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Updated: Oct 12, 2025
Indel Detection following CRISPR/Cas9 Mutagenesis using High-resolution Melt Analysis in the Mosquito Aedes aegypti
Published on: September 10, 2021
Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos
Raghuvir Viswanatha1, Enzo Mameli2,3, Jonathan Rodiger2
1Department of Genetics, Blavatnik Institute, Harvard Medical School, Boston, MA, 02115, USA. ram@genetics.med.harvard.edu.
Abstract:
Mosquito-borne diseases present a worldwide public health burden. Current efforts to understand and counteract them have been aided by the use of cultured mosquito cells. Moreover, application in mammalian cells of forward genetic approaches such as CRISPR screens have identified essential genes and genes required for host-pathogen interactions, and in general, aided in functional annotation of genes. An equivalent approach for genetic screening of mosquito cell lines has been lacking. To develop such an approach, we design a new bioinformatic portal for sgRNA library design in several mosquito genomes, engineer mosquito cell lines to express Cas9 and accept sgRNA at scale, and identify optimal promoters for sgRNA expression in several mosquito species. We then optimize a recombination-mediated cassette exchange system to deliver CRISPR sgRNA and perform pooled CRISPR screens in an Anopheles cell line. Altogether, we provide a platform for high-throughput genome-scale screening in cell lines from disease vector species.
Insights
Researchers developed a new CRISPR screening platform for mosquito cell lines, enabling high-throughput genetic analysis to combat mosquito-borne diseases.
Area of Science:
- Genomics
- Molecular Biology
- Vector Biology
Background:
- Mosquito-borne diseases pose a significant global health challenge.
- Cultured mosquito cells and CRISPR screens in mammalian cells have advanced research.
- A lack of scalable genetic screening tools for mosquito cell lines hinders progress.
Purpose of the Study:
- To develop a high-throughput genome-scale screening platform for mosquito cell lines.
- To enable functional annotation of genes in disease vector species.
Main Methods:
- Designed a bioinformatic portal for sgRNA library design in mosquito genomes.
- Engineered mosquito cell lines for Cas9 expression and sgRNA acceptance.
- Optimized a recombination-mediated cassette exchange system for CRISPR delivery.
- Performed pooled CRISPR screens in an Anopheles cell line.
Main Results:
- Established a functional CRISPR screening platform for mosquito cell lines.
- Identified optimal promoters for sgRNA expression across multiple mosquito species.
- Demonstrated the utility of the platform for genome-scale screening.
Conclusions:
- The developed platform facilitates high-throughput genetic screening in disease vector cell lines.
- This advancement will accelerate research into mosquito-borne diseases and vector control strategies.
