Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum

Amuza Byaruhanga Lucky; Chengqi Wang; Xiaolian Li; Xiaoying Liang; Azhar Muneer; Jun Miao

iScience (Apr 2024)

Transforming the CRISPR/dCas9-based gene regulation technique into a forward screening tool in Plasmodium falciparum

Amuza Byaruhanga Lucky,
Chengqi Wang,
Xiaolian Li,
Xiaoying Liang,
Azhar Muneer,
Jun Miao

Affiliations

Amuza Byaruhanga Lucky: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USA
Chengqi Wang: Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USA
Xiaolian Li: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USA
Xiaoying Liang: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USA
Azhar Muneer: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USA
Jun Miao: Department of Internal Medicine, Morsani College of Medicine, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USA; Center for Global Health and Infectious Diseases Research, College of Public Health, University of South Florida, 3720 Spectrum Boulevard, Tampa, FL 33612, USA; Corresponding author

Journal volume & issue: Vol. 27, no. 4
p. 109602

Abstract

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Summary: It is a significant challenge to assess the functions of many uncharacterized genes in human malaria parasites. Here, we present a genetic screening tool to assess the contribution of essential genes from Plasmodium falciparum by the conditional CRISPR-/deadCas9-based interference and activation (i/a) systems. We screened both CRISPRi and CRISPRa sets, consisting of nine parasite lines per set targeting nine genes via their respective gRNAs. By conducting amplicon sequencing of gRNA loci, we identified the contribution of each targeted gene to parasite fitness upon drug (artemisinin, chloroquine) and stress (starvation, heat shock) treatment. The screening was highly reproducible, and the screening libraries were easily generated by transfection of mixed plasmids expressing different gRNAs. We demonstrated that this screening is straightforward, robust, and can provide a fast and efficient tool to study essential genes that have long presented a bottleneck in assessing their functions using existing genetic tools.

Published in iScience

ISSN: 2589-0042 (Online)
Publisher: Elsevier
Country of publisher: United States
LCC subjects: Science
Website: http://www.cell.com/iscience/home

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