A genome-wide crispr screen in Toxoplasma Identifies Essential Apicomplexan Genes
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Article A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes Graphical Abstract Highlights d CRISPR enables the first genome-wide loss-of-function screen in T. gondii d The screen measures each gene’s impact during infection of human fibroblasts d Functional assays validate the essentiality of formerly uncharacterized proteins d An invasion factor found in all apicomplexans is essential in malarial parasites Authors
Saima M. Sidik, Diego Huet, Suresh M. Ganesan, ..., Vern B. Carruthers, Jacquin C. Niles, Sebastian Lourido Correspondence lourido@wi.mit.edu In Brief
The first genome-wide genetic screen of an apicomplexan parasite identifies an invasion factor essential for the fitness of all parasites in this phylum, including those causing malaria and toxoplasmosis. Sidik et al., 2016, Cell 167, 1–13 September 22, 2016 ª 2016 Elsevier Inc. http://dx.doi.org/10.1016/j.cell.2016.08.019
Article A Genome-wide CRISPR Screen in Toxoplasma Identifies Essential Apicomplexan Genes Saima M. Sidik, 1,7 Diego Huet, 1,7 Suresh M. Ganesan, 2 My-Hang Huynh, 3 Tim Wang, 1,4,5 Armiyaw S. Nasamu, 2 Prathapan Thiru, 1 Jeroen P.J. Saeij, 6 Vern B. Carruthers, 3 Jacquin C. Niles, 2 and Sebastian Lourido 1,8, *
Whitehead Institute for Biomedical Research, Cambridge, MA 02142, USA 2 Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA 3 Department of Microbiology and Immunology, University of Michigan School of Medicine, Ann Arbor, MI 48109, USA 4 Department of Biology, Massachusetts Institute of Technology, Cambridge, MA 02139 USA 5 Broad Institute of MIT and Harvard, Cambridge, MA 02142, USA 6 Department of Pathology, Microbiology and Immunology, University of California, Davis, Davis, CA 95616, USA 7 Co-first author 8 Lead Contact *Correspondence: lourido@wi.mit.edu http://dx.doi.org/10.1016/j.cell.2016.08.019 SUMMARY
Apicomplexan parasites are leading causes of human and livestock diseases such as malaria and toxoplas- mosis, yet most of their genes remain uncharacter- ized. Here, we present the first genome-wide genetic screen of an apicomplexan. We adapted CRISPR/ Cas9 to assess the contribution of each gene from the parasite Toxoplasma gondii during infection of human fibroblasts. Our analysis defines $200 previ- ously
uncharacterized, fitness-conferring genes unique to the phylum, from which 16 were investi- gated, revealing essential functions during infection of human cells. Secondary screens identify as an in- vasion factor the claudin-like apicomplexan micro- neme protein (CLAMP), which resembles mammalian tight-junction proteins and localizes to secretory or- ganelles, making it critical to the initiation of infection. CLAMP is present throughout sequenced apicom- plexan genomes and is essential during the asexual stages of the malaria parasite Plasmodium falcipa- rum. These results provide broad-based functional information on T. gondii genes and will facilitate future approaches to expand the horizon of antipara- sitic interventions. INTRODUCTION Apicomplexans comprise a phylum of over 5,000 obligate para- sites whose hosts span the animal kingdom ( Levine, 1988 ). Several species are leading causes of infant mortality, such as Plasmodium and Cryptosporidium spp., which cause malaria and severe diarrhea, respectively ( Checkley et al., 2015; World Health Organization, 2014 ). Toxoplasma gondii, predicted to establish lifelong infections in a quarter of the world’s population, can cause life-threatening disease in immune-compromised in- dividuals or when contracted congenitally ( Pappas et al., 2009
). Despite their importance to global health, apicomplexans remain enigmatic. Only a handful of species have been studied, and fewer than half of their genes have been functionally anno- tated. The ease with which T. gondii can be cultured, along with the genetic tractability that comes with its balanced nucle- otide composition and high transfection rates, presents compel- ling arguments for using this parasite as a model apicomplexan. Scalable methods to assess gene function in T. gondii could therefore greatly extend our understanding of apicomplexan biology.
