A genome-wide crispr screen in Toxoplasma Identifies Essential Apicomplexan Genes
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in several branches of the Apicomplexa suggest that this organ- elle might serve as a focal point for functions conserved across the phylum, in addition to the currently appreciated genus-spe- cific modifications ( Seeber et al., 2008 ). Two other ICAPs local- ized to apical secretory vesicles called micronemes, which are more typically associated with specific apicomplexan adapta- tions. During the preparation of this paper, the first, ICAP1 (APH), was shown to be required for microneme secretion ( Bullen et al., 2016 ). The second one, ICAP12, was critically important for invasion, as demonstrated by our secondary screens and subsequent analysis. These data argue for an in- depth examination of other ICAPs, which will likely reveal essen- tial processes conserved among apicomplexans. We renamed ICAP12 CLAMP to reflect its subcellular localiza- tion and structural similarity to mammalian claudins. Conditional silencing demonstrated that CLAMP is specifically required dur- ing invasion of host cells and did not affect microneme secretion or dependent processes. CLAMP is thereby distinct from factors that participate in both gliding motility and invasion ( Bargieri
et al., 2014 ). Instead, CLAMP resembles factors involved in the formation of the tight junction—or moving junction—through which parasites enter host cells. Knockdown of the micronemal protein MIC8 resembles CLAMP disruption by blocking secre- tion of the rhoptry neck proteins that anchor the micronemal ad- hesin AMA1 to the host cell membrane ( Kessler et al., 2008 ). Video microscopy showed CLAMP-deficient parasites repeat- edly pushing against the host-cell membrane while failing to initiate invasion, much like P. falciparum parasites unable to form a moving-junction ( Treeck et al., 2009 ). Apposition of the parasite and host-cell membrane is a hallmark of apicomplexan parasitism, mediating discharge of rhoptry contents into host cells and complete or partial invasion ( Bargieri et al., 2014 ). Despite the similarities, previously identified invasion factors are not completely conserved throughout the phylum; MIC8 is restricted to the coccidia, and AMA1 and RON2, which are cen- tral components of the moving junction, are absent from early branching apicomplexans (EupathDB). In contrast, CLAMP ho- mologs are found in all sequenced apicomplexan genomes, including early branching members like cryptosporidians and gregarines. Consistent with its conservation, the ortholog of CLAMP in P. falciparum could be readily identified, and its knockdown leads to a complete inhibition of the asexual cycle, although further work will be necessary to define its precise func- tion. These results argue for a pivotal role of CLAMP in all mem- bers of the phylum. Its structural resemblance to mammalian claudins suggests the physical involvement of CLAMP in tight- junction formation during invasion. Claudins are known to engage in homotypic and heterotypic interactions, as well as the formation of paracellular channels that restrict the flow of ions across the tight junction ( Krause et al., 2008 ). Whether CLAMP participates in similar processes remains speculative, but its study might shed light into one of the most conserved fea- tures of apicomplexan parasitism. By screening for genes that confer parasite fitness during growth in human fibroblasts, we provide a baseline for gene 10 Cell 167, 1–13, September 22, 2016 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019
function under arguably permissive conditions. Future screens will need to define the genes required during other life stages, in different hosts, under varying nutrient conditions, and in response to immune pressures. The potential to obscure or exacerbate deleterious mutations in pooled screens due to competition with wild-type parasites should be mentioned here. However, this screening format has the advantage of simultaneously determining the fate of hundreds of indepen- dently generated mutants for a given gene. As such, they are less influenced by rare compensatory mutations that can confound interpretation of outcomes when using clonal strains ( Lamarque et al., 2014; Ma et al., 2008 ). Studies in yeast have re- vealed that adaptations can overcome the need for $9% of genes previously considered essential ( Liu et al., 2015 ). These is- sues argue for a nuanced view of gene essentiality. Although our experiments demonstrate the strong predictive value of this method, careful follow-up experiments are necessary to fully explore the role of individual genes. Genome-wide functional analyses have transformed the study of many organisms. We demonstrate the power of this approach to identify genes that contribute to T. gondii fitness during infec- tion of human fibroblasts. Although important adaptations distin- guish different parasite genera, this method provides a unique tool to model conserved apicomplexan processes in T. gondii, and its success is demonstrated by the identification of a previ- ously uncharacterized protein essential for the malaria parasite P. falciparum. Coupled with the diverse tools available for ge- netic and chemical manipulation of T. gondii, the genome-wide screens will provide a framework for the systematic examination of genetic interactions. The unconstrained study of apicom- plexan genomes will help us understand their unique biology and broaden the scope of interventions to control these wide- spread parasitic infections. STAR+METHODS Detailed methods are provided in the online version of this paper and include the following: d KEY RESOURCES TABLE d CONTACT FOR REAGENT AND RESOURCE SHARING d EXPERIMENTAL MODEL AND SUBJECT DETAILS d METHOD DETAILS B Plasmid Design and Construction B Library Design and Construction B
