ENGINEERING PLASMODIUM FALCIPARUM TO ENABLE CRISPR-CAS 9 ASSISTED TEMPLATE FREE GENE EDITING

Malaria remains a leading cause of global morbidity and mortality, especially in the developing world. The decrease in antimalarial drug efficacy and lack of effective vaccines against the disease, emphasise the need for the identification of better drug and vaccine targets. However, the paucity of genomic tools coupled with the lack of a classical non-homologous end joining (NHEJ) mechanism in Plasmodium falciparum hinders the scalability of functional genomic studies aimed at identifying novel druggable targets. Our research aims at developing a novel Clustered Regularly Interspaced Short Palindromic Repeat (CRISPR) Cas9 based NHEJ gene editing tool for scalable gene editing in P. falciparum. The benefit of this tool would be its ability to repair double stranded breaks (DSBs) without requiring a homology template, enabling high throughput forward genetic screens. We approached this by engineering DD2attB parasites to express a minimal two component NHEJ system (Ku and Ligase D) from Mycobacterium tuberculosis (Mt) through single copy integration using the BxB1 (attB x attP) integrase system. We regulated the expression of these two proteins using the TetR-DOZI conditional system. We induced DSBs in two non-essential genes by CRISPR Cas9 gene editing and compared the survival of transgenic parasites (containing the Mt NHEJ proteins) to that of wildtype DD2 parasites. Our data indicate that the Mt NHEJ mechanism can constitute a functional NHEJ mechanism in P. falciparum and acts in an error free manner. We also report that the Mt NHEJ mechanism contributes to de novo telomere addition at both chromosomal and non-chromosomal ends. These results serve as a proof-of-concept for a novel CRISPR based NHEJ strategy in P. falciparum and the first evidence of a role played by a bacterial Ku protein in de novo telomere addition