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CRISPR

CRISPR

Genome editing technologies allow scientists to modify an organism’s DNA via the addition, removal, or rearrangement of genetic material at specific genomic locations. These types of techniques could potentially be used to cure genetic disorders such as hemophilia and sickle cell anemia. One popular and widely used DNA-editing research tool that could lead to safe and effective cures for genetic disorders is the CRISPR-Cas9 system. CRISPR-Cas9 stands for Clustered Regularly Interspaced...

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Updated: Jun 29, 2025

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

Efficient PAM-Less Base Editing for Zebrafish Modeling of Human Genetic Disease with zSpRY-ABE8e

Published on: February 17, 2023

Efficient genome editing using modified Cas9 proteins in zebrafish.

Laura Dorner¹, Benedikt Stratmann¹, Laura Bader¹

¹Max Planck Institute for Biology, RG Colour Pattern Evolution, Tuebingen, Max-Planck-Ring 5, 72076 Tuebingen, Germany.

|March 28, 2024

Summary

Researchers enhanced CRISPR gene editing in zebrafish by developing new Cas9 protein variants. These variants improve gene knockout efficiency and enable precise single base pair edits through homology-directed repair (HDR).

Keywords:

albino kcnj13 CRISPR Pigment pattern evolution Zebrafish

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Last Updated: Jun 29, 2025

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Area of Science:

Genetics and Genomics
Molecular Biology
Model Organisms

Background:

Zebrafish (Danio rerio) are crucial model organisms in biomedical research due to their genetic tractability.
CRISPR/Cas systems have revolutionized gene editing, enabling efficient generation of gene knockouts via non-homologous end joining (NHEJ).
Homology-directed repair (HDR) for precise base pair editing in zebrafish is less efficient, often requiring extensive genotyping.

Purpose of the Study:

To evaluate the efficacy of the Cas9 protein variant SpRY for targeting specific sites in the zebrafish genome.
To enhance the efficiency of both gene knockout and homology-directed repair (HDR) in zebrafish gene editing.
To develop cost-effective protocols for generating improved Cas9 variants and facilitating precise genome modification in zebrafish.

Main Methods:

Utilized the Cas9 protein variant SpRY, which has a relaxed PAM (protospacer adjacent motif) requirement.
Incorporated an artificial nuclear localization signal (aNLS) into Cas9 protein variants.
Applied CRISPR/Cas systems for gene editing in zebrafish, focusing on both NHEJ and HDR pathways.

Main Results:

Confirmed that SpRY can target certain sites within the zebrafish genome.
Demonstrated that Cas9 variants with aNLS significantly enhance gene knockout efficiency.
Showed increased frequency of HDR, facilitating efficient single base pair modifications in the zebrafish genome.

Conclusions:

Cas9 protein variants, particularly SpRY and those with aNLS, offer improved tools for zebrafish genome editing.
The developed protocols enable more efficient and precise genetic modifications in zebrafish, including single base pair edits.
These advancements provide a cost-effective guide for generating versatile Cas9 variants and achieving efficient gene editing in this important model organism.