Epigenetic editing by CRISPR/dCas9 inPlasmodium falciparumBo Xiaoa, Shigang Yina, Yang Hua, Maoxin Suna,b, Jieqiong Weia, Zhenghui Huanga, Yuhao Wena, Xueyu Daia,Huiling Chena, Jianbing Muc, Liwang Cuid, and Lubin Jianga,b,1

aUnit of Human Parasite Molecular and Cell Biology, Key Laboratory of Molecular Virology and Immunology, Institut Pasteur of Shanghai, University ofChinese Academy of Sciences, Chinese Academy of Sciences, Shanghai 200031, P.R. China; bSchool of Life Science and Technology, ShanghaiTech University,Shanghai 201210, P.R. China; cLaboratory of Malaria and Vector Research, National Institute of Allergy and Infectious Diseases, National Institutes of Health,Bethesda, MD 20892-8132; and dDepartment of Internal Medicine, Morsani College of Medicine, University of South Florida, Tampa, FL 33612

Edited by L. David Sibley, Washington University School of Medicine, St. Louis, MO, and approved November 26, 2018 (received for review August 9, 2018)

Genetic manipulation remains a major obstacle for understandingthe functional genomics of the deadliest malaria parasite Plasmo-dium falciparum. Although the CRISPR/Cas9 (clustered regularlyinterspaced short palindromic repeat/CRISPR-associated protein9) system has been successfully applied to introduce permanentchanges in the parasite genome, its use is still limited. Here weshow that fusing different epigenetic effector domains to a Cas9null mutant efficiently and specifically reprograms the expressionof target genes in P. falciparum. By precisely writing and erasinghistone acetylation at the transcription start site regions of theinvasion-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 intohuman erythrocytes. By using the epigenetic knockdown sys-tem, we have also characterized the effects of PfSET1, previ-ously identified as an essential gene, on expression of mainlytrophozoite- and schizont-specific genes, and therefore regula-tion of the growth of the mature forms of P. falciparum. Thisepigenetic CRISPR/dCas9 system provides a powerful approachfor regulating gene expression at the transcriptional level inP. falciparum.

malaria parasite | CRISPR/dCas9 | epigenetic editing | invasion change

The most dangerous form of malaria, Plasmodium falciparum,causes almost half a million deaths annually. Efficient ap-

proaches to genome editing in the malaria parasites are urgentlyneeded to better understand fundamental parasite biology, suchas invasion of host cells and pathogenesis. As an excellent com-plement to conventional methods of genetic manipulation inP. falciparum (1–4), CRISPR/Cas9 (clustered regularly inter-spaced short palindromic repeat/CRISPR-associated protein 9)has shown great advantages in genome engineering such as genedisruption, tagging, and allelic replacement in Plasmodium spp(5–7). However, most genetic manipulation systems have majorlimitations in the disruption of essential genes or long noncodingRNAs located within exons or introns in the haploid genome ofthe parasite because of the lack of RNAi in P. falciparum (8).Here we provided a genomic disruption-free system for epige-netically regulating transcription of parasite genes by episomalexpression of dCas9-fusion protein and sgRNA.Dynamic histone modifications within the transcription start

site (TSS) regions of eukaryotic genes are important means oftranscriptional regulation. In P. falciparum, epigenetic enzymesmodifying histone acetylation or methylation play critical roles incontrolling gene expression (9–12). The dCas9 protein fused withthe epigenetic modifier can be efficiently applied to transcrip-tional reprograming of the targeting gene in eukaryotes (13–16).However, the successful application with dCas9 in Plasmodiumparasites has not been reported yet. Here we applied a modi-fied CRISPR/Cas9 system to epigenetically regulate transcrip-tion of parasite invasion-related genes and demonstrated that a

null Cas9 could precisely guide the fused epigenetic regula-tors such as histone acetyltransferase (HAT) or deacetylase(HDAC) domains to the TSS regions of the target genes toup-regulate or down-regulate histone acetylation, leading toefficient gene overexpression or transcriptional knockdownin P. falciparum.

