A selectable, plasmid-based system to generate CRISPR/Cas9 gene edited and knock-in mosquito cell lines

Area of Science:

• Vector Biology
• Genetics
• Molecular Biology

Background:

• Arthropod-borne diseases transmitted by Aedes mosquitoes cause significant global mortality.
• Genetic mutants are crucial for studying vector biology and viral infection mechanisms.
• Limited genetic mutants exist for mosquitoes compared to model organisms, hindering research.

Purpose of the Study:

• To develop and characterize a mosquito-optimized, plasmid-based CRISPR/Cas9 system for use in Aedes aegypti and Aedes albopictus cell lines.
• To establish efficient gene editing methods in mosquito cell lines for creating genetic mutants.
• To facilitate arbovirus research by providing accessible tools for mosquito cell line engineering.

Main Methods:

• Generation and characterization of a plasmid-based CRISPR/Cas9 system tailored for mosquito cells.
• Application of the system in U4.4 (Ae. albopictus) and Aag2 (Ae. aegypti) cell lines.
• Utilizing homology-directed repair for knock-in applications, specifically tagging AGO1.

Main Results:

• Demonstrated highly efficient editing of the AGO1 locus in both cell lines.
• Successfully isolated U4.4 and Aag2 cell lines with reduced AGO1 expression.
• Established Aag2 cell lines with a 3xFLAG-tag at the N-terminus of endogenous AGO1 via homology-directed repair.

Conclusions:

• The developed CRISPR/Cas9 system is versatile, cost-effective, and efficient for editing key mosquito cell lines.
• This system enables the creation of genetic mutants in immune-competent mosquito cell lines widely used in arbovirus studies.
• The tool facilitates fundamental research into mosquito biology and host-pathogen interactions relevant to disease transmission.