Exploring the virulence gene interactome with CRISPR / dC as9 in the human malaria parasite
2020; Springer Nature; Volume: 16; Issue: 8 Linguagem: Inglês
10.15252/msb.20209569
ISSN1744-4292
AutoresJessica M. Bryant, Sebastian Baumgarten, Florent Dingli, Damarys Loew, Ameya Sinha, Aurélie Claës, Peter R. Preiser, Peter C. Dedon, Artur Scherf,
Tópico(s)Insect symbiosis and bacterial influences
ResumoArticle20 August 2020Open Access Source DataTransparent process Exploring the virulence gene interactome with CRISPR/dCas9 in the human malaria parasite Jessica M Bryant Corresponding Author Jessica M Bryant [email protected] orcid.org/0000-0002-2349-8353 Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France INSERM U1201, Paris, France CNRS ERL9195, Paris, France Search for more papers by this author Sebastian Baumgarten Sebastian Baumgarten orcid.org/0000-0003-2646-7699 Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France INSERM U1201, Paris, France CNRS ERL9195, Paris, France Search for more papers by this author Florent Dingli Florent Dingli orcid.org/0000-0002-7715-2446 Institut Curie, PSL Research University, Centre de Recherche, Mass Spectrometry and Proteomics Facility, Paris, France Search for more papers by this author Damarys Loew Damarys Loew orcid.org/0000-0002-9111-8842 Institut Curie, PSL Research University, Centre de Recherche, Mass Spectrometry and Proteomics Facility, Paris, France Search for more papers by this author Ameya Sinha Ameya Sinha orcid.org/0000-0001-7959-075X School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore Search for more papers by this author Aurélie Claës Aurélie Claës Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France INSERM U1201, Paris, France CNRS ERL9195, Paris, France Search for more papers by this author Peter R Preiser Peter R Preiser orcid.org/0000-0003-4331-7000 School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore Search for more papers by this author Peter C Dedon Peter C Dedon orcid.org/0000-0003-0011-3067 Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Artur Scherf Artur Scherf orcid.org/0000-0003-2411-3328 Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France INSERM U1201, Paris, France CNRS ERL9195, Paris, France Search for more papers by this author Jessica M Bryant Corresponding Author Jessica M Bryant [email protected] orcid.org/0000-0002-2349-8353 Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France INSERM U1201, Paris, France CNRS ERL9195, Paris, France Search for more papers by this author Sebastian Baumgarten Sebastian Baumgarten orcid.org/0000-0003-2646-7699 Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France INSERM U1201, Paris, France CNRS ERL9195, Paris, France Search for more papers by this author Florent Dingli Florent Dingli orcid.org/0000-0002-7715-2446 Institut Curie, PSL Research University, Centre de Recherche, Mass Spectrometry and Proteomics Facility, Paris, France Search for more papers by this author Damarys Loew Damarys Loew orcid.org/0000-0002-9111-8842 Institut Curie, PSL Research University, Centre de Recherche, Mass Spectrometry and Proteomics Facility, Paris, France Search for more papers by this author Ameya Sinha Ameya Sinha orcid.org/0000-0001-7959-075X School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore Search for more papers by this author Aurélie Claës Aurélie Claës Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France INSERM U1201, Paris, France CNRS ERL9195, Paris, France Search for more papers by this author Peter R Preiser Peter R Preiser orcid.org/0000-0003-4331-7000 School of Biological Sciences, Nanyang Technological University, Singapore, Singapore Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore Search for more papers by this author Peter C Dedon Peter C Dedon orcid.org/0000-0003-0011-3067 Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA Search for more papers by this author Artur Scherf Artur Scherf orcid.org/0000-0003-2411-3328 Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France INSERM U1201, Paris, France CNRS