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Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum.

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Affiliations

  • 1. Unit of Human Parasite Molecular and Cell Biology, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China.
      Authors
    • Xiao B 1
    • Yin S 1
    • Hu Y 1
    • Sun M 1
    • Wei J 1
    • Huang Z 1
    • Wen Y 1
    • Dai X 1
    • Chen H 1
    (9 authors)
  • 2. Laboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health, Bethesda, MD 20892-8132.
      Authors
    • Mu J 2
    (1 author)
  • 3. Department of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612.
      Authors
    • Cui L 3
    (1 author)
  • 4. Unit of Human Parasite Molecular and Cell Biology, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, University of Chinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China;
      Authors
    • Jiang L 4
    (1 author)

ORCIDs linked to this article

Proceedings of the National Academy of Sciences of the United States of America, 24 Dec 2018, 116(1):255-260
https://doi.org/10.1073/pnas.1813542116 PMID: 30584102 PMCID: PMC6320497

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Abstract 

Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.

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Proc Natl Acad Sci U S A. 2019 Jan 2; 116(1): 255–260.
Published online 2018 Dec 24. https://doi.org/10.1073/pnas.1813542116
PMCID: PMC6320497
PMID: 30584102

Epigenetic editing by CRISPR/dCas9 in Plasmodium falciparum

Bo Xiao,a Shigang Yin,a Yang Hu,a Maoxin Sun,a,b Jieqiong Wei,a Zhenghui Huang,a Yuhao Wen,a Xueyu Dai,a Huiling Chen,a Jianbing Mu,c Liwang Cui,d and Lubin Jianga,b,1
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Supplementary Materials

Significance

As a result of low homologous recombination efficiency and the lack of RNAi in the Plasmodium falciparum genome, feasible gene-editing toolkits are urgently needed. Although various gene editing strategies have been successfully applied to P. falciparum, there are still serious limitations. Here we show that by fusing histone acetyltransferase or deacetylase to a dCas9 mutant in the CRISPR/dCas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system, the expression of target genes could be efficiently and specifically regulated in P. falciparum. The two epigenetic toolkits will expand current applications for functional genetics and epigenetics studies in P. falciparum, and therefore accelerate the understanding of malaria pathogenesis.

Keywords: malaria parasite, CRISPR/dCas9, epigenetic editing, invasion change

Abstract

Genetic manipulation remains a major obstacle for understanding the functional genomics of the deadliest malaria parasite Plasmodium falciparum. Although the CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) system has been successfully applied to introduce permanent changes in the parasite genome, its use is still limited. Here we show that fusing different epigenetic effector domains to a Cas9 null mutant efficiently and specifically reprograms the expression of target genes in P. falciparum. By precisely writing and erasing histone acetylation at the transcription start site regions of the invasion-related genes reticulocyte binding protein homolog 4 (rh4) and erythrocyte binding protein 175 (eba-175), respectively, we achieved significant activation of rh4 and repression of eba-175, leading to the switch of the parasite invasion pathways into human erythrocytes. By using the epigenetic knockdown system, we have also characterized the effects of PfSET1, previously identified as an essential gene, on expression of mainly trophozoite- and schizont-specific genes, and therefore regulation of the growth of the mature forms of P. falciparum. This epigenetic CRISPR/dCas9 system provides a powerful approach for regulating gene expression at the transcriptional level in P. falciparum.

The most dangerous form of malaria, Plasmodium falciparum, causes almost half a million deaths annually. Efficient approaches to genome editing in the malaria parasites are urgently needed to better understand fundamental parasite biology, such as invasion of host cells and pathogenesis. As an excellent complement to conventional methods of genetic manipulation in P. falciparum (1–4), CRISPR/Cas9 (clustered regularly interspaced short palindromic repeat/CRISPR-associated protein 9) has shown great advantages in genome engineering such as gene disruption, tagging, and allelic replacement in Plasmodium spp (5–7). However, most genetic manipulation systems have major limitations in the disruption of essential genes or long noncoding RNAs located within exons or introns in the haploid genome of the parasite because of the lack of RNAi in P. falciparum (8). Here we provided a genomic disruption-free system for epigenetically regulating transcription of parasite genes by episomal expression of dCas9-fusion protein and sgRNA.

