A Rapid Method for Directed Gene Knockout for Screening in G0 Zebrafish

Area of Science:

• Genetics
• Developmental Biology
• Zebrafish Models

Background:

• Zebrafish are valuable for genetic studies, but creating stable mutant lines is time-consuming.
• Existing reverse genetic methods in zebrafish require significant time for stable mutant line generation.

Purpose of the Study:

• To develop a rapid gene knockout system in zebrafish that consistently produces null phenotypes in G0 embryos.
• To create a comprehensive resource for efficient gene targeting in zebrafish.

Main Methods:

• Utilized CRISPR/Cas9 ribonucleoprotein complexes for yolk injection in zebrafish embryos.
• Employed sets of four guides to redundantly target single genes, achieving high efficiency.
• Developed a lookup table for four-guide sets targeting over 21,000 zebrafish genes.

Main Results:

• Achieved germline-transmitted knockout phenotypes in >90% of G0 zebrafish embryos for 8 tested genes.
• Observed both early embryonic and stable adult phenotypes.
• Successfully targeted 50 cardiomyocyte transcriptional regulators, identifying a role for zbtb16a in cardiac development.

Conclusions:

• The described CRISPR/Cas9 system enables rapid generation of knockout phenotypes in G0 zebrafish, significantly reducing time for genetic screening.
• This platform facilitates efficient screening of genes in zebrafish for roles in development, physiology, and disease.
• A validated lookup table of guide sets enhances the accessibility and utility of this rapid gene knockout system.