ERL9195, Paris, France Search for more papers by this author Author Information Jessica M Bryant *,1,2,3, Sebastian Baumgarten1,2,3, Florent Dingli4, Damarys Loew4, Ameya Sinha5,6, Aurélie Claës1,2,3, Peter R Preiser5,6, Peter C Dedon6,7 and Artur Scherf1,2,3 1Biology of Host-Parasite Interactions Unit, Institut Pasteur, Paris, France 2INSERM U1201, Paris, France 3CNRS ERL9195, Paris, France 4Institut Curie, PSL Research University, Centre de Recherche, Mass Spectrometry and Proteomics Facility, Paris, France 5School of Biological Sciences, Nanyang Technological University, Singapore, Singapore 6Antimicrobial Resistance Interdisciplinary Research Group, Singapore-MIT Alliance for Research and Technology, Singapore, Singapore 7Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA, USA *Corresponding author. Tel: +33 1 45 68 86 22; E-mail: [email protected] Molecular Systems Biology (2020)16:e9569https://doi.org/10.15252/msb.20209569 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Mutually exclusive expression of the var multigene family is key to immune evasion and pathogenesis in Plasmodium falciparum, but few factors have been shown to play a direct role. We adapted a CRISPR-based proteomics approach to identify novel factors associated with var genes in their natural chromatin context. Catalytically inactive Cas9 ("dCas9") was targeted to var gene regulatory elements, immunoprecipitated, and analyzed with mass spectrometry. Known and novel factors were enriched including structural proteins, DNA helicases, and chromatin remodelers. Functional characterization of PfISWI, an evolutionarily divergent putative chromatin remodeler enriched at the var gene promoter, revealed a role in transcriptional activation. Proteomics of PfISWI identified several proteins enriched at the var gene promoter such as acetyl-CoA synthetase, a putative MORC protein, and an ApiAP2 transcription factor. These findings validate the CRISPR/dCas9 proteomics method and define a new var gene-associated chromatin complex. This study establishes a tool for targeted chromatin purification of unaltered genomic loci and identifies novel chromatin-associated factors potentially involved in transcriptional control and/or chromatin organization of virulence genes in the human malaria parasite. Synopsis CRISPR/dCas9-based proteomics is used to purify specific DNA regulatory elements in their natural chromatin context and to identify novel chromatin factors associated with virulence genes in the human malaria parasite, Plasmodium falciparum. dCas9 immunoprecipitation and mass spectrometry identify proteins previously implicated in var gene biology in addition to novel factors, including a putative chromatin remodeler, ISWI. Proteomic analysis of ISWI reveals a new var gene-associated complex comprising a putative MORC family protein and an ApiAP2 transcription factor. ISWI binds to promoter regions and plays a role in transcriptional activation of genes, including the active var gene in ring stage parasites. Introduction In the malaria parasite Plasmodium falciparum, antigenic variation is key to evasion of the immune system and persistence of infection in the human host. The P. falciparum variant surface antigen erythrocyte membrane protein 1 (PfEMP1) is a key component in this process and is encoded by the ~ 60-member var gene family in the haploid genome (reviewed in (Scherf et al, 2008)). A system of mutually exclusive expression is used to activate only a single var gene at a time, but occasional switching occurs to facilitate host immune system evasion. Unlike that in other parasites, antigenic variation in P. falciparum is under epigenetic control (reviewed in (Cortés & Deitsch, 2017)). The single active var gene is associated with euchromatin while all other var genes are kept transcriptionally silent via heterochromatin. Functional studies of orthologous histone writers, readers, and erasers have implicated several chromatin-associated proteins in mutually exclusive var gene transcription including heterochromatin protein 1 (HP1), the histone