference between KCNN-injected and WT embryos, however, statistical analysis showed that the observed differences in quantity of movement were not statistically significant.

Conclusion: The data collected in this experiment is not sufficient to conclude whether or not microdeletions in the KCNN2 gene were produced or may have co11tributed to a difference in the expression of movement in zebrafish embryos. Future research should be conducted targeting different guide sequences and using more sophisticated genotypic and phenotypic analyses.

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