CRISPR/Cas9 Genome Editing Reveals That the Intron Is Not Essential for var2csa Gene Activation or Silencing in Plasmodium falciparum

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.IMPORTANCEPlasmodium 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.

Keywords: CRISPR/Cas9; Plasmodium falciparum; antigenic variation; transcriptional regulation; var genes.

FIG 1

Generation of the intronless var gene mutant. (A) Schematic of a representative var gene and all associated RNA: full-length mRNA originating from the 5′ promoter and sense and antisense sterile transcripts originating from the intron (solid gray and dotted gray lines, respectively). var2csa intron knockout mutants were created by transfection with the pUF1_Cas9 and pL7_var2csa_IntKO plasmids. The pL7 plasmid contains the guide RNA (yellow) and DNA homology region KO construct corresponding to the 500 bp of exon I (green) immediately upstream of the intron (orange) fused in frame to the homology region corresponding to the 500 bp of exon II (blue) immediately downstream of the intron. A shield mutation (red) is included in the KO construct. Homologous recombination of the pL7 KO construct with the var2csa gene results in the deletion of the intron. Gel electrophoresis of PCRs performed on genomic DNA from 3D7 WT bulk culture (3D7 WT Bulk), three var2csa intron KO mutant clones (Intron KO A, Intron KO B, and Intron KO C), and one var2csa intron KO telomere mutant clone (Intron KO Tel Mut) demonstrated deletion of the intron. Primers (shown at left as arrowheads labeled “Up,” and “Down”) were designed to detect the presence of the var2csa intron. The marker is indicated (M), and a PCR using only water (H₂O) served as a control. (B) Next-generation sequencing of genomic DNA from WT 3D7 parasites (black), var2csa intron KO clone A (green), and a telomere mutant clone (red) showed that the intron was deleted successfully with CRISPR/Cas9. The top panel shows 57 kb of DNA adjacent to the chromosome 12 telomere (at the right). The bottom panel is an enlargement of the var2csa gene (Pf3D7_1200600). DNA read enrichment is indicated on the y axis, and gene models are shown in gray at the bottom. A schematic of the positions of the telomere repeats (black “Tel”) and homology regions 1 and 2 (HR1 in green and HR2 in blue) within the telomere mutant is shown at the bottom. (C) Stranded sequencing of RNA harvested 12 hpi from WT 3D7 parasites (black), var2csa intron KO clone A (green), and a telomere mutant clone (red) showed that var2csa full-length transcripts were produced in 3D7 WT parasites and the intron KO clone. The telomere mutant clone produced var2csa transcript up to, but not past, the end of the second homology region used for CRISPR/Cas9 editing. Coverage plots are shown at the top of each panel, and read stacks are shown at the bottom, where red lines indicate sense transcription reads, and blue lines indicate antisense transcription reads. Numbers of reads are indicated on the y axis, and the gene model is shown in gray at the top.

FIG 2

var2csa intron deletion results in upregulation of transcription in ring-stage parasites. (A) RNA was isolated from highly synchronized ring-stage parasites (12 hpi) from a CSA-panned FCR3 culture (top panel, yellow), a 3D7 WT bulk culture (second panel, black), an intron KO telomere mutant clone (third panel, red), and six var2csa intron KO mutant clones (fourth, fifth, and sixth panels). RT-qPCR analysis of all var genes was performed, and cDNA levels were normalized to those of serine-tRNA ligase. Intron KO clone A is shown in the fourth panel in green along with three other clones (in gray) showing similar monoallelic transcriptional var profiles. Intron KO clones B (fifth panel in purple) and C (sixth panel in blue) transcribed multiple var genes. A gray dashed box indicates the var2csa gene, Pf3D7_1200600. (B) RNA was isolated from highly synchronized parasites from a CSA-panned FCR3 culture (yellow); a 3D7 WT bulk culture (black); intron KO mutant clones A (green), B (purple), and C (blue); and an intron KO telomere mutant clone (red) at 12 (ring), 24 (trophozoite), and 36 (schizont) hpi. RT-qPCR analysis of Pf3D7_0412700 (upsC var gene) and Pf3D7_1200600 (var2csa) was performed, and var gene cDNA levels were normalized to those of serine-tRNA ligase.

FIG 3

var2csa intronless mutant produces VAR2CSA PfEMP1 protein. (A) Western blot analysis of 30-hpi iRBC membrane extracts performed using antibodies against VAR2CSA PfEMP1 (top left panel) or the PfEMP1 ATS region (top right panel). Extracts were prepared from a CSA-panned FCR3 culture, intron KO clone A, and a 3D7 WT bulk culture. An amido black staining of the Western blot is shown as a loading control. Molecular masses are indicated at the left. (B) Immunofluorescence analysis of live 30-hpi iRBCs containing CSA-panned FCR3, 3D7 WT bulk, intron KO clone A, or intron KO telomere mutant clone parasites. DNA was stained with DAPI (magenta), and iRBCs were surface labeled with specific antibodies against VAR2CSA PfEMP1 (green). Fluorescent images are shown in the top panel, and wide-field merged images are shown in the bottom panel. Scale bar, 10 μm.

FIG 4

An intronless var2csa gene can be silenced via long-term culture or CD36 panning, then reactivated with CSA panning. RNA was isolated from highly synchronized ring-stage parasites (12 hpi) from a 3D7 WT bulk culture (black) and var2csa intron KO mutant clone A (gray). RT-qPCR analysis of all var genes was performed before (“Initial Culture,” top panel) and after (“Long-term Culture,” second panel) 2 months of continuous culture. The initial cultures were also subjected to three rounds of CD36 panning (third panel), followed by three rounds of CSA panning (bottom panel). var gene cDNA levels are normalized to those of serine-tRNA ligase. A gray dotted box indicates the var2csa gene, Pf3D7_1200600.