deacetylases HDA2 and silent information regulator 2 (SIR2a and b), and the histone methyltransferases SET2 and SET10 (Freitas-Junior et al, 2005; Flueck et al, 2009; Pérez-Toledo et al, 2009; Tonkin et al, 2009; Volz et al, 2012; Jiang et al, 2013; Coleman et al, 2014; Ukaegbu et al, 2014). Across the intraerythrocytic developmental cycle (IDC) and other stages, HP1 facilitates transcriptional silencing of all but one var gene via binding to trimethylation of histone H3 at lysine 9 (H3K9me3) (Flueck et al, 2009; Lopez-Rubio et al, 2009; Pérez-Toledo et al, 2009; Fraschka et al, 2018; Zanghì et al, 2018). The histone modifications H3K9ac and H3K4me2/3 and the histone variants H2A.Z and H2B.Z were shown to be enriched at the active var gene promoter (Lopez-Rubio et al, 2007; Petter et al, 2013), but the molecular machinery involved in var gene activation, such as histone-modifying enzymes or nucleosome remodelers, has yet to be elucidated. These epigenetic regulatory proteins may be recruited via DNA regulatory elements. All var genes have the same basic genetic structure: a 5′ upstream promoter followed by exon I (encodes the polymorphic extracellular domain of the PfEMP1), a relatively conserved intron, and exon II (encodes the conserved intracellular domain; Fig 1A). Both the promoter and the intron have been implicated in mutually exclusive transcription control (reviewed in (Guizetti & Scherf, 2013)). Early studies suggested that the var gene promoter could drive transcription unless paired with a downstream var gene intron, which had a repressive effect that was purportedly due to its own bidirectional promoter activity (Calderwood et al, 2003; Gannoun-Zaki et al, 2005; Dzikowski et al, 2006, 2007; Frank et al, 2006; Epp et al, 2009). Conversely, long non-coding RNAs (lncRNA) originating from the var gene intron (Fig 1A) have been implicated in the transcriptional activation of var genes (Amit-Avraham et al, 2015). While deletion of the endogenous var gene intron did not block transcriptional silencing or activation of the targeted var gene, it did lead to higher rates of var gene switching (Bryant et al, 2017). Thus, investigating the protein cohort that binds to either the var gene promoter or intron could provide new mechanistic insight into var gene regulation. Figure 1. Immunoprecipitation of var gene-targeted dCas9 Schematic of a representative var gene with two exons flanking an intron. Transcription originates from the promoter (sense) and intron (sense and antisense). Specific sgRNAs direct dCas9 to either the promoter region (red) or intron (blue). Antibodies were used to isolate the dCas9 and bound genomic regions via a 3xHA tag (yellow star). Schematic of var genes throughout the Plasmodium falciparum genome targeted with intron- (blue), promoter- (red), or both intron and promoter-targeted (green) dCas9. Chromosomes are represented with gray bars, and chromosome numbers are indicated in roman numerals. var gene ups type is indicated on the chromosome, and var gene ID (excluding the preceding chromosome number) is listed to the left of its position on the chromosome. Direction of var gene transcription is indicated with an arrowhead. Western blot analysis of a dCas9 immunoprecipitation experiment in the promoter-targeted strain at ring stage. Levels of dCas9 and histone H3 in the cytoplasmic (Cyt), nuclear (excluding chromatin, Nuc), and chromatin (Ch) fractions are revealed with anti-HA and anti-H3 antibodies, respectively. dCas9 is enriched in the immunoprecipitated fraction (IP) compared to the unbound supernatant (SN) and input (i.e., chromatin fraction) of the IP. Molecular weights are shown to the right. Source data are available online for this figure. Source Data for Figure 1 [msb209569-sup-0016-SDataFig1C.pdf] Download figure Download PowerPoint Coordination of the var genes, located in subtelomeric (upsA and upsB type) or central chromosomal (upsC type) arrays across thirteen chromosomes (Fig 1B), is likely achieved via spatial positioning within the nucleus. Regardless of genome