Presentation Type

Poster

Start Date

5-8-2024 11:00 AM

End Date

5-8-2024 1:00 PM

Subjects

CRISPR (Genetics), Embryology

Advisor

Jason Podrabsky

Student Level

Undergraduate

Abstract

The CRISPR-Cas9 genome editing tool has shown to be successful in knocking out genes in model organisms such as zebrafish, turquoise killifish, and cichlid fish. CRISPR-Cas9 genome editing has been demonstrated in many species of fish, but this technology has not been verified in the annual killifish, Austrofundulus limnaeus. We hypothesize that targeted editing of the tyrosinase gene in embryos of A. limnaeus would lead to the development of fish without the ability to produce melanin, the black/brown pigment molecule. Early embryos (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 low probability for off-target effects. Many injected embryos developed without expressing black pigment. We found for the first time in this species that 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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May 8th, 11:00 AM May 8th, 1:00 PM

Guide RNA Design and Delivery for CRISPR/Cas9 Editing in Annual Killifish

The CRISPR-Cas9 genome editing tool has shown to be successful in knocking out genes in model organisms such as zebrafish, turquoise killifish, and cichlid fish. CRISPR-Cas9 genome editing has been demonstrated in many species of fish, but this technology has not been verified in the annual killifish, Austrofundulus limnaeus. We hypothesize that targeted editing of the tyrosinase gene in embryos of A. limnaeus would lead to the development of fish without the ability to produce melanin, the black/brown pigment molecule. Early embryos (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 low probability for off-target effects. Many injected embryos developed without expressing black pigment. We found for the first time in this species that 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.