B Pooled CRISPR Screens B RT-PCR
B Functional Analysis of ICAPs and Controls B ICAP Tagging B CLAMP Phylogeny and Topology Predictions B CLAMP Conditional Knockdown B Immunofluorescence Microscopy and Immunoblotting B Surveyor Assays B Plaque Formation B Microneme Secretion B Egress Assays B Invasion Assays B Video Microscopy B
d QUANTIFICATION AND STATISTICAL ANALYSIS B Bioinformatic Analysis of the Screening Results B Statistical Testing d DATA AND SOFTWARE AVAILABILITY B Software
B Data Resources SUPPLEMENTAL INFORMATION Supplemental Information includes five figures, three tables, and four movies and can be found with this article online at http://dx.doi.org/10.1016/j.cell. 2016.08.019 AUTHOR CONTRIBUTIONS S.M.S., D.H., and S.L. conceived this study and performed most of the exper- iments. S.M.G., A.S.N., and J.C.N. designed and performed the malaria work. M.-H.H. and V.B.C. provided essential reagents and insight. T.W. provided the scripts to design the library, which P.T. adapted and executed. J.P.J.S. advised the experimental design and drafting of the manuscript. S.M.S., D.H., and S.L. wrote the manuscript, which was read and approved by all authors. ACKNOWLEDGMENTS We thank Emily Shortt for technical support; George Bell for bioinformatics advice; L. David Sibley, Dominique Soldati-Favre, and Lilach Sheiner for the SAG1, MIC8, MYOA, and MYS antibodies; Ke Hu for the NeonGreen plasmid; David S. Roos and Maria Alejandra Diaz-Miranda for the RNA-sequencing data; Markus Meissner for the DiCre strain; and Gail Eskes for naming CLAMP. This work would not have been possible without EupathDB, and we thank all members of the community who have worked to generate this resource. This work was supported by NIGMS Center for Integrative Synthetic Biology Grant P50GM098792, NIH National Research Service Award F31 CA189437 to T.W., NIH Research Project Grant R01AI46675 to V.B.C., and the NIH Director’s New Innovator Award 1DP2OD007124 to J.C.N. and Early Indepen- dence Award 1DP5OD017892 to S.L. Received: May 19, 2016 Revised: July 25, 2016 Accepted: August 5, 2016 Published: September 1, 2016 REFERENCES Achbarou, A., Mercereau-Puijalon, O., Autheman, J.M., Fortier, B., Camus, D., and Dubremetz, J.F. (1991). Characterization of microneme proteins of Toxoplasma gondii. Mol. Biochem. Parasitol. 47, 223–233. 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IDENTIFIER Antibodies Mouse monoclonal anti-SAG1 (clone DG52) ( Burg et al., 1988 ) N/A
Mouse monoclonal anti-TUB1 (clone 12G10) Developmental Studies Hybridoma Bank at the University of Iowa RRID: AB_1157911 Mouse monoclonal anti-MIC2 (clone 6D10) ( Achbarou et al., 1991 ) N/A
Mouse monoclonal anti-Ty1 (clone BB2) ( Bastin et al., 1996 ) N/A
Mouse monoclonal anti-FLAG (clone M2) Sigma-Aldrich Cat#F3165 Mouse monoclonal anti-HA (clone 16B12) BioLegend Cat#901501 Alpaca nanobody anti-CDPK1(clone 1B7) ( Ingram et al., 2015 ) N/A
Rabbit polyclonal anti-MyoA ( Fre´nal et al., 2014 ) N/A
Rabbit polyclonal anti-PLP1 ( Kafsack et al., 2009 ) N/A
Rabbit polyclonal anti-ACT1 ( Dobrowolski et al., 1997 ) N/A
Goat anti-Mouse IgG (H+L) Secondary Antibody, DyLight 488 conjugate Thermo Fisher Cat#35502 Goat anti-Rabbit IgG (H+L) Secondary Antibody, DyLight 594 conjugate Thermo Fisher Cat#35560 Pacific Blue Antibody Labeling Kit Thermo Fisher Cat#P30013 Alexa Fluor 594 Antibody Labeling Kit Thermo Fisher Cat#A20185 Chemicals, Peptides, and Recombinant Proteins Pyrimethamine Sigma-Aldrich Cat#46706 Mycophenolic Acid Sigma-Aldrich Cat#M3536 Xanthine
Sigma-Aldrich Cat#X4002 Gentamicin Thermo Fisher Cat#15710072 5-Fluoro-2 0 -deoxyuridine (FUDR) Sigma-Aldrich Cat#F0503 Rapamycin EMD Millipore Cat#553210 A23187
EMD Millipore Cat#100105 Zaprinast EMD Millipore Cat#684500 Blasticidin S Sigma-Aldrich Cat#15205 WR99210 Sigma Aldrich Cat#W1770 Anhydrotetracycline Takara-Clontech Cat#631310 SYBR Green I Thermo Fisher Cat#S7563 Ampicillin Sigma-Aldrich Cat#A0166 Critical Commercial Assays QuikChange Multi-Site Directed Mutagenesis Kit Agilent Technologies Cat#200514 Gibson Assembly Cloning Kit New England Biolabs Cat#E5510S NucleoBond Xtra Midi Macherey Nagel Cat#740412.50 DNeasy Blood & Tissue Kit QIAGEN Cat#69506 RNeasy Plus Kit QIAGEN
Cat#73404 ProtoScript First Strand cDNA Synthesis Kit New England Biolabs Cat#E6300S Surveyor Mutation Detection Kit Integrated DNA Technologies Cat#706020 Deposited Data T. gondii strain GT1 genome, release 28 ToxoDB