Dynamic histone modifications within the transcription start site (TSS) regions of eukaryotic genes are important means of transcriptional regulation. In P. falciparum, epigenetic enzymes modifying histone acetylation or methylation play critical roles in controlling gene expression (9–12). The dCas9 protein fused with the epigenetic modifier can be efficiently applied to transcriptional reprograming of the targeting gene in eukaryotes (13–16). However, the successful application with dCas9 in Plasmodium parasites has not been reported yet. Here we applied a modified CRISPR/Cas9 system to epigenetically regulate transcription of parasite invasion-related genes and demonstrated that a null Cas9 could precisely guide the fused epigenetic regulators such as histone acetyltransferase (HAT) or deacetylase (HDAC) domains to the TSS regions of the target genes to up-regulate or down-regulate histone acetylation, leading to efficient gene overexpression or transcriptional knockdown in P. falciparum.

Results and Discussion

Recombinant dCas9 Regulators Localize in Parasite Nuclei.

To avoid any DNA cleavages generated by the DNA endonuclease activity of Cas9, a nuclease-deficient Streptococcus pyogenes Cas9 (dead Cas9:dCas9) harboring two mutations (D10A and H840A) in the catalytic domains while preserving the RNA-guided DNA binding ability (17, 18), was selected to recruit HAT or HDAC domains to the TSS regions of target genes (Fig. 1 A and B). A 3×FLAG epitope tag at the N terminus was used for detection of the recombinant dCas9. We first fused the core enzymatic region of PfGCN5 HAT (PF3D7_0823300) (19) to the C terminus of the dCas9 (dCas9GCN5) in the plasmid pUF1-Cas9 to produce pUF1-dCas9GCN5. Successful expression of dCas9GCN5 was determined by Western blot (Fig. 1C). In addition, dCas9GCN5 was highly enriched in parasite nuclei guided by the two nuclear localization sequences (NLS; Fig. 1E). On the contrary, we cloned the coding region of PfSir2a (PF3D7_1328800), a P. falciparum histone deacetylase that can broadly remove acetylation at multiple lysine residues at the N-terminal tail of histone H3 and H4 proteins in a NAD+-dependent manner (20, 21), to the C terminus of dCas9 for generating a recombinant dCas9Sir2a, as described earlier. Our results confirmed the expression of dCas9Sir2a (Fig. 1D) and its nuclear localization (Fig. 1F). As a negative control, a dCas9 fused to GFP was also expressed in the resulting parasite 3D7-dCas9-GFP (Fig. 1D).

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Epigenetic editing of P. falciparum genes by using the CRISPR/dCas9 system. (A and B) Schematic diagram illustrating the strategy of epigenetic gene editing via the CRISPR/dCas9 system, in which an enzymatic domain of PfHAT (PfGCN5) (A) or PfHDAC (PfSir2a) (B) for activating (ON) or repression (OFF) genes is fused in frame to the C terminus of the dCas9 protein, a nuclease-deficient S. pyogenes Cas9 harboring two mutations (D10A and H840A), as shown in the catalytic domains. A 3×FLAG peptide is placed at the N terminus of the recombinant protein, and a nuclear localization signal (NLS) is inserted downstream of the 3×FLAG and dCas9, respectively, to import the recombinant dCas9 into the parasite nucleus. A triple glycine-serine protein domain linker (GS3) is inserted downstream of NLS to allow for an extended conformation and maximal flexibility of the epigenetic enzyme domain. The TSS of the target gene is indicated as a red flag. Ac, histone acetylation. (C and D) Western blot analyses of the recombinant dCas9 proteins in parasite lines Dd2-GCN5-R1 and Dd2-GCN5-R2 (C) and 3D7-Sir2a-E1, 3D7-Sir2a-E2, and 3D7-dCas9-GFP (D), by using an anti-Cas9 antibody. The wild-type Dd2 and 3D7 were tested as negative controls, respectively. The wild-type Cas9 in 3D7-SETvs-KO, in which Cas9 was generated to mediate the PfSETvs gene knockout, was detected to indicate the size of the recombinant dCas9. Actin, as loading control, was also assayed in the same samples by using an anti-actin antibody. (E and F) IFA analyses of the recombinant dCas9 proteins in parasite lines Dd2-GCN5-R1 (E) and 3D7-Sir2a-E1 (F) by using an anti-FLAG antibody showed localization of dCas9GCN5 and dCas9Sir2a in the nucleus, which was visualized by DAPI staining. (Scale bars, 2 μm.)

