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

Author

Listed:
  • Raghuvir Viswanatha

    (Blavatnik Institute, Harvard Medical School)

  • Enzo Mameli

    (Blavatnik Institute, Harvard Medical School
    Boston University School of Medicine, 620 Albany Street)

  • 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
    HHMI, Harvard Medical School)

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.

Suggested Citation

  • Raghuvir Viswanatha & Enzo Mameli & Jonathan Rodiger & Pierre Merckaert & Fabiana Feitosa-Suntheimer & Tonya M. Colpitts & Stephanie E. Mohr & Yanhui Hu & Norbert Perrimon, 2021. "Bioinformatic and cell-based tools for pooled CRISPR knockout screening in mosquitos," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
  • Handle: RePEc:nat:natcom:v:12:y:2021:i:1:d:10.1038_s41467-021-27129-3
    DOI: 10.1038/s41467-021-27129-3
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    References listed on IDEAS

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    1. October M. Sessions & Nicholas J. Barrows & Jayme A. Souza-Neto & Timothy J. Robinson & Christine L. Hershey & Mary A. Rodgers & Jose L. Ramirez & George Dimopoulos & Priscilla L. Yang & James L. Pear, 2009. "Discovery of insect and human dengue virus host factors," Nature, Nature, vol. 458(7241), pages 1047-1050, April.
    2. Xuechun Feng & Víctor López Del Amo & Enzo Mameli & Megan Lee & Alena L. Bishop & Norbert Perrimon & Valentino M. Gantz, 2021. "Optimized CRISPR tools and site-directed transgenesis towards gene drive development in Culex quinquefasciatus mosquitoes," Nature Communications, Nature, vol. 12(1), pages 1-13, December.
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