Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9

Abstract Successful establishment of CRISPR/Cas9 genome editing technology in Plasmodium spp. has provided a powerful tool to transform Plasmodium falciparum into a genetically more tractable organism. Conditional gene regulation approaches are required to study the function of gene products critica...

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Autores principales:	Ellen Knuepfer, Marta Napiorkowska, Christiaan van Ooij, Anthony A. Holder
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2017
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d5e83861321b4d35ab33c4b412310346
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spelling	oai:doaj.org-article:d5e83861321b4d35ab33c4b4123103462021-12-02T12:32:26ZGenerating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas910.1038/s41598-017-03984-32045-2322https://doaj.org/article/d5e83861321b4d35ab33c4b4123103462017-06-01T00:00:00Zhttps://doi.org/10.1038/s41598-017-03984-3https://doaj.org/toc/2045-2322Abstract Successful establishment of CRISPR/Cas9 genome editing technology in Plasmodium spp. has provided a powerful tool to transform Plasmodium falciparum into a genetically more tractable organism. Conditional gene regulation approaches are required to study the function of gene products critical for growth and erythrocyte invasion of blood stage parasites. Here we employ CRISPR/Cas9 to facilitate use of the dimerisable Cre-recombinase (DiCre) that is frequently used to mediate the excision and loss of loxP-flanked DNA sequences in a rapamycin controlled manner. We describe novel CRISPR/Cas9 transfection plasmids and approaches for the speedy, stable and marker-free introduction of transgenes encoding the DiCre recombinase into genomic loci dispensable for blood stage development. Together these plasmids form a toolkit that will allow the rapid generation of transgenic DiCre-expressing P. falciparum lines in any genetic background. Furthermore, the newly developed 3D7-derived parasite lines, constitutively and stably expressing DiCre, generated using this toolkit will prove useful for the analysis of gene products. Lastly, we introduce an improved treatment protocol that uses a lower rapamycin concentration and shorter treatment times, leading to loxP-guided recombination with close to 100% efficiency within the same replication cycle.Ellen KnuepferMarta NapiorkowskaChristiaan van OoijAnthony A. HolderNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 7, Iss 1, Pp 1-12 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ellen Knuepfer Marta Napiorkowska Christiaan van Ooij Anthony A. Holder Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9
description	Abstract Successful establishment of CRISPR/Cas9 genome editing technology in Plasmodium spp. has provided a powerful tool to transform Plasmodium falciparum into a genetically more tractable organism. Conditional gene regulation approaches are required to study the function of gene products critical for growth and erythrocyte invasion of blood stage parasites. Here we employ CRISPR/Cas9 to facilitate use of the dimerisable Cre-recombinase (DiCre) that is frequently used to mediate the excision and loss of loxP-flanked DNA sequences in a rapamycin controlled manner. We describe novel CRISPR/Cas9 transfection plasmids and approaches for the speedy, stable and marker-free introduction of transgenes encoding the DiCre recombinase into genomic loci dispensable for blood stage development. Together these plasmids form a toolkit that will allow the rapid generation of transgenic DiCre-expressing P. falciparum lines in any genetic background. Furthermore, the newly developed 3D7-derived parasite lines, constitutively and stably expressing DiCre, generated using this toolkit will prove useful for the analysis of gene products. Lastly, we introduce an improved treatment protocol that uses a lower rapamycin concentration and shorter treatment times, leading to loxP-guided recombination with close to 100% efficiency within the same replication cycle.
format	article
author	Ellen Knuepfer Marta Napiorkowska Christiaan van Ooij Anthony A. Holder
author_facet	Ellen Knuepfer Marta Napiorkowska Christiaan van Ooij Anthony A. Holder
author_sort	Ellen Knuepfer
title	Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9
title_short	Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9
title_full	Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9
title_fullStr	Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9
title_full_unstemmed	Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9
title_sort	generating conditional gene knockouts in plasmodium – a toolkit to produce stable dicre recombinase-expressing parasite lines using crispr/cas9
publisher	Nature Portfolio
publishDate	2017
url	https://doaj.org/article/d5e83861321b4d35ab33c4b412310346
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Generating conditional gene knockouts in Plasmodium – a toolkit to produce stable DiCre recombinase-expressing parasite lines using CRISPR/Cas9

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