MATERIALS AND METHODSParasites strains and growth conditions. The T. gondii strains used in this study were maintained by growth in human foreskin fibroblasts (HFF) cultured in Dulbecco’s modified Eagle medium (DMEM) supplemented with 10% fetal bovine serum (FBS), 10 µg/ml gentamicin, and 10 mM glutamine (Thermo, Fisher Scientific, Waltham, MA). Wild-type RH and GT1 and the parasite mutant lines RH Δku80 and RH Δku80Δrop18 were described previously (9). Transgenic parasites were obtained by electroporation of constructs into cells and selection with 3 µM pyrimethamine (Sigma-Aldrich, St. Louis, MO) or 10 µM FUDR (Sigma-Aldrich, St. Louis, MO).Plasmid construction. All the plasmids and primers used in this study are listed in Tables S1 and S2 in the supplemental material, and further details on plasmid construction can be found in the supplemental methods in the supplemental material. A map and sequence file for the plasmid expressing CAS9 and a single guide RNA (sgRNA) targeting the UPRT gene in T. gondii can be found in Fig. S1 in the supplemental material. The UPRT-targeting CRISPR plasmid (sgUPRT) was constructed in two steps: first, the U6 promoter from T. gondii driving expression of the UPRT-specific sgRNA was cloned into the backbone plasmid pSAG1-Ble-SAG1, as described in the supplemental methods. This plasmid was further modified to express the CAS9-NLS-GFP cassette under the SAG1 promoter. All other CRISPR/CAS9 plasmids were obtained through Q5 mutagenesis (New England Biolabs, Ipswich, MA) of this plasmid to change the UPRT targeting gRNA to other specific sgRNAs using primers listed in Table S2. To generate a plasmid for inserting DHFR into the UPRT gene, upstream (750-bp) and downstream (900-bp) regions directly adjacent to the sgUPRT target sequence were used to flank DHFR (24). To generate a construct for deleting the entire coding sequence of UPRT, flanking regions 5′ (~1 kb) and 3′ (~1 kb) outside the coding region were used to surround the DHFR cassette. To test the efficiency of short homology regions for site-specific integration, the DHFR cassette was amplified using 20-bp flanking regions from the UPRT locus (surrounding the sgUPRT site) (Table S2) or the DHFR cassette was used as an amplicon generated without homology regions. To generate a construct for disrupting the ROP18 locus, 5′ (820-bp) and 3′ (850-bp) regions flanking the sgROP18 target region were used to surround the DHFR cassette (Table S1). The ROP18 gene was complemented using a previously described plasmid for targeting the type I ROP18 allele to the UPRT locus (23).Site-specific mutagenesis of the UPRT gene. To test the efficiency of targeted mutation at the UPRT locus, RH strain parasites (107 cells) were transfected with indicated CRISPR plasmids (Table S1) by electroporation. Transfected cells were allowed to grow for 48 h in the absence of drug selection and egress naturally. To estimate viability, 200 parasites were subject to plaque assays in HFF monolayers grown for 7 days without FUDR. In parallel, aliquots containing 6,000 or 600,000 parasites were tested for growth in HFF cells in 10 µM FUDR. Monolayers were stained with 0.1% crystal violet, and the number of plaques produced under each condition was recorded. To confirm the mutations in the UPRT gene, we obtained FUDR-resistant clones from transfected parasites by subcloning after 5 to 6 days of selection. DNA from clones was PCR amplified to obtain the 1.2-kb region flanking the target region (i.e., PCR3) and sequenced using the Sanger dideoxy method (Genewiz Inc., South Plainfield, NJ) (Table S2).Targeted disruption and deletion of the UPRT gene. To study the efficiency of CRISPR/CAS9-mediated gene disruption by integration of a selectable marker, sgUPRT or sgROP18 targeting CRISPR/CAS9 plasmids (see Table S1 in the supplemental material) was combined with various amplicons containing DHFR expression cassettes for transfection. Amplicons were PCR amplified using primers listed in Table S2 in the supplemental material, purified by agarose gel electrophoresis, and extracted using the Qiaquick gel extraction kit (Qiagen Inc., Valencia, CA). Recovered DNAs were quantified using a Nanodrop 2000 instrument (Nanodrop Instruments, Wilmington, DE). Mixtures of CRISPR/CAS9 plasmids with the purified DHFR amplicons (5:1 mass ratio) were cotransfected into RH parasites by electroporation. Pyrimethamine-resistant parasites were obtained by selection with 3 µM pyrimethamine, and resistant cells were then used for determination of viability and FUDR resistance by plaque assay as described above. In parallel, single-cell clones were obtained by limiting dilution, and lysates were generated as described previously (4). The specificity of DHFR integration and loss of genes by deletion were tested by PCR using primers listed in Table S2. PCRs were performed using Taq DNA polymerase (New England Biolabs, Ipswich, MA) in a 25-µl reaction mixture containing 2 µl of lysate as the template according to the manufacturer’s directions. In general, reactions were performed for 35 cycles with denaturation at 95°C for 30 s, annealing at 57°C for 45 s, and extension for 90 s at 68°C. Products were analyzed by electrophoresis in agarose gels with ethidium bromide. In some cases, PCR3 was conducted using Q5 DNA polymerase (New England Biolabs, Ipswich, MA) to efficiently amplify the inserted DHFR fragment, giving a band of 4.4 kb, which was inefficiently amplified using Taq