PfGCN5 Activates Expression of rh4 by dCas9-Mediated Histone Hyperacetylation.

First, we tested whether a functional HAT fused to dCas9 could be targeted to a gene of interest, and therefore activate it in P. falciparum. We used an sgRNA targeting the recombinant dCas9GCN5 to the TSS of an rh4 gene (22) (Fig. 2A), which controls the sialic acid-independent invasion pathway of the parasite into human erythrocytes. Rh4 is silent in the P. falciparum strain Dd2, which lacks the ability to invade neuraminidase-treated erythrocytes (23). Plasmids pL6-RH4sgRNA1 and pUF1-dCas9GCN5 were cotransfected into Dd2 under drug selection of WR99210 and Blasticidin S. We next examined whether dCas9GCN5 was specifically targeted to rh4 by RH4sgRNA1. A chromatin immunoprecipitation assay (ChIP) using an anti-FLAG antibody showed a high-level and specific occupancy of dCas9GCN5 at the TSS of rh4 in the transfected parasite clone Dd2-GCN5-R1 (Fig. 2B and SI Appendix, Fig. S1). Consistently, compared with the wild-type Dd2, hyperacetylation of residues lysine 9 and lysine 14 of histone H3 (H3K9/14) along rh4 in Dd2-GCN5-R1 was also confirmed by ChIP-qPCR (Fig. 2C). More important, we demonstrated that rh4 had at least a 113-fold higher level of transcription in Dd2-GCN5-R1 than in Dd2 by RT-qPCR (Fig. 2D). As a control, transcription of another gene ama1 that expresses along with rh4 at the same life stage was measured in parallel. RT-qPCR analysis showed no difference in ama1 expression between the wild-type and the transfected Dd2 lines (Fig. 2D), indicating a specific correlation between rh4 activation and targeting of the dCas9GCN5/RH4sgRNA1 system.

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CRISPR/dCas9GCN5-mediated activation of the P. falciparum rh4 gene. (A) Schematic diagram of the rh4 gene activated by dCas9GCN5 in Dd2. The position of the RH4 sgRNA1 is shown as a black rectangle. The TSS site determined by 5′-RACE (22) is shown as a red flag. P1-12, the 12 primer sets for ChIP-qPCR (see also SI Appendix, Table S1). (B and C) ChIP-qPCR analyses of schizont populations of cloned parasites derived from the Dd2-GCN5-R1 and Dd2-VPR-R1 by using an anti-FLAG antibody (B) and an anti-acetyl-histone H3 antibody (C) respectively. (D) RT-qPCR analysis of the rh4 transcript in Dd2-GCN5-R1 and Dd2-VPR-R1 at 44 h after invasion. A schizont-specific ama1 gene is used as the control. Expression levels of rh4 and ama1 were normalized to expression of a housekeeping gene, seryl-tRNA synthetase (PF3D7_0717700), which is an enzyme involved in the amino acylation of protein translation. (E) Cell invasion assays of various parasite clones. The invasion rates of different parasites into the neuraminidase-treated erythrocytes were normalized to that of the same one into untreated erythrocytes set as 100%. The eukaryotic activation domain VP64-P65-Rta (VPR) is chosen to replace the PfGCN5 domain in the dCas9VPR protein to evaluate the CRISPR/dCas9 system in gene activation. The wild-type Dd2 (Dd2 WT) is used as a negative control in all experiments. Experiments were biologically repeated three times. Error bars represent SEM. P values were obtained using unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

Epigenetic Activation of rh4 Increases a Sialic Acid-Dependent Invasion Pathway in Dd2.

To determine whether epigenetic up-regulation of rh4 at the transcriptional level works in coordination with the biological function of the PfRH4 protein, a parasite invasion assay was carried out by using neuraminidase-treated human erythrocytes. Our result showed that Dd2-GCN5-R1 was highly invasive to neuraminidase-treated erythrocytes, to which the wild-type Dd2 was restricted as expected (Fig. 2E). However, RH4sgRNA2 activation of rh4 at position +767 bp away from the TSS of rh4 and resulting phenotypic changes were not as robust as RH4sgRNA1 (SI Appendix, Fig. S2). The results indicated that gene overexpression could be significantly induced by an sgRNA-recruiting dCas9GCN5 located in the promoter region of the target.

