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
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.
