High-efficiency CRISPR gene editing in C. elegans using Cas9 integrated into the genome

Area of Science:

• Genetics
• Molecular Biology
• Developmental Biology

Background:

• CRISPR-Cas9 gene editing in C. elegans is often inefficient due to germline silencing of plasmid-borne Cas9.
• Producing single gene edits typically requires microinjection of numerous animals.

Purpose of the Study:

• To develop C. elegans strains with constitutive germline expression of Cas9 from integrated transgenes.
• To enhance the efficiency and reduce the workload of plasmid-based CRISPR gene editing in C. elegans.

Main Methods:

• Generated C. elegans strains with integrated, constitutively expressed Cas9 transgenes in the germline.
• Incorporated heatshock-driven Cre recombinase and fluorescence markers for selectable marker removal and outcrossing.
• Tested editing efficiency for GFP insertions and template-guided editing.

Main Results:

• Achieved 50-100% success rates for simple GFP insertions in injected animals.
• Demonstrated stable template-guided editing over multiple generations.
• Integrated Cas9 strains significantly improved editing success and reduced experimental effort.

Conclusions:

• Constitutive germline Cas9 expression from integrated transgenes overcomes major barriers to efficient CRISPR editing in C. elegans.
• These engineered strains streamline genome editing workflows, making it more accessible and efficient.
• The developed strains facilitate rapid generation of specific genetic modifications in C. elegans.