To further corroborate the efficacy of dCas9GCN5 in gene activation, Dd2 was also transfected with pL6-RH4sgRNA1 and pUF1-dCas9VPR carrying another version of dCas9 fused to three eukaryotic activation domains, VP64-P65-Rta (VPR), in a tandem array (13), instead of PfGCN5. The resulting dCas9VPR protein, similar to dCas9GCN5, was still enriched at the TSS of rh4 directed by RH4sgRNA1, resulting in an increase in histone acetylation and expression of rh4, as well as parasite invasion rate (Fig. 2 B–E). Although VPR is functional in the protozoan parasite for gene activation, we found that dCas9GCN5 was much stronger than dCas9VPR in activating P. falciparum genes and the resulting changes of invasion rates (Fig. 2 D and E). Collectively, our data demonstrate implementation of the recombinant dCas9GCN5/gene-specific sgRNA system in efficient activation of rh4 in P. falciparum.

Gene Knockdown of eba-175 by dCas9Sir2a-Mediated Histone Hypoacetylation Alters Invasion Pathway of the Resulting Parasite to Erythrocytes.

Next, we determined whether an HDAC PfSir2a tagged to dCas9 could be used to repress gene expression in P. falciparum. We selected eba-175 as the target for transcriptional knockdown because it is among the most highly expressed erythrocyte invasion-related genes in the P. falciparum strain 3D7. EBA-175 binds erythrocyte receptor glycophorin A to mediate parasite invasion (24). Disruption of eba-175 results in parasites being unable to invade chymotrypsin-treated erythrocytes (25). The TSS of eba-175 was first identified by 5′-rapid amplification of cDNA ends (RACE) analysis. Based on the result, an sgRNA was designed in the plasmid pL6-EBA175sgRNA1 to direct dCas9Sir2a to the TSS region of eba-175 (Fig. 3A). We first examined the binding of dCas9Sir2a at the TSS of eba-175 by ChIP analysis. As expected, dCas9Sir2a was successfully targeted to the DNA sequence containing the TSS of eba-175 directed by EBA175sgRNA1 (Fig. 3B and SI Appendix, Fig. S3). Hypoacetylation of histone H3 was also observed at the same locus of eba-175 (Fig. 3C), consistent with the significant enrichment of dCas9Sir2a (Fig. 3B). Concomitantly, expression of eba-175 was considerably reduced in the transfected parasite clone 3D7-Sir2a-E1. As expected, there were no significant changes in eba-175 transcription between the negative control 3D7-dCas9-GFP and the wild-type 3D7 (Fig. 3D), indicating a functional role of dCas9Sir2a in transcriptional knockdown in P. falciparum. In addition, expression of ama1 was not affected in transfected parasites (Fig. 3D). Functionally, repression of eba-175 restrained 3D7-Sir2a-E1 from invading chymotrypsin-treated erythrocytes, whereas 3D7-dCas9-GFP and wild-type 3D7 were invasive to both chymotrypsin-treated and untreated erythrocytes (Fig. 3E). Taken together, our data show that the dCas9Sir2a/sgRNA system could be applied for efficient transcriptional knockdown without DNA editing in the genome.

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CRISPR/dCas9Sir2a-mediated transcriptional repression of the P. falciparum eba-175 gene. (A) Schematic diagram of the eba-175 gene repressed by dCas9Sir2a in 3D7. The position of the EBA175 sgRNA1 is shown as a black rectangle. The TSS site determined by 5′-RACE is shown as a red flag. P1-7, the seven primer sets for ChIP-qPCR (see also SI Appendix, Table S1). (B and C) ChIP-qPCR analyses of schizont populations of cloned parasites derived from the 3D7-Sir2a-E1 and the wild-type 3D7 (3D7 WT), as a control, by using an anti-FLAG antibody (B) and an anti-acetyl-Histone H3 antibody (C), respectively. (D) RT-qPCR analysis of the transcripts of eba-175 and ama1 in 3D7-Sir2a-E1, 3D7-dCas9-GFP, and the wild-type 3D7 at 44 h after invasion. Expression levels of eba-175 and ama1 were normalized to expression of a housekeeping gene, seryl-tRNA synthetase (PF3D7_0717700). (E) Invasion efficiency of 3D7-Sir2a-E1, 3D7-dCas9-GFP, and the wild-type 3D7 with the different erythrocytes. The invasion rates of various parasite clones into chymotrypsin-treated erythrocytes were normalized to that of the same one into untreated erythrocytes set as 100%. Experiments were biologically repeated three times. Error bars represent SEM. P values were obtained using unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

