Defining multiplicity of vector uptake in transfected Plasmodium parasites

Manuela Carrasquilla, Sophie Adjalley, Theo Sanderson, Alejandro Marin-Menendez, Rachael Coyle, Ruddy Montandon, Julian Rayner, Alena Pance, Marcus CS Lee, Apollo-University Of Cambridge Repository
The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Reverse genetics have facilitated genome-scale knockout screens in Plasmodium berghei and Toxoplasma gondii, in which pooled transfections of multiple vectors were critical to increasing scale and throughput. These approaches have not yet been implemented in human malaria species such as P. falciparum and P. knowlesi, in part
more » ... se the extent to which pooled transfections can be performed in these species remains to be evaluated. Here we use next-generation sequencing to quantitate uptake of a pool of 94 barcoded vectors. The distribution of vector acquisition allowed us to estimate the number of barcodes and DNA molecules taken up by the parasite population. Dilution cloning of P. falciparum transfectants showed that individual clones possess as many as seven episomal barcodes, revealing that an intake of multiple vectors is a frequent event despite the inefficient transfection efficiency. Transfection of three spectrally-distinct fluorescent reporters allowed us to evaluate different transfection methods and revealed that schizont-stage transfection limited the tendency for parasites to take up multiple vectors. In contrast to P. falciparum, we observed that the higher transfection efficiency of P. knowlesi resulted in near complete representation of the library. These findings have important implications for how reverse genetics can be scaled in culturable Plasmodium species. Reverse genetics is a key tool in the global effort to identify drug targets or resistance mechanisms, as well as to explore new biology. Technologies for genetic manipulation of organisms have advanced significantly in the last decade, particularly through site-specific nucleases such as Cas9 1 that can be used to increase the efficiency and specificity of modification. However, using genetics to validate gene function in Plasmodium falciparum, the most virulent of the causative agents of human malaria, has been consistently challenging, for multiple reasons. The high AT content of its genome (> 80%) makes generating large stable plasmids in E. coli difficult, and also limits the potential targets for Cas9 due to its requirement for an NGG protospacer adjacent motif (PAM) sequence, which is much rarer in P. falciparum genomic DNA than most eukaryotes. P. falciparum also has low transfection efficiencies compared with other Plasmodium species 2,3 , despite attempts to generate more efficient protocols 4,5 . These constraints have limited progress in interrogating the genome of the parasite to uncover potential new drug targets and the roles of the many genes of unknown or poorly described function. There are also specific challenges to the application of CRISPR/Cas9 to large-scale genetic screening in P. falciparum, unlike in related apicomplexans such as Toxoplasma 6 . Analysis of the P. falciparum genome 7 indicates that this organism lacks one of the two major DNA repair mechanisms, non-homologous end joining (NHEJ). In principle, this should provide an advantage for genome editing, as the introduction of a double strand break in the parasite DNA coupled with homology templates that provide the desired modification should result in consistent homology-directed repair without competing error-prone events. As a result, the application of CRISPR/ Cas9 in P. falciparum has relied on donor templates and has been used to knockout genes 8,9 , and validate key open 1
doi:10.17863/cam.57160 fatcat:uh75cuogwrcshffw4ygf3dgq5u