polymerase under the conditions described above. When PCR3 was performed with Q5 polymerase, 25-µl reaction mixtures containing 2 µl template were amplified for 35 cycles with denaturation at 95°C for 30 s, annealing at 60°C for 30 s, and extension for 3 min at 68°C.Generation of ROP18 knockout and complemented lines. To disrupt ROP18 in GT1, we cotransfected the sgROP18 CRISPR plasmid along with an amplicon containing ROP18 homology regions surrounding a pyrimethamine-resistant DHFR cassette. Selection by growth for 7 to 8 days in pyrimethamine (3 µM) was used to get stably resistant clones that were subsequently screened by PCR for correct integration of DHFR into the ROP18 locus (see Table S2 in the supplemental material). PCR-positive clones were further analyzed by Western blotting to confirm the loss of ROP18 expression. To complement the ROP18-deficient parasites, we transfected the GT1 rop18::DHFR* parasites with the sgUPRT CRISPR plasmid (mass ratio, 1:5 CRISPR/CAS9 to ROP18 complement) to target integration to the UPRT locus, along with a previously described ROP18-complementing plasmid (Ty-tagged ROP18 driven by the IMC1 promoter) (23). Parasites were selected with 10 µM FUDR for 5 to 6 days, and single clones were screened by immunofluorescence for Ty expression using monoclonal antibody (MAb) BB2 (37). Positive clones were further analyzed by PCR for correct integration at the UPRT locus (see Table S2) and by Western blotting for ROP18 expression.Immunofluorescence microscopy. To estimate the frequency of CRISPR/CAS9-mediated gene disruptions, RH parasites (~107) were transfected with 7.5 µg of indicated CRISPR plasmids (Table S1) and analyzed by immunofluorescence staining for GFP 24 h posttransfection. HFF monolayers were fixed with 4% formaldehyde, permeabilized with 0.1% Triton X-100, and incubated with MAb 3E6 (Life Technologies, Carlsbad, CA) to detect GFP. Parasites were detected with rabbit anti-TgALD (38). Alexa Fluor 594-conjugated goat anti-rabbit IgG and Alexa Fluor 488-conjugated goat anti-mouse IgG secondary antibodies (Life Technologies, Carlsbad, CA) were used to detect primary antibodies. Images were acquired using a Zeiss Axioskop 2 MOT Plus microscope equipped with a 63×, NA (numerical aperture) 1.6 oil immersion lens and an AxioCam MRm monochrome camera (Carl Zeiss, Inc., Thornwood, NJ).Western blotting. To assess ROP18 expression levels, parasite lysates were subjected to Western blot analysis as previously described (39). Rabbit anti-ROP18 (9) was used to stain ROP18, and mouse anti-GRA7 (40) was used as a loading control. IRDye800CW-conjugated goat anti-mouse IgG and IRDye680RD-conjugated goat anti-rabbit IgG were used as secondary antibodies, and the blot was scanned using the Li-Cor Odyssey imaging system (Li-Cor Biosciences, Lincoln, NE).Virulence in laboratory mice. Mice were obtained from The Jackson Laboratory (Bar Harbor, ME) and maintained in an Association for Assessment and Accreditation of Laboratory Animal Care International-approved facility at Washington University School of Medicine. All animal experiments were conducted according to the U.S. Public Health Service Policy on Humane Care and Use of Laboratory Animals, and protocols were approved by the Institutional Care Committee at the School of Medicine, Washington University in St. Louis. To determine the virulence in mice, parasites were injected intraperitoneally (i.p.) into 8- to 10-week-old female CD-1 mice (5 mice/condition). The survival of mice was monitored for 30 days, and blood was drawn from surviving mice at day 30 and tested by enzyme-linked immunosorbent assay (ELISA) to confirm infection. Mice that were seronegative by ELISA were not included in the analysis. Cumulative mortality was plotted as a Kaplan-Meier survival plot and analyzed in Prism (GraphPad Software, Inc., La Jolla, CA).
Toxoplasma gondiihas become a model for studying the phylum Apicomplexa, in part due to the availability of excellent genetic tools. Although reverse genetic tools are available in a few widely utilized laboratory strains, they rely on special genetic backgrounds that are not easily implemented in natural isolates. Recent progress in modifying CRISPR (clustered regularly interspaced short palindromic repeats), a system of DNA recognition used as a defense mechanism in bacteria and archaea, has led to extremely efficient gene disruption in a variety of organisms. Here we utilized a CRISPR/CAS9-based system with single guide RNAs to disrupt genes inT. gondii. CRISPR/CAS9 provided an extremely efficient system for targeted gene disruption and for site-specific insertion of selectable markers through homologous recombination. CRISPR/CAS9 also facilitated site-specific insertion in the absence of homology, thus increasing the utility of this approach over existing technology. We then tested whether CRISPR/CAS9 would enable efficient transformation of a natural isolate. Using CRISPR/CAS9, we were able to rapidly generate bothrop18knockouts and complemented lines in the type I GT1 strain, which has been used for forward genetic crosses but which remains refractory to reverse genetic approaches. Assessment of their phenotypesin vivorevealed that ROP18 contributed a greater proportion to acute pathogenesis in GT1 than in the laboratory type I RH strain. Thus, CRISPR/CAS9 extends reverse genetic techniques to diverse isolates ofT. gondii, allowing exploration of a much wider spectrum of biological diversity.