In a parallel experiment, another sgRNA (EBA175sgRNA2) matching a dCas9-binding site 1,234 bp upstream the TSS of eba-175 was tested in 3D7 (SI Appendix, Fig. S4A). Although enrichment of dCas9Sir2a and histone acetylation were observed at the target region (SI Appendix, Fig. S4 B and C), it could not repress eba-175 expression as efficiently as that targeting the TSS region (SI Appendix, Fig. S4 D and E). This indicates that the dCas9Sir2a system could significantly repress expression of eba-175 by designing an sgRNA to the promoter region of the target. Considering the efficacy of different tested sgRNAs, designing the guided site closer to the TSS of targets would allow more effective regulation by the CRISPR/dCas9 system.

Epigenetic Gene Editing by CRISPR/dCas9 Comprises Few Off-Target Effects.

To evaluate possible off-target effects, transcriptomes of Dd2-GCN5-R1 and 3D7-Sir2a-E1 were measured by RNA-seq analysis (SI Appendix, Fig. S5) and subsequently authenticated by RT-qPCR. As expected, both RNA-seq and RT-qPCR analyses demonstrated rh4 activation and eba-175 repression in the resulting parasites. It is worthy to note that, similar to rh4, a pseudo gene pebl was also activated in all three transgenic lines Dd2-GCN5-R1, Dd2-GCN5-R2, and Dd2-VPR-R1 (SI Appendix, Fig. S6). It has been reported that pebl shares a bidirectional promoter with rh4 (22), suggesting that dCas9-mediated epigenetic editing would have an effect on the untargeted genes that share the same bidirectional promoter with the target. In contrast, this indicates that the epigenetic editing systems could also be applied to regulating noncoding RNAs, as pebl is a pseudo gene. Taken together, our data show that the CRISPR/dCas9-mediated epigenetic gene editing systems are specific to the target by sgRNA.

Repression of an Essential Gene PfSET1 by dCas9Sir2a Delays Growth of Mature Trophozoites.

To further evaluate whether the CRISPR/dCas9 system could be applied to manipulate essential genes in P. falciparum, we constructed a transfected parasite line 3D7-Sir2a-G1, in which a histone methyltransferase gene PfSET1 was targeted by the dCas9Sir2a system. PfSET1 is essential to the growth of the P. falciparum asexual stage, and its function remains largely unknown (11). As expected, the expression of PfSET1 was significantly repressed at schizont stages of 3D7-Sir2a-G1 (Fig. 4A) compared with that in 3D7-dCas9-GFP and wild-type 3D7. RNA-seq analysis indicated that a total of 322 parasite genes were transcriptionally down-regulated because of repression of PfSET1 (Dataset S1). We found that 50% of these gene orthologs were essential, and an additional 19% of gene orthologs were associated with reduced growth of the asexual blood stages in Plasmodium berghei (26) (Fig. 4B). Further bioinformatic analysis showed that 72% of the essential genes were highly expressed in mature trophozoites and early schizonts (Fig. 4C). More important, transcriptional repression of PfSET1 coincides with a delay in parasite growth starting at the trophozoite stage, as evaluated by flow cytometry (Fig. 4D) and by microscopy (SI Appendix, Fig. S7). In contrast, this type of growth delay was not observed in the negative control 3D7-dCas9-GFP and the wild-type 3D7, indicating that transcriptional repression of PfSET1 specifically led to the phenotypic changes in parasite growth. This indicates that this CRISPR/dCas9Sir2a system could be applied to repression of essential genes.