http://toxodb.org/toxo/ Ortholog Groups of Protein Sequences, version 5 OrthoMCL DB http://orthomcl.org/orthomcl/ (Continued on next page) e1 Cell 167, 1–13.e1–e7, September 22, 2016 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019
CONTACT FOR REAGENT AND RESOURCE SHARING Further information and requests for reagents may be directed to, and will be fulfilled by the corresponding author Sebastian Lourido ( lourido@wi.mit.edu ). EXPERIMENTAL MODEL AND SUBJECT DETAILS T. gondii tachyzoites from the strain RH and derived strains were maintained at 37
C with 5% CO 2 growing in human foreskin fibro- blasts (HFFs) cultured in DMEM supplemented with 10% heat-inactivated fetal bovine serum and 10 mg/ml gentamicin. When appro- priate, chloramphenicol was used at 40 mM and pyrimethamine at 3 mM (1.5 mM for plaque assays). Continued REAGENT or RESOURCE SOURCE IDENTIFIER Experimental Models: Cell Lines Human Foreskin Fibroblasts (HFFs) ATCC SCRC-1041 Human Erythrocytes Research Blood Components, LLC N/A Experimental Models: Organisms/Strains T. gondii: strain RH ATCC
50838 T. gondii: strain RH/Cas9 This paper N/A
ATCC
PRA-319 T. gondii: strain RH/DKU80/DRAB4 This paper N/A
( Sheiner et al., 2011 ) N/A
T. gondii: strain RH/Cas9/H2B-YFP This paper N/A
flox
-YFP ( Pieperhoff et al., 2015 ) N/A
T. gondii: strain DiCre/CLAMP cKD This paper N/A
attB
Laboratory of David A. Fidock N/A
P. falciparum: strain PfCLAMP cKD This paper N/A Recombinant DNA pU6-Universal Addgene
Cat#52694 pCas9/decoy This paper N/A
pH2B-YFP/decoy This paper N/A pU6-DHFR
This paper; Addgene Cat#80329 pLIC-HA-FLAG-(3 0 UTR SAG1 -pDHFR-HXGPRT- 5 0
DHFR ) flox -4xU1 ( Pieperhoff et al., 2015 ) N/A
pPfCLAMP-cKD/TetR-DOZI ( Ganesan et al., 2016 ) N/A
pU6[sgSAG1]-DHFR This paper; Addgene Cat#80322 pU6-Decoy This paper; Addgene Cat#80324 pCas9/CAT This paper; Addgene Cat#80323 Genome-wide sgRNA library Addgene Cat#80636 Sequence-Based Reagents All primers and oligonucleotides are listed in Table S2
This paper N/A
Software and Algorithms ClustalW
( Larkin et al., 2007 ) http://www.clustal.org/clustal2/ Bioperl, version 1.5.2 BioPerl
http://bioperl.org/ R, version 3.2.3 R Foundation for Statistical Computing https://www.R-project.org/ Prism, version 6.0 GraphPad
http://www.graphpad.com FIJI running ImageJ, version 2.0 ( Schindelin et al., 2012 ) http://imagej.net/Welcome sgRNA library design scripts ( Wang et al., 2014 ) N/A
Screen analysis scripts This paper N/A Other
Genome-wide screening data This paper Table S3 Cell 167, 1–13.e1–e7, September 22, 2016 e2 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019 P. falciparum parasites of the strain NF54 attB
(kindly provided by David Fidock) and the derived strain were grown in human eryth- rocytes (Research Blood Components) at 5% hematocrit under 5% O 2 and 5% CO 2 in RPMI 1640 media supplemented with 5 mg/ml Albumax II (Life Technologies), 2 mg/ml NaHCO 3 , 25 mM HEPES (pH 7.4), 1 mM hypoxanthine and 50 mg/ml gentamicin. METHOD DETAILS Plasmid Design and Construction To construct pCas9/CAT, the chloramphenicol acetyltransferase (CAT) gene, under the TUB1 promoter and SAG1 3 0 UTR ( Soldati and Boothroyd, 1993 ), was amplified using primers P1 and P2 (see Table S2
for a complete list of primers and oligonucleotides), and ligated into the PciI and XbaI sites of pU6-Universal (Addgene, #52694). To generate pCas9/decoy, oligonucleotides P3 and P4 were hybridized and cloned into the BsaI sites of pU6-Universal. H2B-YFP ( Hu et al., 2004 ) was excised with NsiI and NotI, and used to replace Cas9 in pCas9/decoy by ligating the fragment into the same restriction sites to generate pH2B-YFP/decoy. The plasmid for sgRNA expression was constructed by amplifying the pyrimethamine-resistance cassette ( Donald and Roos, 1993 ) with primers P5 and P6, and cloning it into the NsiI and SbfI sites of pU6-Universal. Three BsaI sites in DHFR were eliminated using the QuikChange multi-site directed mutagenesis kit (Agilent Technologies) with primers P7, P8, and P9 resulting in the plasmid pU6-DHFR. The SAG1 protospacer, encoded by primers P10 and P11, was cloned into pU6-DHFR as described above for the decoy protospacer. Guides for gene disruption (P14–P63) and ICAP tagging (P80–P113) were synthesized for Gibson Assembly (New En- gland Biolabs) into pU6-DHFR or pU6-Universal, respectively. Such guides and their reverse complements were hybridized, and Gibson cloned into their respective vectors linearized with BsaI. Guides to knock out RAB4 were similarly generated by cloning sgRNAs against the 5 0 (P160 and P161) and 3 0 (P162 and P163) ends of the coding sequence into pU6-Universal. For C-terminal HA-FLAG epitope tagging and U1 mediated knockdown, a 3 0 flank of the CLAMP gene, upstream of the stop codon, was amplified by PCR with primers P148 and P149 and inserted into pLIC-HA-FLAG-(3 0 UTR SAG1 -pDHFR-HXGPRT-5 0 UTR
DHFR ) flox - 4xU1 (