Results and DiscussionRecombinant dCas9 Regulators Localize in Parasite Nuclei. To avoidany DNA cleavages generated by the DNA endonuclease activityof Cas9, a nuclease-deficient Streptococcus pyogenes Cas9 (deadCas9:dCas9) harboring two mutations (D10A and H840A) in thecatalytic domains while preserving the RNA-guided DNA bind-ing ability (17, 18), was selected to recruit HAT or HDAC do-mains to the TSS regions of target genes (Fig. 1 A and B). A3×FLAG epitope tag at the N terminus was used for detection ofthe recombinant dCas9. We first fused the core enzymatic regionof PfGCN5 HAT (PF3D7_0823300) (19) to the C terminus ofthe dCas9 (dCas9GCN5) in the plasmid pUF1-Cas9 to producepUF1-dCas9GCN5. Successful expression of dCas9GCN5 wasdetermined by Western blot (Fig. 1C). In addition, dCas9GCN5was highly enriched in parasite nuclei guided by the two nuclearlocalization sequences (NLS; Fig. 1E). On the contrary, we clonedthe coding region of PfSir2a (PF3D7_1328800), a P. falciparum

Significance

As a result of low homologous recombination efficiency andthe lack of RNAi in the Plasmodium falciparum genome, feasi-ble gene-editing toolkits are urgently needed. Although vari-ous gene editing strategies have been successfully applied to P.falciparum, there are still serious limitations. Here we showthat by fusing histone acetyltransferase or deacetylase to adCas9 mutant in the CRISPR/dCas9 (clustered regularly inter-spaced short palindromic repeat/CRISPR-associated protein 9)system, the expression of target genes could be efficiently andspecifically regulated in P. falciparum. The two epigenetictoolkits will expand current applications for functional geneticsand epigenetics studies in P. falciparum, and therefore accel-erate the understanding of malaria pathogenesis.

Author contributions: B.X. and L.J. designed research; B.X., Y.H., M.S., J.W., Z.H., X.D., andH.C. performed research; B.X., S.Y., Y.W., J.M., L.C., and L.J. analyzed data; and B.X., S.Y.,J.M., L.C., and L.J. wrote the paper.

The authors declare no conflict of interest.

This article is a PNAS Direct Submission.

Published under the PNAS license.

Data deposition: The newly generated RNA-seq data reported in this paper have beendeposited in the Sequence Read Archive, https://www.ncbi.nlm.nih.gov/sra (accession no.PRJNA493217, deposited September 26, 2018).1To whom correspondence should be addressed. Email: lbjiang@ips.ac.cn.

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

Published online December 24, 2018.

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histone deacetylase that can broadly remove acetylation at mul-tiple lysine residues at the N-terminal tail of histone H3 and H4proteins in a NAD+-dependent manner (20, 21), to the C terminusof dCas9 for generating a recombinant dCas9Sir2a, as describedearlier. Our results confirmed the expression of dCas9Sir2a (Fig.1D) and its nuclear localization (Fig. 1F). As a negative control, adCas9 fused to GFP was also expressed in the resulting parasite3D7-dCas9-GFP (Fig. 1D).

PfGCN5 Activates Expression of rh4 by dCas9-Mediated HistoneHyperacetylation. First, we tested whether a functional HATfused to dCas9 could be targeted to a gene of interest, andtherefore activate it in P. falciparum. We used an sgRNA tar-geting the recombinant dCas9GCN5 to the TSS of an rh4 gene(22) (Fig. 2A), which controls the sialic acid-independent in-vasion pathway of the parasite into human erythrocytes. Rh4 issilent in the P. falciparum strain Dd2, which lacks the ability toinvade neuraminidase-treated erythrocytes (23). Plasmids pL6-RH4sgRNA1 and pUF1-dCas9GCN5 were cotransfected intoDd2 under drug selection of WR99210 and Blasticidin S. Wenext examined whether dCas9GCN5 was specifically targeted torh4 by RH4sgRNA1. A chromatin immunoprecipitation assay(ChIP) using an anti-FLAG antibody showed a high-level andspecific occupancy of dCas9GCN5 at the TSS of rh4 in thetransfected parasite clone Dd2-GCN5-R1 (Fig. 2B and SI Ap-pendix, 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 byChIP-qPCR (Fig. 2C). More important, we demonstrated thatrh4 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 withrh4 at the same life stage was measured in parallel. RT-qPCRanalysis showed no difference in ama1 expression between thewild-type and the transfected Dd2 lines (Fig. 2D), indicating a

specific correlation between rh4 activation and targeting of thedCas9GCN5/RH4sgRNA1 system.

