Highly Efficient CRISPR-Cas9-Based Methods for Generating Deletion Mutations and F0 Embryos that Lack Gene Function...

Area of Science:

• Molecular Biology
• Genetics
• Developmental Biology

Background:

• CRISPR-Cas9 technology is crucial for gene editing.
• Inconsistent CRISPR-Cas9 activity in zebrafish embryos has limited its application.
• The presence of extra guanine nucleotides in single guide RNAs (sgRNAs) has been identified as a cause of reduced efficiency.

Purpose of the Study:

• To identify the cause of inconsistent CRISPR-Cas9 activity in zebrafish.
• To develop a highly efficient CRISPR-Cas9 system for zebrafish gene editing.
• To enable robust functional genetic studies in zebrafish embryos.

Main Methods:

• Utilizing Cas9 ribonucleoproteins (RNPs) with precisely matched sgRNAs or synthetic crRNA:tracrRNA duplexes.
• Injecting engineered RNPs into zebrafish eggs.
• Analyzing induced indel mutations and gene function in F0 embryos.

Main Results:

• Cas9 RNPs with perfectly matching guide RNAs achieve consistent and highly efficient gene targeting in zebrafish embryos.
• Nearly all targeted loci in injected embryos contained indel mutations, leading to complete gene function loss.
• Low mosaicism in F0 embryos facilitated the study of cell non-autonomous gene functions and gene redundancy.
• Heritable deletions of up to 50 kbp were induced using paired guide RNPs.

Conclusions:

• Optimized Cas9 RNP delivery overcomes previous limitations of CRISPR-Cas9 in zebrafish.
• This enhanced system provides a powerful tool for precise gene editing and functional genomics in zebrafish.
• The method allows for efficient generation of knockout zebrafish and the study of complex genetic interactions.