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Repression of an essential P. falciparum gene PfSET1 by CRISPR/dCas9Sir2a. (A) RT-qPCR analysis of the transcription of PfSET1 in the resulting parasite 3D7-Sir2a-G1, 3D7-dCas9-GFP, and the wild-type 3D7 at 44 h after invasion. Transcriptional level of PfSET1 was normalized to that of a housekeeping gene, seryl-tRNA synthetase (PF3D7_0717700). Experiments were biologically repeated three times. Error bars represent SEM. P values were obtained using unpaired two-tailed Student’s t test. **P < 0.01. (B) Frequency distribution of down-regulated genes (Log2FC < −1 & P value < 0.05) resulted by repression of PfSET1 based on published phenotypes in P. berghei (26). Genes associated with dispensable, slow-growth, and essential phenotypes are indicated by the light blue, yellow, and red boxes, respectively. There are 68 down-regulated genes in total with essential phenotypes. (C) The transcription pattern of the slow-growth, dispensable and essential genes. Heat map representation of the relative transcription activity of the 42 dispensable, 25 slow-growth, and 68 essential genes at eight points during the asexual erythrocytic growth, according to the public database (GEO accession number: GSE23787). Among them, 72% of the essential genes that are highly expressed in the trophozoite and schizont stages are boxed by red dotted lines. Expression level of each gene was normalized by z-score from −2 (lowest, blue) to +2 (highest, yellow). (D) Flow cytometry analysis of parasite growth progression in 3D7-Sir2a-G1, 3D7-dCas9-GFP, and the wild-type 3D7 (3D7 WT) at five consecutive points from a single time course experiment. n, number of gated infected red blood cells, the x axis indicates the SYBR green intensity.

Genome-Wide Scanning of Potential dCas9 Sites Indicates Enough Targets of Histone Acetylation for Editing the P. falciparum Genome.

The CRISPR/Cas9 system requires the NGG or NGA PAM sequences at the target site. Bioinformatic analysis identified 261,196 NGG and 727,817 NGA PAMs located from −1,500 bp to +500 bp relative to the start codon (the potential promoter region) of each P. falciparum gene, presenting more than 173-fold coverage of the entire 5,712 parasite genes (SI Appendix, Fig. S8). PfSir2a not only contributes to histone deacetylation-related repression of either protein-coding genes (27) or noncoding genes (28) in P. falciparum, but its HDAC activity also has been demonstrated in other eukaryotic cells (29). This suggests a possible use of dCas9Sir2a for epigenetic repression in other eukaryotes. Meanwhile, PfGCN5 as a well-characterized HAT is broadly associated with acetylation of histone H3K9 and H3K14 in P. falciparum genes (30). Given that transcription activity of P. falciparum genes are associated with dynamic alterations of histone H3K9 acetylation from −1,500 bp to +500 bp relative to the start codon of each gene in the entire asexual blood stages (SI Appendix, Fig. S9) (31), our bioinformatic analyses suggest that virtually all parasite genes could be targeted by the CRISPR/dCas9GCN5 or dCas9Sir2a systems.

It is worth noting that dCas9GCN5 could be targeted to either nonprotein- or protein-coding genes, including those of large sizes, even though this system might not be able to further activate the genes with high basal-level expression as a result of existing high enrichment of histone acetylation at the TSS of these genes (13). The effect of dCas9GCN5 on pebl suggests epigenetic regulation of dCas9Sir2a on noncoding genes. Interestingly, with low levels of expression or possible cleavage of the dCas9 proteins in some cases (Fig. 1 C and D), these epigenetic editing systems could still achieve substantial gene regulation via well-designed sgRNAs that recruit the dCas9 to the promoter regions of the target genes. To improve the design of the sgRNA, it is also worth performing ChIP-seq analysis to measure the dynamics and fine distribution of histone acetylation along the target genes regulated by the epigenetic editing systems. Another CRISPR system based on an endonuclease Cpf1 has been used to achieve multiplex gene editing guided by multiple sgRNAs transcribed by a single promotor in mammalian cells (32). The usefulness of the CRISPR/Cpf1 system for epigenetic editing in P. falciparum, especially for simultaneously targeting multigene families, remains to be evaluated. Altogether, the two complementary epigenome editing systems developed in this study greatly expand our current toolkits for functional genetic and epigenetic studies in P. falciparum. In addition, they could be further customized, which is a major advantage for genome-wide screening of P. falciparum in pathogenesis of malaria.

Materials and Methods

Plasmids Construction.

