Efficient genome editing using modified Cas9 proteins in zebrafish.

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.