Dataset Information

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

ABSTRACT: 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.

SUBMITTER: Lucky AB

PROVIDER: S-EPMC11015506 | biostudies-literature | 2024 Apr

REPOSITORIES: biostudies-literature

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

Lucky Amuza Byaruhanga AB Wang Chengqi C Li Xiaolian X Liang Xiaoying X Muneer Azhar A Miao Jun J

iScience 20240327 4

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 <i>Plasmodium falciparum</i> 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, ...[more]

PMID: 38617559

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Project description:BackgroundDiagnosis of infectious diseases now benefits from advancing technology to perform multiplex analysis of a growing number of variables. These advances enable simultaneous surveillance of markers characterizing species and strain complexity, mutations associated with drug susceptibility, and antigen-based polymorphisms in relation to evaluation of vaccine effectiveness. We have recently developed assays detecting single nucleotide polymorphisms (SNPs) in the P. falciparum genome that take advantage of post-PCR ligation detection reaction and fluorescent microsphere labeling strategies. Data from these assays produce a spectrum of outcomes showing that infections result from single to multiple strains. Traditional methods for distinguishing true positive signal from background can cause false positive diagnoses leading to incorrect interpretation of outcomes associated with disease treatment.ResultsFollowing analysis of Plasmodium falciparum dihydrofolate reductase SNPs associated with resistance to a commonly used antimalarial drug, Fansidar (Sulfadoxine/pyrimethamine), and presumably neutral SNPs for parasite strain differentiation, we first evaluated our data after setting a background signal based on the mean plus three standard deviations for known negative control samples. Our analysis of single allelic controls suggested that background for the absent allele increased as the concentration of the target allele increased. To address this problem, we introduced a simple change of variables from customary (X,Y) (Cartesian) coordinates to planar polar coordinates (X = rcos(theta), Y = rsin(theta)). Classification of multidimensional fluorescence signals based on histograms of angular and radial data distributions proved more effective than classification based on Cartesian thresholds. Comparison with known diallelic dilution controls suggests that histogram-based classification is effective for major:minor allele concentration ratios as high as 10:1.ConclusionWe have observed that the diallelic SNP data resulting from analysis of P. falciparum mutations is more accurately diagnosed when a simple polar transform of the (X,Y) data into (r,theta) is used. The development of high through-put methods for genotyping P. falciparum SNPs and the refinement of analytical approaches for evaluating these molecular diagnostic results significantly advance the evaluation of parasite population diversity and antimalarial drug resistance.

Dataset Information

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

Publications

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

Similar Datasets

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