First Advisor

Jason Podrabsky

Date of Award

Winter 3-2024

Document Type

Thesis

Degree Name

Bachelor of Science (B.S.) in Biology and University Honors

Department

Biology

Language

English

Subjects

CRISPR-Cas9, Tyrosinase, embryo, fish, mutant, melanin

DOI

10.15760/honors.1466

Abstract

CRISPR-Cas9 genome editing has been used successfully to knock out genes in model organisms such as zebrafish, turquoise killifish, and cichlid fish. CRISPR-Cas9 genome editing has not been verified in the annual killifish, Austrofundulus limnaeus. We hypothesize that targeted editing of the tyrosinase gene in embryos of A. limnaeus will lead to fish without the ability to produce black pigment. Embryos at the 1-cell stage were injected with a Cas9 cocktail containing a mix of guide RNA molecules that target the genomic sequence of the tyrosinase gene and either an mRNA coding for the Cas9 protein or Cas9 protein. Guide RNAs were designed using ChopChop, and two guides were selected for injection based on a high predicted percent efficiency for binding with a low probability for off-target effects. When using Cas-9 protein, many injected embryos developed without expressing black pigment. Injections with Cas9 mRNA failed to exhibit an edited phenotype. We found for the first time in this species that CRISPR-Cas9 can be successfully used to knockout the tyrosinase gene. In the future, we plan to establish a breeding line of non-pigmented killifish to aid in embryological studies of this species.

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Persistent Identifier

https://archives.pdx.edu/ds/psu/41492

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