Epigenetic Activation of rh4 Increases a Sialic Acid-DependentInvasion Pathway in Dd2. To determine whether epigenetic up-regulation of rh4 at the transcriptional level works in coordinationwith the biological function of the PfRH4 protein, a parasite in-vasion assay was carried out by using neuraminidase-treated humanerythrocytes. Our result showed that Dd2-GCN5-R1 was highlyinvasive to neuraminidase-treated erythrocytes, to which the wild-typeDd2 was restricted as expected (Fig. 2E). However, RH4sgRNA2activation of rh4 at position +767 bp away from the TSS of rh4 andresulting phenotypic changes were not as robust as RH4sgRNA1(SI Appendix, Fig. S2). The results indicated that gene over-expression could be significantly induced by an sgRNA-recruitingdCas9GCN5 located in the promoter region of the target.To further corroborate the efficacy of dCas9GCN5 in gene acti-

vation, Dd2 was also transfected with pL6-RH4sgRNA1 and pUF1-dCas9VPR carrying another version of dCas9 fused to threeeukaryotic activation domains, VP64-P65-Rta (VPR), in a tandemarray (13), instead of PfGCN5. The resulting dCas9VPR protein,similar to dCas9GCN5, was still enriched at the TSS of rh4 directedby RH4sgRNA1, resulting in an increase in histone acetylation andexpression of rh4, as well as parasite invasion rate (Fig. 2 B–E). Al-though VPR is functional in the protozoan parasite for gene activa-tion, we found that dCas9GCN5 was much stronger than dCas9VPRin activating P. falciparum genes and the resulting changes of in-vasion rates (Fig. 2 D and E). Collectively, our data demonstrateimplementation of the recombinant dCas9GCN5/gene-specificsgRNA system in efficient activation of rh4 in P. falciparum.

Gene Knockdown of eba-175 by dCas9Sir2a-Mediated HistoneHypoacetylation Alters Invasion Pathway of the Resulting Parasite toErythrocytes. Next, we determined whether an HDAC PfSir2a taggedto dCas9 could be used to repress gene expression in P. falciparum.

Fig. 1. Epigenetic editing of P. falciparum genes byusing the CRISPR/dCas9 system. (A and B) Schematicdiagram illustrating the strategy of epigenetic geneediting via the CRISPR/dCas9 system, in which an en-zymatic 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 dCas9protein, a nuclease-deficient S. pyogenes Cas9 harbor-ing two mutations (D10A and H840A), as shown in thecatalytic domains. A 3×FLAG peptide is placed at theN terminus of the recombinant protein, and a nu-clear localization signal (NLS) is inserted downstream ofthe 3×FLAG and dCas9, respectively, to import therecombinant dCas9 into the parasite nucleus. A tripleglycine-serine protein domain linker (GS3) is inserteddownstream of NLS to allow for an extended confor-mation and maximal flexibility of the epigenetic en-zyme domain. The TSS of the target gene is indicatedas a red flag. Ac, histone acetylation. (C and D) Westernblot analyses of the recombinant dCas9 proteins inparasite lines Dd2-GCN5-R1 and Dd2-GCN5-R2 (C) and3D7-Sir2a-E1, 3D7-Sir2a-E2, and 3D7-dCas9-GFP (D), byusing an anti-Cas9 antibody. The wild-type Dd2 and3D7 were tested as negative controls, respectively. Thewild-type Cas9 in 3D7-SETvs-KO, in which Cas9 wasgenerated to mediate the PfSETvs gene knockout, wasdetected to indicate the size of the recombinant dCas9.Actin, as loading control, was also assayed in the samesamples by using an anti-actin antibody. (E and F) IFAanalyses of the recombinant dCas9 proteins in parasitelines Dd2-GCN5-R1 (E) and 3D7-Sir2a-E1 (F) by using ananti-FLAG antibody showed localization of dCas9GCN5and dCas9Sir2a in the nucleus, which was visualized byDAPI staining. (Scale bars, 2 μm.)

