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

Raghuvir Viswanatha (), Enzo Mameli, Jonathan Rodiger, Pierre Merckaert, Fabiana Feitosa-Suntheimer, Tonya M. Colpitts, Stephanie E. Mohr, Yanhui Hu and Norbert Perrimon ()
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Raghuvir Viswanatha: Blavatnik Institute, Harvard Medical School
Enzo Mameli: Blavatnik Institute, Harvard Medical School
Jonathan Rodiger: Blavatnik Institute, Harvard Medical School
Pierre Merckaert: Blavatnik Institute, Harvard Medical School
Fabiana Feitosa-Suntheimer: Boston University School of Medicine, 620 Albany Street
Tonya M. Colpitts: Boston University School of Medicine, 620 Albany Street
Stephanie E. Mohr: Blavatnik Institute, Harvard Medical School
Yanhui Hu: Blavatnik Institute, Harvard Medical School
Norbert Perrimon: Blavatnik Institute, Harvard Medical School

Nature Communications, 2021, vol. 12, issue 1, 1-13

Abstract: 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.

Date: 2021
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DOI: 10.1038/s41467-021-27129-3

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