location, var genes form heterochromatic clusters at the nuclear periphery, with the single active var gene spatially separated from the rest (Duraisingh et al, 2005; Ralph et al, 2005; Lopez-Rubio et al, 2009; Lemieux et al, 2013; Ay et al, 2014). This clustering may also facilitate the recombination observed among the polymorphic members of the var gene family during the asexual replicative cycle, which generates antigenic diversity via the formation of chimeric genes (Bopp et al, 2013; Claessens et al, 2014). While the RecQ DNA helicase PfWRN was shown to be important for promoting genome stability and preventing aberrant recombination between var genes, the molecular mechanism behind normal mitotic recombination of var gene members has not been elucidated (Claessens et al, 2018). The telomere repeat-binding zinc finger protein (TRZ) and some members of the DNA/RNA-binding Alba family of proteins or the Apetala2 (ApiAP2) family of transcription factors have been shown to bind to telomeres, subtelomeric regions, or potential var gene regulatory elements; however, a direct role for these proteins in transcriptional control, organization, or recombination of var genes has not been demonstrated (Flueck et al, 2010; Zhang et al, 2011; Chêne et al, 2012; Goyal et al, 2012; Bertschi et al, 2017; Martins et al, 2017; Sierra-Miranda et al, 2017). In addition, actin has been linked to the nuclear positioning and thus transcriptional regulation of var genes via binding to the conserved var gene intron (Zhang et al, 2011). Importantly, proteins such as CCCTC binding factor (CTCF) or lamins, involved in higher-order chromosome organization in metazoans, are absent in P. falciparum (Batsios et al, 2012; Heger et al, 2012). Most studies of var gene biology have relied on in silico identification of factors found to be important for epigenetic regulation in other eukaryotic systems; however, P. falciparum has already shown important differences to other model organisms with regard to epigenetics (reviewed in (Cortés & Deitsch, 2017)). In addition, most studies investigating proteins that bind to specific DNA motifs have used oligo-based in vitro approaches such as gel shift or immunoprecipitation. Thus, a new, unbiased approach is needed to identify novel var gene-interacting factors that contribute to transcriptional regulation and organization of var genes. To this end, we adapted a recently developed CRISPR technology for isolating specific genomic loci with a tagged, nuclease-deficient Cas9 (dCas9) (Fujita & Fujii, 2013; Waldrip et al, 2014; Liu et al, 2017; Myers et al, 2018). Importantly, this technique identifies interactions taking place in a natural spatio-temporal chromatin context and does not require genetic modification of the targeted locus. In this study, we show specific dCas9 enrichment at targeted var gene DNA regulatory elements with chromatin immunoprecipitation followed by sequencing (ChIP-seq). dCas9 immunoprecipitation followed by liquid chromatography-mass spectrometry (IP LC-MS/MS) confirmed previously identified var gene interactors and revealed enrichment of several novel chromatin-associated factors possibly involved in transcriptional control and/or chromatin organization of var genes. Using inducible knockdown, ChIP-seq, and proteomics, we functionally characterize the promoter-associated putative nucleosome remodeler, PfISWI, and demonstrate its role in var gene transcriptional activation. Results Targeting dCas9 to var genes To identify factors associated with putative var gene DNA regulatory elements in an unbiased manner, we adapted the CRISPR/dCas9-based engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP) method developed in Fujita and Fujii (2013) to P. falciparum. As both the var promoter and intron likely contain regulatory protein-binding elements, a tagged dCas9 was directed by an optimized single guide RNA (sgRNA) to either feature (Fig 1A). As a consequence of (i) the need for a GC-rich sequence and (ii) the homology of the promoter and intronic sequences across the var gene