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We selected eba-175 as the target for transcriptional knockdownbecause it is among the most highly expressed erythrocyteinvasion-related genes in the P. falciparum strain 3D7. EBA-175binds erythrocyte receptor glycophorin A to mediate parasiteinvasion (24). Disruption of eba-175 results in parasites beingunable to invade chymotrypsin-treated erythrocytes (25). TheTSS of eba-175 was first identified by 5′-rapid amplification ofcDNA ends (RACE) analysis. Based on the result, an sgRNAwas designed in the plasmid pL6-EBA175sgRNA1 to directdCas9Sir2a to the TSS region of eba-175 (Fig. 3A). We firstexamined the binding of dCas9Sir2a at the TSS of eba-175 byChIP analysis. As expected, dCas9Sir2a was successfully targetedto the DNA sequence containing the TSS of eba-175 directedby EBA175sgRNA1 (Fig. 3B and SI Appendix, Fig. S3). Hypo-acetylation of histone H3 was also observed at the same locus ofeba-175 (Fig. 3C), consistent with the significant enrichment ofdCas9Sir2a (Fig. 3B). Concomitantly, expression of eba-175 wasconsiderably reduced in the transfected parasite clone 3D7-Sir2a-E1. As expected, there were no significant changes ineba-175 transcription between the negative control 3D7-dCas9-GFP and the wild-type 3D7 (Fig. 3D), indicating a functionalrole 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 restrained3D7-Sir2a-E1 from invading chymotrypsin-treated erythrocytes,whereas 3D7-dCas9-GFP and wild-type 3D7 were invasive to bothchymotrypsin-treated and untreated erythrocytes (Fig. 3E). Takentogether, our data show that the dCas9Sir2a/sgRNA system couldbe applied for efficient transcriptional knockdown without DNAediting in the genome.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 en-richment of dCas9Sir2a and histone acetylation were observed atthe target region (SI Appendix, Fig. S4 B and C), it could notrepress eba-175 expression as efficiently as that targeting the TSSregion (SI Appendix, Fig. S4 D and E). This indicates that thedCas9Sir2a 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, designingthe guided site closer to the TSS of targets would allow moreeffective regulation by the CRISPR/dCas9 system.

Epigenetic Gene Editing by CRISPR/dCas9 Comprises Few Off-TargetEffects. To evaluate possible off-target effects, transcriptomes ofDd2-GCN5-R1 and 3D7-Sir2a-E1 were measured by RNA-seq

Fig. 2. CRISPR/dCas9GCN5-mediated activation of the P. falciparum rh4 gene. (A) Schematic diagram of the rh4 gene activated by dCas9GCN5 in Dd2. Theposition 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 andDd2-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 ahousekeeping gene, seryl-tRNA synthetase (PF3D7_0717700), which is an enzyme involved in the amino acylation of protein translation. (E) Cell invasionassays of various parasite clones. The invasion rates of different parasites into the neuraminidase-treated erythrocytes were normalized to that of the sameone into untreated erythrocytes set as 100%. The eukaryotic activation domain VP64-P65-Rta (VPR) is chosen to replace the PfGCN5 domain in the dCas9VPRprotein to evaluate the CRISPR/dCas9 system in gene activation. The wild-type Dd2 (Dd2 WT) is used as a negative control in all experiments. Experimentswere 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.

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analysis (SI Appendix, Fig. S5) and subsequently authenticated byRT-qPCR. As expected, both RNA-seq and RT-qPCR analysesdemonstrated rh4 activation and eba-175 repression in theresulting parasites. It is worthy to note that, similar to rh4, apseudo gene pebl was also activated in all three transgenic linesDd2-GCN5-R1, Dd2-GCN5-R2, and Dd2-VPR-R1 (SI Appen-dix, Fig. S6). It has been reported that pebl shares a bidirectionalpromoter with rh4 (22), suggesting that dCas9-mediated epige-netic editing would have an effect on the untargeted genes thatshare the same bidirectional promoter with the target. In con-trast, this indicates that the epigenetic editing systems could alsobe applied to regulating noncoding RNAs, as pebl is a pseudogene. Taken together, our data show that the CRISPR/dCas9-mediated epigenetic gene editing systems are specific to thetarget by sgRNA.