Genetic crosses have long been used to identify loci respon- sible for phenotypes ranging from drug resistance in Plasmo- dium falciparum ( Wellems et al., 1991 ) to virulence in T. gondii ( Saeij et al., 2006; Taylor et al., 2006 ). However, completing the sexual cycles of T. gondii or Plasmodium spp. in cats or mosqui- toes is challenging, and the traits examined must vary within the species. Spontaneous mutations, or those induced chemically or by transposition, can sample a wider range of phenotypes ( Crabb et al., 2011; Farrell et al., 2014; Flannery et al., 2013 ), but the population size required to achieve saturation is imprac- tical, and causal mutations are often difficult to identify. Gene deletion collections, such as those available for fungi ( Winzeler et al., 1999 ), can aid functional analysis of eukaryotic genomes. With this aim, large-scale efforts have generated col- lections of knockout vectors for P. falciparum ( Maier et al., 2008 ) and Plasmodium berghei ( Gomes et al., 2015 ), which
have led to the functional annotation of dozens of genes in both species. However, similar approaches have not been adapted to T. gondii, despite the advantage of both high trans- fection rates and a continuous culture system. The recent adaptation of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 has further enhanced the genetic trac- tability of T. gondii ( Shen et al., 2014; Sidik et al., 2014 ). This technology has the advantage of being easily reprogrammable by changing the 20 bp of homology between the single guide RNA (sgRNA, or guide) and the genomic target (reviewed in Sander and Joung, 2014 ). The endogenously high rates of non-homologous end-joining (NHEJ) in T. gondii make it well suited to CRISPR-mediated gene disruption by efficiently creating frame-shift mutations and insertions at the cleavage site ( Sidik et al., 2014 ). Cell 167, 1–13, September 22, 2016 ª 2016 Elsevier Inc. 1 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
Several studies have developed CRISPR/Cas9-based genome- wide genetic screens for mammalian cells ( Koike-Yusa et al., 2014; Shalem et al., 2014; Wang et al., 2014 ). These screens use lentiviral libraries of sgRNAs to generate pools of mutants that can be exposed to selective pressures. The integrated sgRNAs can be used as barcodes to measure the contribution of targeted genes to cell fitness. Despite the lack of viral transduction, we adapted CRISPR/Cas9 for pooled screening in T. gondii. We pre- sent the first genome-wide genetic screen performed in any api- complexan. We demonstrate the power of this approach using both positive and negative selection strategies. This approach provides the first complete survey of contributions to parasite fitness, cataloguing the $40% of genes needed during infection of human fibroblasts. Based on this analysis, we were able to pinpoint previously uncharacterized conserved apicomplexan proteins necessary for the T. gondii lytic cycle. We demonstrate that one of these proteins acts as an essential invasion factor and is also required by the malaria parasite P. falciparum to com- plete its asexual replication cycle. This protein is conserved throughout the phylum, providing an important molecular link to the invasion process of distantly related apicomplexans. Our anal- ysis demonstrates the potential of genetic screens in T. gondii to uncover conserved biological processes and provides a transfor- mative tool for parasitology. RESULTS
Constitutive Cas9 Expression Maximizes Gene Disruption in T. gondii Highly efficient gene disruption and stable integration of the sgRNA are necessary to develop large-scale CRISPR screens. Transient expression of SpCas9 and an sgRNA in T. gondii can disrupt a targeted gene in $20% of parasites ( Sidik et al., 2014 ). We reasoned that constitutive Cas9 expression, prior to introducing the sgRNA, might increase the likelihood of gene disruption. We transfected parasites with a Cas9-expression plasmid carrying a chloramphenicol acetyltransferase (CAT) selectable marker (pCas9/CAT; Figure 1 A). However, repeated attempts failed to isolate Cas9-expressing parasites, suggesting that Cas9 expression is detrimental to T. gondii, as has been suggested for other microorganisms ( Jiang et al., 2014; Peng et al., 2014 ). We hypothesized that expression of a ‘‘decoy’’ sgRNA (pCas9/decoy; Figure 1
A) could prevent toxicity that might arise from unintended Cas9 activity directed by Figure 1. Expression of Cas9 Maximizes Gene Disruption in T. gondii (A) Constructs used to constitutively express Cas9 in T. gondii. The sequence of the decoy sgRNA is highlighted (blue), followed by the Cas9-binding scaffold (orange). (B) Immunoblot showing expression of FLAG-tag- ged Cas9 (green) in the strain constitutively ex- pressing the transgene. ACT1 serves as a loading control (red). (C) Cas9 localizes to the parasite nucleus. ACT1 provides a counterstain and DAPI stains for host-cell and parasite nuclei. Scale bar, 10 mm.