family, we designed an sgRNA for either genetic feature that targets multiple var loci. For the promoter sgRNA, we identified a 20-base-pair (bp) sequence flanking a protospacer adjacent motif (PAM) that was ~ 40 bp upstream of the translation start site of 19 var genes (Table 1, Table EV1, and Fig EV1A). For the intron sgRNA, we identified a 20-bp sequence flanking a PAM that was in region III (Calderwood et al, 2003) of the intron of 13 var genes (Table 1, Table EV2, and Fig EV1C). For the promoter and intron sgRNAs, the target sequences were exclusive to upsA/B, upsB, upsB/C, and upsC type var genes (Fig 1B and Tables EV1 and EV2). There was only a single off-target sequence predicted for the intron sgRNA in the upstream region of PF3D7_0400200, which is a var pseudogene with a var exon II-like sequence. Table 1. sgRNA characteristics for dCas9 targeting. Shown are the sequences (5′–3′), targeted DNA strand, GC content, and number of on-target (intended) and off-target sequences predicted for each sgRNA sgRNA Sequence Target strand GC content Predicted On-target Predicted Off-target Promoter GGTTTATATCGTGGCATACA Coding 40% 19 0 Intron GTAAATGTGTATGTAGTTAT Coding 25% 16 1 Control GCTCGCGATGCTGCCCGACA — 70% 0 0 Click here to expand this figure. Figure EV1. Supplementary information for immunoprecipitation of non-targeted and var gene-targeted dCas9 A–C. Alignments of the var gene promoter (A) and intron (C) non-coding strand sequences, with sgRNA sequences highlighted in red. The presumed off-target sequence for the var promoter-targeted dCas9 is shown in (B). Mismatches are highlighted in gray. Corresponding data for the promoter or intron sgRNAs can be found in Tables EV1 and EV2, respectively. D. Western blot analysis of cell fractions from ring stage parasites of the non-targeted dCas9 control strain. Levels of dCas9-3HA and histone H3 in the cytoplasmic (Cyt), nuclear (excluding chromatin, Nuc), and chromatin (Ch) fractions are revealed with anti-HA and anti-H3 antibodies, respectively. Molecular weights are shown to the right. Source data are available online for this figure. Download figure Download PowerPoint To create P. falciparum strains expressing var gene-targeted dCas9, we generated two types of plasmids—pUF1_dCas9-3HA and pL7_var_IP—based on the two-plasmid system originally designed for CRISPR/Cas9 in P. falciparum (Ghorbal et al, 2014). pUF1_dCas9-3HA leads to expression of a hemagglutinin-tagged (HA) catalytically inactive Cas9 containing the RuvC and HNH mutations originally described in Qi et al (2013). The pL7 plasmid encodes a sequence-optimized sgRNA described in Dang et al (2015). A control plasmid (pL7_Control_IP) was used to determine background dCas9 binding to the genome, as it generates a non-specific sgRNA that does not have a predicted target site in the P. falciparum 3D7 genome (Table 1). A bulk culture of 3D7 parasites was transfected with pUF_dCas9-3HA and either pL7_Control_IP, pL7_varPromoter_IP, or pL7_varIntron_IP. Western blot analysis of the cytoplasmic, nuclear (non-chromatin), and chromatin fractions showed that targeted dCas9 was most enriched in the chromatin fraction, and immunoprecipitation of dCas9 from the chromatin fraction with an anti-HA antibody resulted in a robust enrichment of the tagged dCas9 (Fig 1C). The non-targeted dCas9 was present in the nuclear and chromatin fractions (Fig EV1D). Genome-wide binding of intron- and promoter-targeted dCas9 is specific and robust To investigate the specificity of var gene-targeted dCas9 binding, we performed ChIP-seq in synchronized parasites cross-linked at 14 h post-invasion (hpi) of the host red blood cells, the ring stage during which var gene transcription is highest. Analysis of the ChIP-seq data revealed that var promoter and intron sgRNAs led to specific and significant dCas9 enrichment at most intended target sequences (Fig 2A and Tables EV1 and EV2) while the non-targeted dCas9 control showed no specific enrichment (Fig EV2A). For the promoter sgRNA, a significant peak of dCas9 was found at 17 out of 19 predicted