Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos

Raghuvir Viswanatha,Stephanie E Mohr,Yanhui Hu,Jonathan Rodiger,Pierre Merckaert,Fabiana Feitosa-Suntheimer,Enzo Mameli,Tonya M Colpitts,Norbert Perrimon

doi:10.1038/s41467-021-27129-3

Raghuvir Viswanatha, Stephanie E Mohr + Show 7 more

Open Access

https://doi.org/10.1038/s41467-021-27129-3

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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.

Highlights

Mosquito-borne diseases present a worldwide public health burden
To facilitate CRISPR-based genome engineering in mosquitos and provide a batch-mode design resource for pooled CRISPR knockout (KO) screening targeting protein-coding genes, we developed CRISPR GuideXpress, an online resource with a number of features
The sgRNA designs are accompanied by pre-computed sets of parameters that are displayed alongside sgRNA sequences and the total search output can be downloaded in table format

Summary

Introduction

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. Current efforts to fight malaria and other mosquito-transmitted pathogens such as dengue, Zika, Chikungunya and West Nile viruses rely on control of vector populations, mostly by means of insecticides[2,3]. A key advantage of the introduction of CRISPR-Cas[9] technology was the ability to generate large pools of sgRNAs and simultaneously test their effect in mammalian cells This approach has transformed several areas of cell biology and revealed the function of previously unannotated genes[15]. 20 mosquito cell lines from Aedes, Culex, and Anopheles genera have been established over the last 50 years[24] These cells are most widely used to propagate and characterize mosquito-borne viruses, including dengue, yellow fever, La. Crosse, Japanese encephalitis virus, West Nile, Rift Valley, o’nyong-nyong, Sindbis, and Zika viruses[24]. Cell lines provide a platform to propagate viruses or intracellular pathogens[30], and permit in vitro characterization of mosquitospecific drugs[31], toxins[32], viruses[33], and Wolbachia[8,34], supporting the development of biocontrol strategies

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