(D) Chromatogram showing the presence of the decoy in the Cas9-expressing strain. (E) The sgRNA expression construct with the py- rimethamine-resistance selectable marker (DHFR). The targeting sequence of the SAG1 sgRNA is highlighted. The timeline indicates the period of pyrimethamine (pyr) selection (if applied), passaging to new host cells (P1), and the immu- nofluorescence assay (IFA). (F) Representative micrographs showing intra- cellular parasites 3 days post-transfection. Parasites were stained for SAG1 (green) and ACT1 (red).
Host-cell and
parasite nuclei
were stained with DAPI (blue). Scale bar, 60 mm. The efficiency of SAG1 disruption in wild-type and Cas9-expressing parasites was measured following different treatments. Mean ± SD for n = 2 independent experi- ments; **p < 0.005. wt, wild-type. n.d., not detected. 2 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 endogenous RNAs. For this purpose, we used an sgRNA that ap- peared non-functional against the 3 0 UTR of NHE1. Co-transfec- tion of pCas9/CAT and pCas9/decoy readily yielded Cas9-ex- pressing parasites, confirmed by immunoblotting ( Figure 1 B) and immunofluorescence ( Figure 1 C). As predicted, the Cas9- expressing strain retained the decoy locus ( Figure 1
D), reinforc- ing its requirement for constitutive Cas9 expression. We assessed the efficiency of gene disruption in the Cas9-ex- pressing strain by expressing an sgRNA against the surface antigen SAG1. Pyrimethamine treatment of the population selected for stable integration of the sgRNA expression vector (pU6-DHFR), which carries the resistant allele of dihydro- folate reductase (DHFR; Figure 1 E). SAG1 provides a reliable measure of gene disruption, because it is dispensable yet stably maintained in cultured parasites ( Kim and Boothroyd, 1995 ). 3 days after transfection with the sgRNA construct, 70% of Cas9-expressing parasites had lost SAG1 expression. Pyrimeth- amine selection further improved SAG1 disruption to 97% over the same time period ( Figure 1
F). The high efficiency of CRISPR-mediated gene disruption in Cas9-expressing para- sites provided the platform for large-scale genetic screens in
A Genome-scale Genetic Screen Identifies Genes Involved in Drug Sensitivity We designed a library of sgRNAs containing ten guides against each of the 8,158 predicted T. gondii protein-coding genes us- ing previously described criteria ( Wang et al., 2014 ). The library was cloned into the sgRNA expression vector ( Figure 1
E). 40% of the parasites that survived transfection integrated the vector into their genomes (data not shown). We could therefore mea- sure the relative abundance of each integrated sgRNA by next-generation sequencing. Since the frequency of a given sgRNA corresponds to the relative abundance of parasites car- rying the targeted disruption, the change in relative abundance from the composition of the plasmid library before transfection indicates the enrichment or depletion of a given mutant. We defined the average log 2 fold change in abundance for sgRNAs targeting a given gene as the ‘‘phenotype’’ score for that gene ( Figure 2 A). To determine whether we could maintain diversity over time, we transfected the library into both wild-type and Cas9-expressing parasites and sampled the populations after each of three lytic cycles ( Figure 2 B, left). The representation of guides against all genes remained stable over the course of the experiment in the absence of Cas9. In contrast, sgRNAs against specific genes were lost from the Cas9-expressing population ( Figure 2 C), indicating that a diverse set of mutants had been generated. To investigate the compatibility of our screen with positive-se- lection strategies, we treated pools of mutants with 5-fluoro- deoxyuridine (FUDR), which is toxic to parasites through its incorporation into pyrimidine pools. Three lytic cycles after trans- fection with the library, we split the Cas9-expressing parasites into cultures with or without FUDR ( Figure 2
B, right). As ex- pected, FUDR-treated cultures recovered slowly, and untreated cultures were passaged two or three times over the same period. Measuring the sgRNAs in the two populations revealed that FUDR strongly selected against uracil phosphoribosyltransfer- ase (UPRT) activity, observed as an increased abundance of sgRNAs against UPRT and the highly reproducible phenotype score for the gene ( Figures 2 D and 2E). Since loss of UPRT—a component of the pyrimidine salvage pathway—is known to confer FUDR resistance ( Donald and Roos, 1995 ), this experi- ment demonstrates the power of this approach to rapidly and efficiently identify positively selected mutants from a T. gondii population. A Genome-scale Genetic Screen Identifies Fitness-Conferring Genes in T. gondii Loss of sgRNAs from a population of mutants can serve to iden- tify genes that contribute to cellular fitness ( Wang et al., 2015 ). In the context of our T. gondii screen, the changes in sgRNA repre- sentation observed after the third lytic cycle provided a conve- nient measure of a gene’s contribution to fitness. This time point resembled the gene rankings of later cycles ( Figure 2
