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.
