Defining multiplicity of vector uptake in transfected Plasmodium parasites

Manuela Carrasquilla,Ruddy Montandon,Marcus C S Lee,Rachael Coyle,Sophie Adjalley,Alena Pance,Julian C Rayner,Theo Sanderson,Alejandro Marin-Menendez

doi:10.1038/s41598-020-67791-z

Abstract

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 because 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.

Highlights

The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets
Reverse genetics is a key tool in the global effort to identify drug targets or resistance mechanisms, as well as to explore new biology
We examined the transfection properties of a different Plasmodium species, P. knowlesi, which is reported to possess a considerably higher transfection efficiency than P. falciparum[3], to determine if this system would yield a richer representation of the barcode pool

Summary

Introduction

The recurrent emergence of drug resistance in Plasmodium falciparum increases the urgency to genetically validate drug resistance mechanisms and identify new targets. Developed CRISPR-interference and CRISPR-activation approaches[14,15,16,17], which use a nuclease-dead version of Cas[9] as a DNA-binding protein to either repress or activate gene expression, might allow multiplexed screening even in the absence of NHEJ machinery Such approaches will rely on achieving relatively high transfection efficiencies, and an understanding of how many unique plasmids (containing different gRNAs) are taken up by each parasite. We examined the transfection properties of a different Plasmodium species, P. knowlesi, which is reported to possess a considerably higher transfection efficiency than P. falciparum[3], to determine if this system would yield a richer representation of the barcode pool The data from these collective approaches help us to understand the limitations of the species and the transfection method applied, and have important implications for the design of future library-based genetic experiments

Methods

Results

Conclusion