Pieperhoff et al., 2015 ) by ligation-independent cloning ( Huynh and Carruthers, 2009 ) to generate pCLAMP-U1. The plasmid for conditional knockdown of CLAMP in P. falciparum was generated by amplifying the 5 0 and 3 0 homology regions from the CLAMP locus using primers P152–P155. P155 included an sgRNA targeting the 3 0 end of the CLAMP locus, placed under the regulation of the T7 promoter in the final construct. The fragments were cloned by Gibson assembly into the plasmid containing the aptamers, and the Renilla luciferase-blasticidin deaminase fusion separated from the TetR-DOZI fusion by a T2A self-cleaving peptide, to generate pPfCLAMP-cKD/TetR-DOZI ( Ganesan et al., 2016 ). Library Design and Construction Ten guides were designed against each gene in the 14 annotated chromosomes of the T. gondii GT1 genome (release 28, ToxoDB.
org ), according to published guidelines ( Wang et al., 2014 ). Briefly, selection of sgRNAs was weighted based on targeting of exons, number of potential off-target sequences, and overlap between sgRNAs for a given gene. A ‘G’ was prepended to any sgRNAs that did not start with one to ensure proper RNA polymerase III initiation. Guides against DHFR and HXGPRT were removed from the li- brary to preclude interference with drug-resistance markers. The guide library was synthesized by CustomArray, and includes guides flanked by sequences for Gibson Assembly into pU6-DHFR. The sgRNA library was amplified using primers P12 and P13, and Gibson cloned into pU6-DHFR linearized with BsaI. Assembled constructs were transformed into Mega-X DH10B electrocompetent Escher-
isolation (Macherey Nagel) or frozen storage. Cloning and electroporation efficiencies were monitored to ensure proper library coverage. T. gondii Strain Generation All transfections were performed as described previously with a square-wave electroporator ( Sidik et al., 2014 ). RH/Cas9 and RH/ Cas9/H2B-YFP were generated by co-transfecting RH with 50 mg each of pCas9/CAT and pCas9/decoy, or pH2B-YFP/decoy and pCas9/CAT, respectively. Stable transgenic strains were selected with 40 mM chloramphenicol. Single clones were isolated by limiting dilution and screened for the presence of Cas9 using immunofluorescence and immunoblotting against the triple- FLAG tag. The decoy protospacer was amplified by PCR from RH/Cas9 genomic DNA using primers P64 and P65, and confirmed by sequencing. To generate the RAB4 knockout, the pyrimethamine-resistance cassette was amplified from pU6-DHFR with primers P164 and P165 to contain homology regions to replace the entire open reading frame. The amplicon was cotransfected along with two pU6-Universal plasmids carrying guides against the 5 0 and 3 0 end of the coding sequence into RH/DKU80 parasites ( Huynh and Car- ruthers, 2009 ). Stable transformants were selected for with pyrimethamine and clones were isolated by limiting dilution. Correct inte- gration of the pyrimethamine-resistance cassette into the RAB4 locus was confirmed using P166 and P169 to amplify the 5 0 junction, and P167 and P168 to amplify 3 0 junction. Deletion of the RAB4 locus was assessed using P170 and P171 to amplify a portion of the open reading frame. Fitness of the RAB4 knockout was determined by plaque assay, as described below. To generate the CLAMP conditional knockdown (DiCre/CLAMP), 25 mg of pCLAMP-U1 were linearized with MfeI for efficient ho- mologous recombination and transfected into DiCre/Dku80/KillerRed
Pieperhoff et al., 2015 ). Following selection with 25 mg/ml mycophenolic acid and 50 mg/ml xanthine, individual clones were obtained by limiting dilution. e3 Cell 167, 1–13.e1–e7, September 22, 2016 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019
Pooled CRISPR Screens For each biological replicate, 400 mg of the sgRNA library were linearized with AseI, dialyzed against water, and transfected into approximately 4 3 10
8 RH or RH/Cas9 parasites divided between 8 separate cuvettes. Transfections were used to infect 8 T-175 flasks with confluent HFF monolayers, and pyrimethamine was added 24 hr later. The parasites were allowed to egress naturally from host cells two days after infection, isolated by filtration, and 1.5 3 10 8
fresh monolayers. The remaining parasites ( $10
7 ) were pelleted and stored at À80
again 5 days and 7 days post-transfection. For the drug-resistance screens, 5 mM 5-fluorodeoxyuridine (FUDR; Sigma) was added to 1.2 3 10 7
mutant pools were maintained in parallel for the duration of FUDR selection. Parasite DNA was extracted using the DNeasy Blood and Tissue kit (QIAGEN) and integrated sgRNA constructs were amplified using a nested PCR with primers P74 and P75 followed by P76 and P77. The resulting libraries were sequenced on a HiSeq 2500 (Illumina) with single-end reads using primers P150 and P151. RT-PCR
using the RNeasy Plus Kit (QIAGEN). The ProtoScript First Strand cDNA Synthesis Kit (New England Biolabs) was used to prepare cDNA, and reverse transcriptase (RT) was excluded from the control reactions. PCR was performed on the cDNA samples with primers specific for RAB4 (P174 and P175) or the ACT1 control (P172 and P173). Functional Analysis of ICAPs and Controls 1.25