Repression of an Essential Gene PfSET1 by dCas9Sir2a Delays Growthof Mature Trophozoites. To further evaluate whether the CRISPR/dCas9 system could be applied to manipulate essential genes inP. falciparum, we constructed a transfected parasite line 3D7-Sir2a-G1, in which a histone methyltransferase gene PfSET1was targeted by the dCas9Sir2a system. PfSET1 is essential to thegrowth of the P. falciparum asexual stage, and its function re-mains largely unknown (11). As expected, the expression ofPfSET1 was significantly repressed at schizont stages of 3D7-Sir2a-G1 (Fig. 4A) compared with that in 3D7-dCas9-GFP andwild-type 3D7. RNA-seq analysis indicated that a total of 322parasite genes were transcriptionally down-regulated because ofrepression of PfSET1 (Dataset S1). We found that 50% of thesegene orthologs were essential, and an additional 19% of gene

orthologs were associated with reduced growth of the asexualblood stages in Plasmodium berghei (26) (Fig. 4B). Further bio-informatic analysis showed that 72% of the essential genes werehighly expressed in mature trophozoites and early schizonts (Fig.4C). More important, transcriptional repression of PfSET1 co-incides with a delay in parasite growth starting at the trophozoitestage, as evaluated by flow cytometry (Fig. 4D) and by micros-copy (SI Appendix, Fig. S7). In contrast, this type of growth delaywas not observed in the negative control 3D7-dCas9-GFP andthe wild-type 3D7, indicating that transcriptional repression ofPfSET1 specifically led to the phenotypic changes in parasitegrowth. This indicates that this CRISPR/dCas9Sir2a systemcould be applied to repression of essential genes.

Genome-Wide Scanning of Potential dCas9 Sites Indicates EnoughTargets of Histone Acetylation for Editing the P. falciparum Genome.The CRISPR/Cas9 system requires the NGG or NGA PAMsequences at the target site. Bioinformatic analysis identified261,196 NGG and 727,817 NGA PAMs located from −1,500 bpto +500 bp relative to the start codon (the potential promoterregion) 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 non-coding genes (28) in P. falciparum, but its HDAC activity also hasbeen demonstrated in other eukaryotic cells (29). This suggests apossible use of dCas9Sir2a for epigenetic repression in othereukaryotes. Meanwhile, PfGCN5 as a well-characterized HAT isbroadly associated with acetylation of histone H3K9 and H3K14in P. falciparum genes (30). Given that transcription activity of

Fig. 3. CRISPR/dCas9Sir2a-mediated transcriptional repression of the P. falciparum eba-175 gene. (A) Schematic diagram of the eba-175 gene repressed bydCas9Sir2a 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 sevenprimer 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 werenormalized to that of the same one into untreated erythrocytes set as 100%. Experiments were biologically repeated three times. Error bars represent SEM. Pvalues were obtained using unpaired two-tailed Student’s t test. *P < 0.05; **P < 0.01; ***P < 0.001; NS, not significant.

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P. falciparum genes are associated with dynamic alterations ofhistone H3K9 acetylation from −1,500 bp to +500 bp relative tothe start codon of each gene in the entire asexual blood stages(SI Appendix, Fig. S9) (31), our bioinformatic analyses suggestthat 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 largesizes, even though this system might not be able to further ac-tivate the genes with high basal-level expression as a result ofexisting high enrichment of histone acetylation at the TSS of

these genes (13). The effect of dCas9GCN5 on pebl suggestsepigenetic regulation of dCas9Sir2a on noncoding genes. In-terestingly, with low levels of expression or possible cleavage ofthe dCas9 proteins in some cases (Fig. 1 C and D), these epi-genetic editing systems could still achieve substantial gene reg-ulation via well-designed sgRNAs that recruit the dCas9 to thepromoter regions of the target genes. To improve the design ofthe sgRNA, it is also worth performing ChIP-seq analysis tomeasure the dynamics and fine distribution of histone acetylationalong the target genes regulated by the epigenetic editing sys-tems. Another CRISPR system based on an endonuclease Cpf1

Fig. 4. Repression of an essential P. falciparum gene PfSET1 by CRISPR/dCas9Sir2a. (A) RT-qPCR analysis of the transcription of PfSET1 in the resulting parasite3D7-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 un-paired 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 ofPfSET1 based on published phenotypes in P. berghei (26). Genes associated with dispensable, slow-growth, and essential phenotypes are indicated by the lightblue, 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 es-sential 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 wasnormalized 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 axisindicates the SYBR green intensity.

