Dataset Information

Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction.

ABSTRACT: The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of their subcellular compartmentalization. Induction and repression of gene expression are rapid, homogeneous and stable over prolonged periods. To demonstrate practical application of our system, we used it to reveal direct links between antimalarial drugs and their native parasite molecular target. This is an important outcome given the rapid spread of resistance, and intensified efforts to efficiently discover and optimize new antimalarial drugs. Overall, the studies presented highlight the utility of our system for broadly controlling gene expression and performing functional genetics in P. falciparum.

SUBMITTER: Goldfless SJ

PROVIDER: S-EPMC4223869 | biostudies-literature | 2014 Nov

REPOSITORIES: biostudies-literature

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Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction.

Goldfless Stephen J SJ Wagner Jeffrey C JC Niles Jacquin C JC

Nature communications 20141105

The available tools for conditional gene expression in Plasmodium falciparum are limited. Here, to enable reliable control of target gene expression, we build a system to efficiently modulate translation. We overcame several problems associated with other approaches for regulating gene expression in P. falciparum. Specifically, our system functions predictably across several native and engineered promoter contexts, and affords control over reporter and native parasite proteins irrespective of th ...[more]

PMID: 25370483

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Project description:By fusing catalytically dead Cas9 (dCas9) to active domains of histone deacetylase (Sir2a) or acetyltransferase (GCN5), this CRISPR interference/activation (CRISPRi/a) system allows gene regulation at the transcriptional level without causing permanent changes in the parasite genome. However, the constitutive expression of dCas9 poses a challenge for studying essential genes, which may lead to adaptive changes in the parasite, masking the true phenotypes. Here, we developed a leak-free inducible CRISPRi/a system by integrating the DiCre/loxP regulon to allow the expression of dCas9-GCN5/-Sir2a upon transient induction with rapamycin, which allows convenient transcriptional regulation of a gene of interest by introducing a guide RNA targeting its transcription start region. Using eight genes that are either silent or expressed from low to high levels during asexual erythrocytic development, we evaluated the robustness and versatility of this system in the asexual parasites. For most genes analyzed, this inducible CRISPRi/a system led to 1.5- to 3-fold up-or downregulation of the target genes at the mRNA level. Alteration in the expression of PfK13 and PfMYST resulted in altered sensitivities to artemisinin. For autophagy-related protein 18, an essential gene related to artemisinin resistance, a >2-fold up- or downregulation was obtained by inducible CRISPRi/a, leading to growth retardation. For the master regulator of gametocytogenesis, PfAP2-G, a >10-fold increase of the PfAP2-G transcripts was obtained by CRISPRa, resulting in >4-fold higher gametocytemia in the induced parasites. Additionally, inducible CRISPRi/a could also regulate gene expression in gametocytes. This inducible epigenetic regulation system offers a fast way of studying gene functions in Plasmodium falciparum. IMPORTANCE Understanding the fundamental biology of malaria parasites through functional genetic/genomic studies is critical for identifying novel targets for antimalarial development. Conditional knockout/knockdown systems are required to study essential genes in the haploid blood stages of the parasite. In this study, we developed an inducible CRISPRi/a system via the integration of DiCre/loxP. We evaluated the robustness and versatility of this system by activating or repressing eight selected genes and achieved up- and downregulation of the targeted genes located in both the euchromatin and heterochromatin regions. This system offers the malaria research community another tool for functional genetic studies.

Dataset Information

Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction.

Publications

Versatile control of Plasmodium falciparum gene expression with an inducible protein-RNA interaction.

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