3 10 7 RH/Cas9 or RH/Cas9/H2B-YFP parasites were transfected with 50–100 mg of pU6-DHFR containing guides against different ICAPs or controls. If aiming for integration, plasmids were linearized with AseI and dialyzed against water prior to transfec- tion. Transfected parasites were seeded on HFFs at a MOI of 10, and pyrimethamine was added 24 hr after transfection. Parasites transected with sgSAG1 were allowed to egress from host cells two days after transfection, and used to infect host cells seeded on coverslips. SAG1 loss was quantified by immunofluorescence 24 hr after infecting coverslips, which was equivalent to three days after transfection. To screen for ICAPs that participate in invasion, parasites were released from host cells 24 hr after transfection by passage through a 27.5-gauge needle, and used to infect fresh monolayers, thus extending the time allowed for protein depletion. To test invasion, freshly lysed parasites were suspended in invasion media (DMEM supplemented with 1% FBS, 20 mM HEPES [pH 7.4]). 2 3 10 5
3 g for 5 min. Invasion was allowed to proceed for 10 min at 37
C, before the monolayers were fixed in 4% formaldehyde for 20 min on ice. Extracellular parasites were stained using mouse-anti-SAG1 ( Burg et al., 1988 ) conjugated to Alexa-Fluor-594 (Life Technologies), and host cell nuclei were stained using Hoechst (Santa Cruz). Images were acquired using a Cytation 3 imager (BioTek), and analyzed using custom FIJI ( Schindelin et al., 2012 ) macros to count the number of parasites and host-cell nuclei. ICAP Tagging CRISPR-mediated C-terminal Ty tagging was performed as previously described ( Sidik et al., 2014 ). 30 mg of a repair oligonucleotide containing an in-frame Ty epitope ( Bastin et al., 1996 ) flanked by homology regions to the C terminus of each gene (P114–P147) were co-transfected with 100 mg of pU6-Universal carrying the appropriate sgRNA into TATi/DKU80 parasites ( Sheiner et al., 2011 ). Trans- fected parasites were cultured until their first lysis and used to infect confluent HFF monolayers grown on coverslips. Localization of the Ty-tagged ICAPs was determined 24 post infection by immunofluorescence microscopy. C-terminal tagging of CLAMP with mNeonGreen, was accomplished by amplifying the fluorescent protein coding sequence with primers P78 and P79 from a template plasmid (kindly provided by Ke Hu). 30 mg of the resulting product were co-transfected into TATi/DKU80 parasites along with 100 mg of pU6-Universal carrying an sgRNA against the C-terminal sequence of the endogenous CLAMP locus. Fluorescent parasites were isolated by FACS two days post transfection, and cloned by limiting dilution. Correct inte- gration of mNeonGreen into the CLAMP locus was confirmed by sequencing. CLAMP Phylogeny and Topology Predictions CLAMP homologs were readily identified by BLAST searches against all sequenced apicomplexan genomes ( EupathDB.org ). Sequences were curated for Babesia and Theileria spp. to correct errors in the gene models. Alignment was performed using ClustalW ( Larkin et al., 2007 ) and the phylogenetic tree was generated by neighbor-joining excluding positions with gaps. Boot- strap values were calculated for 10,000 trials. A hidden Markov model-based search was performed for the alignment using HHpred ( Meier and So¨ding, 2015 ). Significant structural similarity was found between CLAMP and Claudin-19 (p = 4.1 3 10 À9 ), Claudin-15 (p = 7.3 3 10 À9
À10 ), which all belong to the same tetraspan family ( Simske, 2013 ). Structural similarity between CLAMP and Claudin-19 (95% confidence) was also found using Phyre2 ( Kelley et al., 2015 ). Topology prediction was performed against several representative orthologs with CCTOP ( Dobson et al., 2015 ) and in all cases arrived at the same prediction of four transmembrane domains with cytoplasmic N and C termini ( Figure S4 ). Cell 167, 1–13.e1–e7, September 22, 2016 e4 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019 CLAMP Conditional Knockdown DiCre/CLAMP parasites were treated with 50 nM rapamycin or vehicle control for 2 hr, then cultured for 48 hr before phenotypic anal- ysis by immunoblot, plaque formation, MIC2 secretion, or invasion assay. Immunofluorescence Microscopy and Immunoblotting Mouse monoclonal antibodies were used to detect SAG1 (clone DG52; Burg et al., 1988 ), TUB1 (clone 12G10, Developmental Studies Hybridoma Bank at the University of Iowa), MIC2 (clone 6D10; Achbarou et al., 1991 ), Ty-tagged proteins (clone BB2; Bastin et al., 1996 ), FLAG-tagged proteins (clone M2; Sigma-Aldrich), and HA-tagged proteins (clone 16B12; BioLegend). CDPK1 was de- tected using the alpaca-derived nanobody 1B7 ( Ingram et al., 2015 ). Rabbit polyclonal sera were used to detect MyoA ( Fre´nal et al., 2014
), PLP1 ( Kafsack et al., 2009 ) and ACT1 ( Dobrowolski et al., 1997 ). Prior to immunoblotting, DiCre and DiCre/CLAMP parasites were suspended in lysis buffer (137 mM NaCl, 10 mM MgCl 2 , 1% Triton X-100, Halt protease inhibitors [Thermo Fisher], 20 mM HEPES [pH 7.5]). An equal volume of 2X Laemmli buffer (4% SDS, 20% glyc- erol, 5% b-mercaptoethanol, 0.02% bromophenol blue, 120 mM Tris-HCl [pH 6.8]) was added, and the samples were heated to 37
C
