GeneWeld: Efficient Targeted Integration Directed by Short Homology in Zebrafish

Area of Science:

• Molecular Biology
• Genetics
• Zebrafish Model Organisms

Background:

• Efficient genome engineering demands high-frequency and specific DNA integration at target sites.
• Previous zebrafish gene targeting strategies exhibit variable germline integration frequencies.
• Streamlining precision gene targeting in zebrafish is crucial for genetic research.

Purpose of the Study:

• To introduce the GeneWeld protocol and pGTag vector series for efficient zebrafish gene targeting.
• To establish a streamlined, cost-effective method for precision genome engineering in zebrafish.
• To improve the frequency and reliability of germline integration alleles in zebrafish.

Main Methods:

• Utilized short homology arms (24-48 bp) for homology-mediated end joining (HMEJ).
• Employed CRISPR/Cas9 to induce targeted DNA double-strand breaks.
• Designed pGTag vectors with reporters, a universal CRISPR sgRNA sequence, and homology arms for in vivo cassette liberation and integration.

Main Results:

• Achieved high-frequency targeted integration of DNA reporter cassettes.
• Reported germline transmission rates for precision-targeted integration alleles ranging from 22% to 100%.
• Demonstrated a streamlined and straightforward approach for gene targeting applications.

Conclusions:

• The GeneWeld protocol and pGTag vectors offer a robust system for high-efficiency gene targeting in zebrafish.
• This method provides a cost-effective and reliable solution for precision genome engineering.
• The system facilitates enhanced germline transmission rates for targeted integration alleles.