Simultaneous multiple allelic replacement in the malaria parasite enables dissection of PKG function.

Konstantinos Koussis,Michael J Blackman,David A Baker,Chrislaine Withers-Martinez

doi:10.26508/lsa.201900626

Abstract

Over recent years, a plethora of new genetic tools has transformed conditional engineering of the malaria parasite genome, allowing functional dissection of essential genes in the asexual and sexual blood stages that cause pathology or are required for disease transmission, respectively. Important challenges remain, including the desirability to complement conditional mutants with a correctly regulated second gene copy to confirm that observed phenotypes are due solely to loss of gene function and to analyse structure-function relationships. To meet this challenge, here we combine the dimerisable Cre (DiCre) system with the use of multiple lox sites to simultaneously generate multiple recombination events of the same gene. We focused on the Plasmodium falciparum cGMP-dependent protein kinase (PKG), creating in parallel conditional disruption of the gene plus up to two allelic replacements. We use the approach to demonstrate that PKG has no scaffolding or adaptor role in intraerythrocytic development, acting solely at merozoite egress. We also show that a phosphorylation-deficient PKG is functionally incompetent. Our method provides valuable new tools for analysis of gene function in the malaria parasite.

Highlights

From the early documentation of targeted gene disruption in yeast by homologous recombination [1] to the use of site-specific recombinases [2] and the development of gene-editing tools such as CRISPR [3, 4], the ability to modify DNA has revolutionised understanding of gene function in model organisms and pathogens
This problem was solved with the adaptation of the dimerisable Cre (DiCre) system initially for Toxoplasma and subsequently for P. falciparum blood stages [10, 11]
Over the past 5 yr, additional modifications have been made to the P. falciparum DiCre system, including installation of the DiCre cassette into alternative chromosomal loci and use of different P. falciparum strains, and the approach has been exploited for the functional analysis of many essential genes [17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29]

Summary

Introduction

From the early documentation of targeted gene disruption in yeast by homologous recombination [1] to the use of site-specific recombinases [2] and the development of gene-editing tools such as CRISPR [3, 4], the ability to modify DNA has revolutionised understanding of gene function in model organisms and pathogens. Conditional deletion or rearrangement of DNA segments through activation of site-specific recombinases such as Cre has been the gold-standard system for gene editing in many model organisms, but attempts to adapt the Cre-lox system to blood stages of P. falciparum initially failed because of difficulties in suppressing constitutive activity of the recombinase [8, 9]. This problem was solved with the adaptation of the dimerisable Cre (DiCre) system initially for Toxoplasma and subsequently for P. falciparum blood stages [10, 11]. Application of the system for the simultaneous generation of both loss-of-function and genetically complemented parasite lines has remained technically challenging

Methods

Results

Conclusion