A versatile strategy for rapid conditional genome engineering using loxP sites in a small synthetic intron in Plasmodium falciparum

Scientific Reports
Matthew L JonesMoritz Treeck

Abstract

Conditional genome engineering in the human malaria pathogen Plasmodium falciparum remains highly challenging. Here we describe a strategy for facile and rapid functional analysis of genes using an approach based on the Cre/lox system and tailored for organisms with short and few introns. Our method allows the conditional, site-specific removal of genomic sequences of essential and non-essential genes by placing loxP sites into a short synthetic intron to produce a module (loxPint) can be placed anywhere in open reading frames without compromising protein expression. When duplicated, the loxPint module serves as an intragenic recombineering point that can be used for the fusion of gene elements to reporters or the conditional introduction of point mutations. We demonstrate the robustness and versatility of the system by targeting the P. falciparum merozoite surface protein 1 gene (msp1), which has previously proven refractory to genetic interrogation, and the parasite exported kinase FIKK10.1.

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Citations

Mar 14, 2017·Nature Methods·Jakob BirnbaumTobias Spielmann
Mar 29, 2018·Parasites & Vectors·Holly MatthewsCatherine J Merrick
Dec 6, 2018·PloS One·Nonlawat BoonyalaiMichael J Blackman
Jan 17, 2020·The Biochemical Journal·Sarah J TarrMichael J Blackman
Jul 25, 2019·Nature Communications·Julien Robert-PaganinAnne Houdusse
Oct 28, 2019·British Journal of Haematology·Ghizal SiddiquiBrian M Cooke
Oct 20, 2018·Parasites & Vectors·Praveen Balabaskaran-Nina, Sanjay A Desai
Mar 18, 2020·Life Science Alliance·Konstantinos KoussisMichael J Blackman
Jul 1, 2020·Proceedings of the National Academy of Sciences of the United States of America·Joachim M MatzMichael J Blackman
Feb 16, 2021·Frontiers in Cellular and Infection Microbiology·Annemarie Voorberg-van der WelAnne-Marie Zeeman
Apr 23, 2021·PLoS Pathogens·Heather M KudybaVasant Muralidharan
May 12, 2021·Molecular and Biochemical Parasitology·Silvia HaaseJake Baum

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Methods Mentioned

BETA
transfection
genetic modification
RAP
gene modification
PCR
gene knock-out

Software Mentioned

Geneart®
AxioVision
DiCre
PlasmoDB

Related Concepts

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