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H2020Individual fellowship2018–2020

REACT · Uncovering the role of cis genetic elements in antigenic variation of Plasmodium falciparum using the CRISPR-Cas9 genome editing technology

Horizon 2020 — Marie Skłodowska-Curie Actions

Duration
2018-01-01 → 2020-02-28
EU contribution
€185,076
Participants
1
Scheme
MSCA-IF-EF-ST

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Results in brief

Uncovering the role of cis genetic elements in antigenic variation of Plasmodium falciparum using the CRISPR-Cas9 genome editing technology

The objectives of the project REACT were focused on the use of CRISPR-Cas9 technology to find out the role of cis-genetic elements on the var gene family expression in Plasmodium falciparum. Direct deletion of such elements proved to be challenging, mainly due to the high degree of similarity between different var genes and high AT-richness in intergenic regions characteristic of P. falciparum. This hindered the design of a specific guide for a given var gene. We succeeded generating lines with dCas9 fused to some epigenetic effectors as GCN5 and Sir2A, although no effect was observed in the transcriptional level of the targeted var gene. CRISPR-Cas9 technology was then applied to study other DNA elements suspected to be involved in var gene activation, so-called RUF6 (RNA of unknown function 6), whose global depletion (simultaneous downregulation by means of CRISPR interference -CRISPRi-) led to a complete loss of var gene expression. Additional research, still in progress, points that these elements may be involved in the relocation of internal var gene clusters to the nuclear periphery and/or in 3D nuclear organisation by means of long-range interactions among them. In parallel, we studied the heterochromatin biology of the parasite, an epigenetic mechanism responsible for the by-default repression of more than 400 genes in P. falciparum including all the gene families submitted to antigenic variation, as var genes. To do so, we explored a particular gene, the ap2-g, searching for DNA elements adjacent to the limit between euchromatic and heterochromatic domains, the same strategy previously used to find out the cis-genetic DNA elements found in var genes. This gene provided an excellent model for such purpose, as it could be easily targeted for genetic manipulation, is of outstanding relevance for parasite transmission cycle (master regulator for sexual commitment) and it is a single-gene heterochromatin cluster. Specific DNA-protein interaction was detected only in the terminal region of ap2-g by electrophoretic motility shift assay (EMSA), and its replacement using CRISPR-Cas9 tool resulted in the shift of the boundary between chromatin domains up to 2 kb downstream the stop codon, where it is normally located. Proteomic data pointed to involvement of RNA binding proteins in this process. Our results on this topic supposed the first description of a DNA element potentially acting as boundary element and highlighted the possible role of RNA binding proteins in the triggering of sexual development in P. falciparum or establishment of a boundary between chromatin domains.

Data: CORDIS, © European Union

Project objective

Malaria is an infectious human disease caused by parasitic protozoans of the Plasmodium type. P. falciparum causes the most lethal form with about 400.000 deaths annually. Pathogenesis involves expression of clonally variant molecules encoded by multi gene families at the surface of the infected host cells (erythrocyte). The most important virulence factor is called PfEMP1, which undergoes antigenic variation and is encoded by 60 var gene members. Despite the major scientific efforts, the molecular mechanisms orchestrating antigenic variation are still elusive. The reason is the lack of tools to perform genome editing in the natural epigenetic context, which is crucial in this important immune escape process.We aim to investigate antigenic variation of P. falciparum, by means of the recently developed CRISPR-Cas9 tool. This genome editing technology allows the in situ genetic manipulation of var gene regulatory elements in a native chromatin context. Virtually, any locus can be targeted by a RNA guide to insert, exchange or delete specific DNA regions including generation of point mutations without introducing a selective marker. Alternatively, we will use a dead Cas9 version (dCas9), which consist on the targeting of the desired locus by an inactive Cas9. This dCas9 can be fused to protein tags or effectors. We will use these techniques to i) delete cis-regulatory elements of var genes, ii) to identify factors associated to cis-regulatory elements by Cas9 targeted immunoprecipitations and iii) to engineer a tool to perform activation/silencing of specific var genes by fusing the dCas9 with epigenetic effector molecules (silencing and activators).This project will combine state-of-the-art techniques in genome editing to study malaria parasite virulence and provide reliable, accurate and focused data about the implication of epigenetics in the process of antigenic variation. The new tools developed in this project will be highly relevant for other human pathogens.

Original text from CORDIS.

Participants

  • INSTITUT PASTEUR · ParisCoordinatorFrance

Links

Data: CORDIS, © European Union