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has been used to achieve multiplex gene editing guided bymultiple sgRNAs transcribed by a single promotor in mamma-lian cells (32). The usefulness of the CRISPR/Cpf1 system forepigenetic editing in P. falciparum, especially for simultaneouslytargeting multigene families, remains to be evaluated. Alto-gether, the two complementary epigenome editing systems de-veloped in this study greatly expand our current toolkits forfunctional genetic and epigenetic studies in P. falciparum. Inaddition, they could be further customized, which is a majoradvantage for genome-wide screening of P. falciparum in path-ogenesis of malaria.

Materials and MethodsPlasmids Construction. The related plasmids were modified based on theplasmids pUF1-Cas9 and pL7-egfp (5). The yfcu negative selection marker inpL7-egfp was removed with the NotI/AflII restriction enzyme digestion togenerate the pL6 plasmid for transcription of sgRNA. The dhodh gene inpUF1-Cas9 was first replaced at the restriction sites EcoRI/AflII by the bsdresistance gene cassette, using bsd F/bsd R primers to construct pUF1-BSD-Cas9. Subsequently, two point mutations, D10A and H840A, were in-troduced into the wild-type Cas9 gene to create the nuclease-null variantsdCas9 using two primer sets, D10A F/D10A R and H840A F/H840A R, re-spectively. The activation (PfGCN5: PF3D7_0823300) and repression (PfSir2a:PF3D7_1328800) domains were amplified from the P. falciparum 3D7 ge-nome, 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 usingan In-Fusion kit according to the product manual (Vazyme), to generate thepUF1-dCas9GCN5 and pUF1-dCas9Sir2a, respectively. The eukaryotic activa-tion domains VPR (VP64, p65, and RTA) (13) were synthesized and subclonedby using the same strategy to generate pUF1-dCas9VPR. The control plasmidpUF1-dCas9GFP was also constructed in the same manner by fusing a GFPcoding sequence to the C-terminal of dCas9. All sgRNA sequences for tar-geting rh4, eba-175, and Pfset1 and a control sgRNA targeting the telomereregion were listed in the SI Appendix, Table S1.

Parasite Culture and Transfection. P. falciparum parasites were propagatedunder the standard conditions (11). Schizont parasites were isolated by thePercoll/Sorbitol (GE Healthcare) enrichment. Plasmids were introduced intoparasites via electroporation, as described previously (33). Drug selections ofWR99210 (Jacobus Pharmaceuticals) at 5 nM and Blasticidin S (Life Tech-nologies) at 2.5 μg/mL for 3D7 and 5 μg/mL for Dd2 were applied 24 hposttransfection. Detailed information describing protocols for parasitesample treatment and experiment of 5′RACE, RT-qPCR, Western blot, para-site invasion assay, RNA-seq analysis, flow cytometry, and statistical analysesis provided in SI Appendix, Supplementary Methods.

Chromatin Immunoprecipitation. The ChIP assay was performed as describedpreviously (11). Briefly, 1 × 108 schizont parasites were fixed by 1% para-formaldehyde for 10 min at room temperature, followed by 0.125 M glycinefor stopping the fixation. Chromatin was sheared by sonication in a Bio-ruptor UCD-200 (Diagenode) for 8 min with 30-s interval/min. The shearedchromatin samples (mainly 500 bp in size) were incubated at 4 °C overnightwith 5 μg monoclonal anti-FLAG M2 antibody (Sigma) for immunoprecipita-tion of the dCas9 recombinant proteins or with 5 μg of an anti-acetyl-HistoneH3 antibody (Millipore) for enriching acetylated histone H3 proteins. DNAfragments purified from the ChIP-ed samples were further processed toqPCR analyses for detecting specific distribution of the target proteins onvarious genetic loci by using different primer sets (SI Appendix, Table S1).

ACKNOWLEDGMENTS. We thank J. J. Lopez-Rubio in University Montpellier1&2 for providing the pUF1-Cas9 and pL7-egfp plasmids. We also thankCarolina Barillas-Mury at the National Institutes of Health for critical sugges-tions on the manuscript, and Jiping Han for providing the technical supportof in vitro parasite culture. This research was supported by the National KeyR&D Program of China (2018YFA0507300), National Science and TechnologyMajor Project (2018ZX10101004003001), National Natural Science Founda-tion of China (31771455, 81271863, 81361120405), and National Institute ofAllergy and Infectious Diseases Grants R01AI116466 and U19AI089672. Allauthors have reviewed and agreed on the content of the manuscript.

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