incubated in stripping buffer (100 mM b-mercaptoethanol, 2% SDS, 62.5 mM Tris-HCl [pH 6.8]) for 15 min at 50
C, then washed twice in TBS-T (20 mM Tris-HCl, 138 mM NaCl, 0.1% Tween-20 [pH 7.5]). Samples for all other blots were prepared similarly, but boiled 10 min prior to separation by SDS-PAGE, and membranes were not incubated in stripping buffer. b-mercaptoethanol was not included when probing for SAG1. Intracellular parasites were fixed with either methanol at 4
C for 2 min, or 4% formaldehyde for 10 min. Staining was performed with the antibodies described above and detected with Alexa-Fluor-labeled secondary antibodies. Formaldehyde-fixed samples were permeablized with 0.25% Triton X-100 in PBS for 8 min. Nuclei were stained with Hoechst (Santa Cruz) or DAPI (Life Technologies) and coverslips were mounted in Prolong Diamond (Thermo Fisher). Images were acquired using an Eclipse Ti epifluorescence mi- croscope (Nikon) using the NIS elements imaging software. FIJI was used for image analysis, and Adobe Photoshop for image processing. Surveyor Assays Pools of mutant parasites were suspended in PBS containing 200 mg/ml Proteinase K (Sigma-Aldrich) and 1X Taq PCR Buffer (Sigma- Aldrich) and heated to 37
C for 1 hr, 50 C for 2 hr, and 95
C for 15 min. Mutated regions were then amplified using primers for the SAG1 locus (P66 and P67), the MyoA locus (P68 and P69), the PLP1 locus (P70 and P71) or the CDPK1 locus (P72 and P73). Surveyor reactions were performed using a kit according to the manufacturer’s instructions (Integrated DNA Technologies). Plaque Formation 4 3 10 6 RH/Cas9/H2B-YFP parasites were transfected with 50 mg of pU6-DHFR encoding guides against ICAPs or controls. 2,000 transfected parasites were added to HFF monolayers in 6-well plates. 1.5 mM pyrimethamine was added one day post-transfection. Ten days post transfection, the monolayers were rinsed with PBS, fixed in 95% ethanol for 10 min and stained with crystal violet (2% crystal violet, 0.8% ammonium oxalate, 20% ethanol) for 5 min. 500 parasites per well were used to analyze the effect of CLAMP or RAB4 on plaque formation over the course of 8 days. Microneme Secretion Microneme secretion assays were performed as previously described ( Lourido et al., 2012 ). 2
3 10 7 DiCre or DiCre/CLAMP parasites were suspended in DMEM, then treated with 3% FBS with or without 1% ethanol for 10 min at 37
C, 5% CO 2 . Supernatants were collected by centrifugation 10 min at 400 g, 4
C. Proteins were separated by SDS-PAGE, and secreted MIC2 was quantified and normalized to MIC2 levels in total lysates measured by immunoblotting. Egress Assays 4 3 10
6 DiCre or DiCre/CLAMP parasites were treated with 50 nM rapamycin or vehicle upon infection of HFF monolayers in 96-well plates. Rapamycin was removed after two hours, and parasites were allowed to grow for 24–30 hr. Egress was induced with 1 mM A23187 (EMD Millipore) for 10 min. Knockdown parasites in the rapamycin-treated samples were identified by expression of the YFP reporter. The number of intact vacuoles before or after the induction of egress was quantified. Only YFP-expressing vacuoles were counted for the rapamycin-treated samples. Invasion Assays DiCre and DiCre/CLAMP parasites were suspended in invasion media and 5 3 10 6
containing HFF monolayers seeded on coverslips. Invasion was allowed to proceed at 37
C with 5% CO 2 for 20 min. HFF cells and parasites were fixed with 4% formaldehyde for 20 min on ice. Extracellular parasites were stained with Pacific Blue-conjugated anti- SAG1 prior to permeabilization, and all parasites were stained with Alexa-Fluor-594-conjugated anti-SAG1 after permeabilization with 0.25% Triton. The average number of host cells per field of view was obtained from coverslips prepared and processed in par- allel and stained with Hoechst. The number of invaded parasites per fields were manually counted and normalized to the number of e5 Cell 167, 1–13.e1–e7, September 22, 2016 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019
host cells in the same area. CLAMP-mNeonGreen parasites were prepared similarly and allowed to invade for 30 min before being fixed with 4% formaldehyde for 20 min on ice. Extracellular parasites were stained with Alexa-Fluor-594-conjugated anti-SAG1. Video Microscopy To capture egress, DiCre or DiCre/CLAMP parasites were prepared as described for the egress assays in glass-bottom 35 mm dishes (MatTek). Parasites were stimulated to egress with 1 mM A23187 or 500 mM zaprinast, and recorded at 2–5 frames per second for ten minutes, using an Eclipse Ti microscope (Nikon) with an enclosure heated to 37
C. The same setup was used to capture in- vasion, with the exception that freshly lysed parasites were added directly to monolayers under observation. P. falciparum Strain Generation and Analysis The P. falciparum NF54 attB