target sites, with only one major unpredicted off-target binding event at PF3D7_1209900 (Figs 2A and EV1B, and Table EV1). For the intron sgRNA, a significant peak of dCas9 was found at 13 out of 16 predicted target sites and the predicted off-target sequence PF3D7_0400200 (Fig 2A and Table EV2). Two additional peaks of dCas9 were detected with the var intron-targeted sgRNA at unpredicted upsB var gene introns (PF3D7_0500100 and PF3D7_1300100) with similar sequences (one or two mismatches; Figs 2A and EV1C, and Table EV2). Figure 2. ChIP sequencing shows specific enrichment of dCas9 at targeted var gene introns and promoters Circos plots of ChIP-seq data showing genome-wide enrichment of dCas9 in ring stage parasites. The 14 chromosomes are represented circularly by the outer gray bars, with chromosome number indicated in roman numerals and chromosome distances indicated in Arabic numerals (Mbp). Enrichment for intron- or promoter-targeted dCas9 (normalized to non-targeted dCas9) is shown as average reads per million (RPM) over bins of 1,000 nt. The maximum y-axis value is 3,000 RPM for the promoter-targeted dCas9 (rings represent increments of 500) and 400 RPM for the intron-targeted dCas9 (rings represent increments of 66.7). var genes are represented by red bars. An asterisk indicates an unintended binding event. One replicate was performed for each strain. Peak quantification for promoter- and intron-targeted dCas9 ChIP-seq can be found in Tables EV1 and EV2, respectively. ChIP-seq data show enrichment of dCas9 in strains at ring stage expressing non-targeted "Control" (gray), var intron-targeted (blue), or var promoter-targeted (red) sgRNAs. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted ChIP enrichment. q-values for promoter-targeted dCas9 peaks shown are 2.22 × 10−289 for PF3D7_0413100 and 7.07 × 10−257 for PF3D7_1240400. q-values for intron-targeted dCas9 peaks shown are 2.03 × 10−76 for PF3D7_412700, 6.81 × 10−46 for PF3D7_0412900, 1.53 × 10−62 for PF3D7_0413100, and 8.37 × 10−4 for PF3D7_1240900. One replicate was performed for each strain. Peak quantification for promoter- and intron-targeted dCas9 ChIP-seq can be found in Tables EV1 and EV2, respectively. ChIP-seq (red) and RIP-seq (green) data show enrichment of DNA and RNA, respectively, from var genes in the promoter-targeted and non-targeted "Control" dCas9 immunoprecipitation from non-clonal bulk population of ring stage parasites. Genome location is indicated at the top of each panel. The x-axis is DNA sequence, with genes represented by black boxes indented to delineate introns and labeled with white arrowheads to indicate transcription direction. The y-axis is input-subtracted dCas9 ChIP enrichment in the var gene promoter-targeted dCas9 strain (red), dCas9 RIP enrichment normalized to IgG control in the var gene promoter-targeted dCas9 strain or non-targeted "Control" dCas9 strain (green), or var gene transcript levels in the promoter-targeted dCas9 strain (RPKM, gray). One replicate was performed for ChIP-seq and RNA-seq, and one replicate was performed for HA and IgG control RIP-seq. ChIP-seq peak quantification can be found in Table EV1, and RIP-seq quantification can be found in Table EV3. Download figure Download PowerPoint Click here to expand this figure. Figure EV2. var gene transcription in non-targeted and var gene-targeted dCas9 strains Circos plot of ChIP-seq data showing genome-wide enrichment of non-targeted dCas9 in ring stage parasites. The 14 chromosomes are represented circularly by the outer gray bars, with chromosome number indicated in roman numerals and chromosome distances indicated in Arabic numerals (Mbp). Enrichment (ChIP normalized to input) is shown as average reads per million (RPM) over bins of 1,000 nt. The maximum y-axis value is 1,000 RPM (rings represent increments of 167). var genes are represented by red bars. One replicate was used for the dCas9 ChIP-seq. RNA-seq data from clonal parasite populations of promoter- (red) or intron-targeted (blue) dCas9 strains show transcript abundance (y-axis = RPKM) for all var genes (x-axis) at 14 hpi. One replicate was performed for each clone. Cell cycle progression estimation of synchronized non-clonal bulk parasite cultures of the promoter- (red), intron- (blue), and non-targeted (gray) dCas9 strains at 14 hpi. RNA-seq data from these parasites were compared to microarray data from Bozdech et al (2003) (Data ref: Bozdech et al, 2003) as in Lemieux et al (2009). RNA-seq data from non-clonal bulk parasite cultures of the promoter- (red), intron- (blue), and non-targeted (gray) dCas9 strains at 14 hpi show transcript abundance (y-axis = RPKM) for all var genes (x-axis). The gray dotted boxes indicate var genes bound by dCas9 in the promoter-targeted strain (top graph) or intron-targeted strain (bottom graph). One replicate was performed for each strain. Circos plot of dCas9 ChIP-seq (outer ring in red) and RIP-seq (inner ring in green) data showing genome-wide DNA and RNA enrichment, respectively, in var gene promoter-targeted dCas9 immunoprecipitation at 14 hpi. The 14 chromosomes are represented circularly by the outer gray bars, with chromosome number indicated in roman numerals and chromosome distances indicated in Arabic numerals (Mbp). dCas9 ChIP enrichment (input-subtracted and normalized to the corresponding value for the non-targeted dCas9 control) is shown as average RPM over bins of 1,000 nt. dCas9 RIP enrichment (IgG-subtracted and normalized to the corresponding value for the non-targeted dCas9 control) is shown as average enrichment per gene. The maximum y-axis value is 3,000 for ChIP-seq, and 3 for RIP-seq. var genes are represented by red bars. One replicate was performed for ChIP-seq, and one replicate was performed for HA and IgG control and promoter-targeted dCas9 RIP-seq. ChIP-seq peak quantification can be found in Table EV1, and RIP-seq quantification can be found in Table EV3. Download figure Download PowerPoint Comparison of the ChIP-seq data of the var gene-targeted dCas9 and non-targeted dCas9 control highlights the specificity of the method from the most statistically significant dCas9 peak (enrichment ratio = 123.9, q = 2.22 × 10−289 at the promoter of PF3D7_0413100) to the least significant dCas9 peak (enrichment ratio = 42.5, q = 8.37 × 10−4 at the intron of PF3D7_1240900; Fig 2B and Tables EV1 and EV2). dCas9 enrichment was approximately two-fold higher at promoter sgRNA-targeted sites (average ChIP/Input = 20.18) than intron sgRNA-targeted sites (average ChIP/Input = 8.9; Tables EV1 and EV2). However, because the var gene intronic sequence is AT-rich relative to the var gene promoter sequence (Table 1 and Fig EV1A and C), it could be under-represented in the sequencing data. Regardless, the dCas9 ChIP-seq protocol we present here could be used to determine genome-wide specificity and efficacy of sgRNAs for use in traditional CRISPR/Cas9 genome editing. To determine the var gene transcriptional profile in the strains expressing dCas9, we performed sequencing of mRNA (RNA-seq) from clones and non-clonal bulk parasite cultures at 14 hpi. In clones of the intron- and promoter-targeted dCas9 strains, a single var gene was predominantly transcribed, suggesting that mutually exclusive expression of the var gene family was unaffected by dCas9 binding (Fig EV2B). RNA-seq of non-clonal bulk parasite cultures of the promoter-, intron-, and non-targeted dCas9 strains showed synchronicity at 14 hpi (Fig EV2C) and that multiple var genes, both targeted and untargeted, were transcribed in the population (Fig EV2D). mRNA from the var genes targeted at their promoters with dCas9 was, in general, present at higher levels in the promoter-targeted dCas9 strain than in the non-targeted dCas9 strain, suggesting that parasites in which dCas9 was able to more readily bind to the euchromatic promoter were selected for during transfection (Fig EV2D, top). To determine if mRNA was transcribed from var genes bound by dCas9 at their promoter, we performed RNA immunoprecipitation (RIP) with dCas9 in the pr
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