Functional Characterisation of Actin Binding Protein Formin 1 in P.falciparum (360G-Wellcome-211858_Z_18_Z)

Actin and actin-binding proteins are highly conserved across eukaryotes, however, Apicomplexa lack genes for a number of actin regulators including the Arp2/3 complex, a nucleator of actin filaments. In Plasmodium falciparum, Formin 1 is a likely candidate for actin-filament nucleation during invasion, however a clear demonstration of Formin 1 function has yet to be fully achieved. The aim of this project is to use CRISPR/Cas9 in conjunction with the dimerisable Cre-recombinase (DiCre) system to generate conditional knockouts of formin 1 in P. falciparum. CRISPR/Cas9 will be utilised to simultaneously epitope tag Formin1 and introduce loxP sites within and downstream of the gene in a DiCre expressing strain. Cas9 will be directed by designed guide-RNAs to generate specific double strand breaks in the formin 1 gene which will be repaired by a rescue plasmid containing a synthetic gene with internal loxP sites and flanking DNA segments homologous to target DNA. Epitope tagged Formin 1 will be fully characterised for localisation by immunofluorescence assay and western blotting. Resulting phenotypes from Formin1 knockdown will be analysed via growth curves, video microscopy and super resolution microscopy. This will provide insights into the function of Formin 1 and its potential as a novel drug target.