strain (kindly provided by David Fidock) was modified to generate a strain NF54 Cas9+T7 Polymerase , which contained Cas9 and T7 RNA polymerase expression cassettes integrated into the attB site. Linearized pPfCLAMP-cKD/TetR-DOZI was transfected into NF54 Cas9+T7 Polymerase . 50 mg of plasmid were used per 200 ml packed red blood cells (RBCs), adjusted to 50% hematocrit, and electroporated as previously described ( Ganesan et al., 2016 ). Transfected parasites were selected with a combi- nation of 2.5 mg/ml Blasticidin S and 2.5 nM WR99210 beginning 4 days after transfection. The PfCLAMP cKD strain was maintained in 0.5 mM anhydrotetracycline (aTc). Correct integration of the construct was confirmed by PCR and sequencing using primer pairs P156/P157 and P158/P159. To analyze P. falciparum growth upon CLAMP downregulation, parasites were synchronized to rings using 0.5 M alanine in 10 mM HEPES (pH 7.4), adjusted to 1% parasitemia, and seeded in triplicate wells of a 12-well plate at 2% hematocrit in 5 ml of media with or without aTc. The parental and PfCLAMP cKD strains were treated in parallel. Expansion was measured over six lytic cycles. Samples were collected after each cycle to measure parasitemia by incubating the cells with a 1:5000 dilution of SYBR Green I (Thermo Fisher) for 15 min at 37
C, prior to flow-cytometry on an Accuri C6 instrument (BD Biosciences). Following the analysis, all cultures were subcultured using the same dilution factor, as required to maintain the pre-invasion parasitemia of the parental lines at 1%, and avoid over-expansion of the cultures. After subculturing, the pre-invasion parasitemia was directly measured as indicated above. Parasite growth was expressed as percent parasitemia at the start and end of each lytic cycle. QUANTIFICATION AND STATISTICAL ANALYSIS Bioinformatic Analysis of the Screening Results Sequencing reads were aligned to the sgRNA library. The abundance of each sgRNA was calculated and normalized to the total num- ber of aligned reads. Guides that were not found were assigned a pseudo-count corresponding to 90% of the lowest value in that sample. Only guides whose abundance was above the 5 th percentile in the original plasmid preparation of the sgRNA library were taken into account for subsequent analyses. For FUDR selection experiments, the log 2 fold change between treated and untreated samples was calculated for each sgRNA, whereas negative selection experiments used the plasmid preparation for comparison. The ‘‘phenotype’’ score for each gene was calculated as the mean log 2 fold change for the top five scoring guides, which minimized the effect of stochastic losses and decreased the error between biological replicates. The mean phenotype score for each gene in four replicates of the screen is reported. Fitness-conferring genes were identified by comparison to 40 control genes known to be dispensable for parasite growth in fibroblast (see Table S1
). For a given gene, the four biological replicates were compared to the mean phenotype of the controls using a one-sided t test, and the log 2 fold changes for the sgRNAs against that gene were compared to the sgRNAs against the controls using a one-sided Mann-Whitney U test. The p values for each test were corrected using the Ben- jamini-Hochberg method. Genes were considered fitness-conferring if they met a significance threshold of 0.05 for both tests. 10-fold cross-validation was performed using the set of previously described essential and dispensable genes ( Table S1
). In each trial, the test sample was compared using the statistical tests described to the control genes in the training set. The cross-validation was per- formed 100 times to estimate the error rate. Gene-set enrichment analysis was performed as previously described ( Subramanian et al., 2005 ), using gene sets specifically curated for T. gondii ( Croken et al., 2014 ). Gene expression data (kindly provided by David S. Roos and Maria Alejandra Diaz-Miranda, available through ToxoDB) consisted of RNA samples collected at several time points following infection with T. gondii strain GT1 ta- chyzoites, and analyzed by RNaseq. The maximum log 2 (RPKM + 1) was used for the analysis and compared to the mean phenotype score for each gene. To compare the phenotype scores with the rate of evolution, syntenic orthologs that did not display copy-number variation were obtained from ToxoDB release 26 for T. gondii GT1, Neospora caninum Liverpool, and Hammondia hammondi HH34. d N
S ratios were determined as previously described ( Lorenzi et al., 2016 ). Briefly, average d N /d
values were calculated according to Goldman & Yang ( Goldman and Yang, 1994 ), following alignment of cDNA sequences based on their protein sequence by ClustalW ( Larkin et al., 2007 ). The distribution of phenotype scores in the top and bottom third of the d N /d S distribution were compared by a Kolmogorov-Smirnov test. Depth of conservation of T. gondii genes was estimated using ortholog groupings of 79 eukaryotic ge- nomes available through OrthoMCL DB release 5 ( Chen et al., 2006 ). Assignment to the different levels of conservation was performed by asking whether an ortholog was present in at least one of the genomes from a neighboring branch. For simplicity, genes that did not conform to a simple assignment were excluded from this analysis (Other) and specific losses in either hematozoa (piroplasmida and