The dimerisable Cre recombinase allows conditional genome editing in the mosquito stages of Plasmodium berghei.

Priyanka Fernandes,Bénédicte Hoareau-Coudert,Delphine Patarot,Manon Loubens,Sylvie Briquet,Olivier Silvie,Tobias Spielmann

doi:10.1371/journal.pone.0236616

Abstract

Asexual blood stages of the malaria parasite are readily amenable to genetic modification via homologous recombination, allowing functional studies of parasite genes that are not essential in this part of the life cycle. However, conventional reverse genetics cannot be applied for the functional analysis of genes that are essential during asexual blood-stage replication. Various strategies have been developed for conditional mutagenesis of Plasmodium, including recombinase-based gene deletion, regulatable promoters, and mRNA or protein destabilization systems. Among these, the dimerisable Cre (DiCre) recombinase system has emerged as a powerful approach for conditional gene deletion in P. falciparum. In this system, the bacteriophage Cre is expressed in the form of two separate, enzymatically inactive polypeptides, each fused to a different rapamycin-binding protein. Rapamycin-induced heterodimerization of the two components restores recombinase activity. We have implemented the DiCre system in the rodent malaria parasite P. berghei, and show that rapamycin-induced excision of floxed DNA sequences can be achieved with very high efficiency in both mammalian and mosquito parasite stages. This tool can be used to investigate the function of essential genes not only in asexual blood stages, but also in other parts of the malaria parasite life cycle.

Highlights

The life cycle of Plasmodium parasites is complex, and involves several stages of development and differentiation within the vertebrate host and Anopheles vector
The two components were placed under control of the constitutive bidirectional promoter eEF1alpha, and followed by the 3’ untranslated region (UTR) from PfCAM and PbHSP70, respectively
We first generated a markerfree fluorescent parasite line in P. berghei, which constitutively expresses both components of the dimerisable Cre recombinase [14], by introducing components of the DiCre along with an mCherry cassette into a non-essential P230p locus [24]

Summary

Introduction

The life cycle of Plasmodium parasites is complex, and involves several stages of development and differentiation within the vertebrate host and Anopheles vector. Infection begins when motile forms of the parasite known as sporozoites are transmitted by infected Anopheles mosquitoes to the host. These sporozoites first go through an obligatory intrahepatic step, where they multiply into thousands of merozoites that are released into the blood stream, where they begin the round of replication within erythrocytes. Some merozoites develop into gametocytes which fuse to form a motile ookinete in the gut of the mosquito.

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Conclusion