The related plasmids were modified based on the plasmids pUF1-Cas9 and pL7-egfp (5). The yfcu negative selection marker in pL7-egfp was removed with the NotI/AflII restriction enzyme digestion to generate the pL6 plasmid for transcription of sgRNA. The dhodh gene in pUF1-Cas9 was first replaced at the restriction sites EcoRI/AflII by the bsd resistance gene cassette, using bsd F/bsd R primers to construct pUF1-BSD-Cas9. Subsequently, two point mutations, D10A and H840A, were introduced into the wild-type Cas9 gene to create the nuclease-null variants dCas9 using two primer sets, D10A F/D10A R and H840A F/H840A R, respectively. The activation (PfGCN5: PF3D7_0823300) and repression (PfSir2a: PF3D7_1328800) domains were amplified from the P. falciparum 3D7 genome, using the primer sets GCN5 F/GCN5 R and Sir2a F/Sir2a R, respectively, and subcloned into the pUF1-BSD-dCas9 vector at the SmaI/SalI sites by using an In-Fusion kit according to the product manual (Vazyme), to generate the pUF1-dCas9GCN5 and pUF1-dCas9Sir2a, respectively. The eukaryotic activation domains VPR (VP64, p65, and RTA) (13) were synthesized and subcloned by using the same strategy to generate pUF1-dCas9VPR. The control plasmid pUF1-dCas9GFP was also constructed in the same manner by fusing a GFP coding sequence to the C-terminal of dCas9. All sgRNA sequences for targeting rh4, eba-175, and Pfset1 and a control sgRNA targeting the telomere region were listed in the SI Appendix, Table S1.

Parasite Culture and Transfection.

P. falciparum parasites were propagated under the standard conditions (11). Schizont parasites were isolated by the Percoll/Sorbitol (GE Healthcare) enrichment. Plasmids were introduced into parasites via electroporation, as described previously (33). Drug selections of WR99210 (Jacobus Pharmaceuticals) at 5 nM and Blasticidin S (Life Technologies) at 2.5 µg/mL for 3D7 and 5 µg/mL for Dd2 were applied 24 h posttransfection. Detailed information describing protocols for parasite sample treatment and experiment of 5′RACE, RT-qPCR, Western blot, parasite invasion assay, RNA-seq analysis, flow cytometry, and statistical analyses is provided in SI Appendix, Supplementary Methods.

Chromatin Immunoprecipitation.

The ChIP assay was performed as described previously (11). Briefly, 1 × 108 schizont parasites were fixed by 1% paraformaldehyde for 10 min at room temperature, followed by 0.125 M glycine for stopping the fixation. Chromatin was sheared by sonication in a Bioruptor UCD-200 (Diagenode) for 8 min with 30-s interval/min. The sheared chromatin samples (mainly 500 bp in size) were incubated at 4 °C overnight with 5 μg monoclonal anti-FLAG M2 antibody (Sigma) for immunoprecipitation of the dCas9 recombinant proteins or with 5 μg of an anti-acetyl-Histone H3 antibody (Millipore) for enriching acetylated histone H3 proteins. DNA fragments purified from the ChIP-ed samples were further processed to qPCR analyses for detecting specific distribution of the target proteins on various genetic loci by using different primer sets (SI Appendix, Table S1).

Supplementary Material

Supplementary File

Supplementary File

Acknowledgments

We thank J. J. Lopez-Rubio in University Montpellier 1&2 for providing the pUF1-Cas9 and pL7-egfp plasmids. We also thank Carolina Barillas-Mury at the National Institutes of Health for critical suggestions on the manuscript, and Jiping Han for providing the technical support of in vitro parasite culture. This research was supported by the National Key R&D Program of China (2018YFA0507300), National Science and Technology Major Project (2018ZX10101004003001), National Natural Science Foundation of China (31771455, 81271863, 81361120405), and National Institute of Allergy and Infectious Diseases Grants R01AI116466 and U19AI089672. All authors have reviewed and agreed on the content of the manuscript.

Footnotes

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Data deposition: The newly generated RNA-seq data reported in this paper have been deposited in the Sequence Read Archive, https://www.ncbi.nlm.nih.gov/sra (accession no. PRJNA493217, deposited September 26, 2018).

This article contains supporting information online at www.pnas.org/lookup/suppl/10.1073/pnas.1813542116/-/DCSupplemental.

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