Plasmodium spp.) or cryptosporidia were allowed when assigning a gene to the apicomplexan category. Cell 167, 1–13.e1–e7, September 22, 2016 e6 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019 Statistical Testing The ratio of plaquing efficiency following mutation of each ICAP relative to SAG1 mutation was compared to a mean of 1 using one- tailed t tests, and the resulting p values were corrected using the Benjamini-Hochberg method. Based on the small sample size, mu- tants with a corrected p value lower than 0.1 were considered significant. ICAP mutant invasion efficiency was analyzed similarly us- ing PLP1 as the control. The effects of inducing CLAMP knockdown on invasion, egress, and MIC2 secretion were analyzed in Prism (GraphPad) using two-tailed t tests with a p value of 0.05 or lower indicating significance. Where appropriate, statistical parameters including the exact value of n, the definition of center, dispersion, and precision measures and statistical significance are reported in the figures and corresponding legends. DATA AND SOFTWARE AVAILABILITY Software
Guide selection and screen analysis were performed using custom software that will be provided upon request. Statistical analyses were performed in R ( http://www.R-project.org ) using built-in packages unless otherwise indicated. Data Resources All data from the CRISPR screens is available in Table S3 , and will be integrated into an upcoming release of ToxoDB ( http://ToxoDB. org
). e7 Cell 167, 1–13.e1–e7, September 22, 2016 CELL 9138 Please cite this article in press as: Sidik et al., A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes, Cell (2016), http://dx.doi.org/10.1016/j.cell.2016.08.019 Supplemental Figures Figure S1. RAB4 Is Not Essential for T. gondii Growth in Human Fibroblasts, Related to Figure 3 (A) Diagram of the RAB4 locus illustrating the knockout strategy and position of the primers used for validation. Specific primers were used to amplify the 5 0 (PCR1) and 3 0 (PCR2) junctions, and a segment of the coding sequence (PCR3). (B) PCR assay for integration of the pyrimethamine-resistance cassette into the RAB4 locus and deletion of the RAB4 coding sequence. (C) RT-PCR probing for expression of RAB4 or the control ACT1 on cDNA samples prepared with or without reverse transcriptase (RT). (D) Plaque assay demonstrating normal growth of the RAB4 knockout in human fibroblasts.
Figure S2. Correlation of Phenotype Scores to Selective Pressure, Related to Figure 3
(A) Distribution of published d N /d S values for 16 reference T. gondii genomes ( Lorenzi et al., 2016 ). The lowest and highest third of the dataset is highlighted in orange and blue, respectively. (B) Genes binned according to d N /d
show higher phenotype scores for genes under positive selection (blue), compared to those under negative selection (orange). Bars indicate the group median. The distributions were compared using a Kolmogorov-Smirnov test. Figure S3. Construction and Validation of the Cas9 Strain Expressing H2B-YFP, Related to Figure 5
(A) Diagram of the constructs used to generate the T. gondii strain expressing H2B-YFP and Cas9. The sequence of the decoy sgRNA is highlighted in the top construct, followed by the Cas9-binding sequence (orange). (B) Expression of H2B-YFP in the nucleus of the clonal parasites detected by live fluorescence video microscopy. (C) Representative micrographs showing intracellular parasites three days post transfection. Parasites were stained for SAG1 (red), and TgACT1 (ACT; green). Host-cell and parasite nuclei were stained with DAPI (blue). Scale bar, 10 mm. SAG1 retention in wild-type (wt) and Cas9-expressing parasites was measured following the different treatments. Figure S4. Alignment of CLAMP Orthologs, Related to Figure 6
CLAMP orthologs representing the entire breadth of the apicomplexan radiation were aligned using Clustal X. Bars indicate the topology of four representative sequences determined by CCTOP ( Dobson et al., 2015 ). The predicted transmembrane domains (TMs), extracellular loops (ECLs) and proline-rich domain (PRD) are indicated.
Figure S5. Extended Analysis of CLAMP in T. gondii and P. falciparum, Related to Figure 6
(A) Localization of CLAMP After Invasion. CLAMP-mNeonGreen parasites were fixed following invasion. Extracellular parasites were stained for the surface antigen SAG1 (red) prior to permeabilization. Arrowheads indicate puncta of mNeonGreen at the posterior of various parasites. Several sections have been magnified to highlight the mNeonGreen localization in invaded (i. and ii.) and non-invaded (iii. and iv.) parasites. (B and C) Generation and Testing of the PfCLAMP cKD. Diagram of the CLAMP locus for wild-type (wt) and conditional knockdown (cKD) parasites (B). Specific primer pairs (P156/P157 and P158/P159) were used to test for the integration sites (C) and fully sequence them. (D) Growth curves of the P. falciparum parental strain (left) or the cKD (right) ± aTc. Means ± SD for n = 3 technical replicates. This is an independent replicate of the experiment shown in Figure 6
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