CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for <italic toggle="yes">var2csa</italic> Gene Activation or Silencing in <italic toggle="yes">Plasmodium falciparum</italic>

ABSTRACT Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epige...

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Autores principales:	Jessica M. Bryant, Clément Regnault, Christine Scheidig-Benatar, Sebastian Baumgarten, Julien Guizetti, Artur Scherf
Formato:	article
Lenguaje:	EN
Publicado:	American Society for Microbiology 2017
Materias:	CRISPR/Cas9 Plasmodium falciparum antigenic variation transcriptional regulation var genes Microbiology QR1-502
Acceso en línea:	https://doaj.org/article/91d89e87606c4bb78714e1eb9743529d
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spelling	oai:doaj.org-article:91d89e87606c4bb78714e1eb9743529d2021-11-15T15:51:43ZCRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for <italic toggle="yes">var2csa</italic> Gene Activation or Silencing in <italic toggle="yes">Plasmodium falciparum</italic>10.1128/mBio.00729-172150-7511https://doaj.org/article/91d89e87606c4bb78714e1eb9743529d2017-09-01T00:00:00Zhttps://journals.asm.org/doi/10.1128/mBio.00729-17https://doaj.org/toc/2150-7511ABSTRACT Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes—the intron—in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression.Jessica M. BryantClément RegnaultChristine Scheidig-BenatarSebastian BaumgartenJulien GuizettiArtur ScherfAmerican Society for MicrobiologyarticleCRISPR/Cas9Plasmodium falciparumantigenic variationtranscriptional regulationvar genesMicrobiologyQR1-502ENmBio, Vol 8, Iss 4 (2017)
institution	DOAJ
collection	DOAJ
language	EN
topic	CRISPR/Cas9 Plasmodium falciparum antigenic variation transcriptional regulation var genes Microbiology QR1-502
spellingShingle	CRISPR/Cas9 Plasmodium falciparum antigenic variation transcriptional regulation var genes Microbiology QR1-502 Jessica M. Bryant Clément Regnault Christine Scheidig-Benatar Sebastian Baumgarten Julien Guizetti Artur Scherf CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for <italic toggle="yes">var2csa</italic> Gene Activation or Silencing in <italic toggle="yes">Plasmodium falciparum</italic>
description	ABSTRACT Plasmodium falciparum relies on monoallelic expression of 1 of 60 var virulence genes for antigenic variation and host immune evasion. Each var gene contains a conserved intron which has been implicated in previous studies in both activation and repression of transcription via several epigenetic mechanisms, including interaction with the var promoter, production of long noncoding RNAs (lncRNAs), and localization to repressive perinuclear sites. However, functional studies have relied primarily on artificial expression constructs. Using the recently developed P. falciparum clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system, we directly deleted the var2csa P. falciparum 3D7_1200600 (Pf3D7_1200600) endogenous intron, resulting in an intronless var gene in a natural, marker-free chromosomal context. Deletion of the var2csa intron resulted in an upregulation of transcription of the var2csa gene in ring-stage parasites and subsequent expression of the PfEMP1 protein in late-stage parasites. Intron deletion did not affect the normal temporal regulation and subsequent transcriptional silencing of the var gene in trophozoites but did result in increased rates of var gene switching in some mutant clones. Transcriptional repression of the intronless var2csa gene could be achieved via long-term culture or panning with the CD36 receptor, after which reactivation was possible with chondroitin sulfate A (CSA) panning. These data suggest that the var2csa intron is not required for silencing or activation in ring-stage parasites but point to a subtle role in regulation of switching within the var gene family. IMPORTANCE Plasmodium falciparum is the most virulent species of malaria parasite, causing high rates of morbidity and mortality in those infected. Chronic infection depends on an immune evasion mechanism termed antigenic variation, which in turn relies on monoallelic expression of 1 of ~60 var genes. Understanding antigenic variation and the transcriptional regulation of monoallelic expression is important for developing drugs and/or vaccines. The var gene family encodes the antigenic surface proteins that decorate infected erythrocytes. Until recently, studying the underlying genetic elements that regulate monoallelic expression in P. falciparum was difficult, and most studies relied on artificial systems such as episomal reporter genes. Our study was the first to use CRISPR/Cas9 genome editing for the functional study of an important, conserved genetic element of var genes—the intron—in an endogenous, episome-free manner. Our findings shed light on the role of the var gene intron in transcriptional regulation of monoallelic expression.
format	article
author	Jessica M. Bryant Clément Regnault Christine Scheidig-Benatar Sebastian Baumgarten Julien Guizetti Artur Scherf
author_facet	Jessica M. Bryant Clément Regnault Christine Scheidig-Benatar Sebastian Baumgarten Julien Guizetti Artur Scherf
author_sort	Jessica M. Bryant
title	CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for <italic toggle="yes">var2csa</italic> Gene Activation or Silencing in <italic toggle="yes">Plasmodium falciparum</italic>
title_short	CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for <italic toggle="yes">var2csa</italic> Gene Activation or Silencing in <italic toggle="yes">Plasmodium falciparum</italic>
title_full	CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for <italic toggle="yes">var2csa</italic> Gene Activation or Silencing in <italic toggle="yes">Plasmodium falciparum</italic>
title_fullStr	CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for <italic toggle="yes">var2csa</italic> Gene Activation or Silencing in <italic toggle="yes">Plasmodium falciparum</italic>
title_full_unstemmed	CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for <italic toggle="yes">var2csa</italic> Gene Activation or Silencing in <italic toggle="yes">Plasmodium falciparum</italic>
title_sort	crispr/cas9 genome editing reveals that the intron is not essential for <italic toggle="yes">var2csa</italic> gene activation or silencing in <italic toggle="yes">plasmodium falciparum</italic>
publisher	American Society for Microbiology
publishDate	2017
url	https://doaj.org/article/91d89e87606c4bb78714e1eb9743529d
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CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for <italic toggle="yes">var2csa</italic> Gene Activation or Silencing in <italic toggle="yes">Plasmodium falciparum</italic>

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