F) while minimizing the chance of stochastic guide loss. We calculated the mean phenotype score for each parasite gene from four biological replicates of the screen. Genes that contribute to para- site fitness, represented by negative scores, were distributed throughout the genome and did not segregate by gene length or position on the chromosome ( Figure 3
A). Gene set enrichment analysis (GSEA) ( Croken et al., 2014; Subramanian et al., 2005 ) showed that genes predicted to be essential, like those encoding ribosomal and proteasomal constituents, were enriched in low phenotype scores ( Figure 3 B). Genes that encode components of the apicoplast—a plastid common to most apicomplex- ans—showed a similar enrichment, in accordance with the essential metabolic functions performed by this organelle ( Seeber and Soldati-Favre, 2010 ). In contrast, specialized secre- tory organelles like the micronemes, dense granules, and rhop- tries had fewer genes with low phenotype scores, possibly re- flecting functional redundancy or dispensability in cell culture ( Figure 3
C). We analyzed the screen results for 81 genes previously re- ported to be either dispensable or essential for T. gondii growth in human fibroblasts ( Table S1 ). The two groups of genes were clearly segregated on the basis of their phenotype scores (p = 6.7
3 10 À16
), with lower scores for the essential genes ( Fig-
ure 3 D). The most prominent outlier was RAB4, which appeared to be essential based on overexpression of a dominant-negative allele (
Kremer et al., 2013 ). However, we readily obtained RAB4 knockouts that grew normally ( Figure S1 ), demonstrating its dispensability in cell culture. We therefore excluded RAB4 and, for consistency, other genes classified by overexpression exper- iments from subsequent analyses. To predict which genes might contribute to parasite fitness, we compared the phenotype score and distribution of sgRNAs for each gene to the values of 40 known dispensable genes. Using 10-fold cross validation on the set of control genes, we estimate this method can classify genes with >95% accuracy. Based on these results, we expect $40% of T. gondii genes significantly contribute to parasite fitness under the conditions tested. To further classify the fitness-conferring genes, we compared our predictions to other measures of gene function. Genes that are not expressed during the examined developmental stage are more likely to appear dispensable. Accordingly, only 6.9% Cell 167, 1–13, September 22, 2016 3 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 of the fitness-conferring genes were found in the lowest quartile of expression, in contrast to 38.6% of genes predicted to be dispensable ( Figure 3
E). Genes under purifying selection are also more likely to be essential ( Jordan et al., 2002 ). Low ratios of non-synonymous to synonymous mutation rates (d N /d S ) are
consistent with purifying selection. Comparison of syntenic genes between related species or other T. gondii strains re- vealed the expected enrichment for low phenotype scores among genes with low d N /d
values ( Figures 3 F and S2
extension of the same principle, genes found in a greater propor- tion of eukaryotic genomes are more likely to be essential. To test this prediction, we assessed the depth of conservation of
nomes available through OrthoMCL DB ( Chen et al., 2006 ). The
distribution of phenotype scores within each category followed the predicted trend, which correlated depth of conservation with contribution to fitness and functional annotation ( Figure 3
G). The strong agreement of our results with published observations and expected trends allows us to confidently predict which genes will contribute to parasite fitness. Functional Characterization of Fitness-Conferring Genes Conserved in Apicomplexans We focused our efforts on the $200 fitness-conferring genes that lacked functional annotation and were only present in apicom- plexans, which we called indispensable conserved apicom- plexan proteins (ICAPs). We examined the subcellular localiza- tion of 28 ICAPs using CRISPR-mediated endogenous tagging to introduce a C-terminal Ty epitope into each targeted gene ( Figure 4 A). 3 days post-transfection, $60% of the populations Figure 2. Using Pooled Screens to Identify Genes Responsible for Drug Sensitivity (A) Schematic depiction of the pooled CRISPR screen. Cas9-expressing parasites are transfected with the sgRNA library and grown in human foreskin fibroblasts (HFFs). At various time points, sgRNAs are amplified and enumerated by sequencing to determine relative abundance and phenotype scores for individual genes. Yüklə 8 Mb. Dostları ilə paylaş:
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