Here, we describe a protocol to introduce a gene knockout into the extracellular amastigote of Trypanosoma cruzi, using the CRISPR/Cas9 system. The growth phenotype can be followed up either by cell counting of axenic amastigote culture or by proliferation of intracellular amastigotes after host cell invasion.
Trypanosoma cruzi is a pathogenic protozoan parasite that causes Chagas’ disease mainly in Latin America. In order to identify a novel drug target against T. cruzi, it is important to validate the essentiality of the target gene in the mammalian stage of the parasite, the amastigote. Amastigotes of T. cruzi replicate inside the host cell; thus, it is difficult to conduct a knockout experiment without going through other developmental stages. Recently, our group reported a growth condition in which the amastigote can replicate axenically for up to 10 days without losing its amastigote-like properties. By using this temporal axenic amastigote culture, we successfully introduced gRNAs directly into the Cas9-expressing amastigote to cause gene knockouts and analyzed their phenotypes exclusively in the amastigote stage. In this report, we describe a detailed protocol to produce in vitro derived extracellular amastigotes, and to utilize the axenic culture in a CRISPR/Cas9-mediated knockout experiment. The growth phenotype of knockout amastigotes can be evaluated either by cell counts of the axenic culture, or by replication of intracellular amastigote after host cell invasion. This method bypasses the parasite stage differentiation normally involved in producing a transgenic or a knockout amastigote. Utilization of the temporal axenic amastigote culture has the potential to expand the experimental freedom of stage-specific studies in T. cruzi.
Trypanosoma cruzi is the causative agent of Chagas’ disease, which is prevalent mainly in Latin America1. T. cruzi has distinctive life cycle stages as it travels between an insect vector and a mammalian host2. T. cruzi replicates as an epimastigote in the midgut of a blood-sucking triatomine bug and differentiates into an infectious metacyclic trypomastigote in its hindgut before being deposited on a human or animal host. Once the trypomastigote gets into the host body through the bite site or through a mucous membrane, the parasite invades a host cell and transforms into a flagella-less round form called an amastigote. The amastigote replicates within the host cell and eventually differentiates into trypomastigote, which bursts out of the host cell and enters the blood stream to infect another host cell.
Since currently available chemotherapeutic agents, benznidazole and nifurtimox, cause adverse side effects and are ineffective in the chronic phase of the disease3, it is of a great interest to identify novel drug targets against T. cruzi. In recent years, the CRISPR/Cas9 system has become a powerful tool to effectively perform gene knockout in T. cruzi, either by transfection of separate or single plasmid(s) containing gRNA and Cas94, by stable expression of Cas9 and subsequent introduction of gRNA5,6,7 or transcription template of gRNA8, or by electroporation of the pre-formed gRNA/Cas9 RNP complex7,9. This technological advancement is highly anticipated to accelerate the drug target research in Chagas’ disease.
To proceed with the drug development, it is crucial to validate the essentiality of the target gene or efficacy of drug candidate compounds in the amastigote of T. cruzi, as it is the replication stage of the parasite in the mammalian host. However, this is a challenging task, because amastigotes cannot be directly manipulated due to the presence of an obstructive host cell. In Leishmania, a closely related protozoan parasite to T. cruzi, an axenic amastigote culturing method was developed and has been utilized in drug screening assays10,11,12,13. Although there are some discrepancies in susceptibility to compounds between axenic amastigotes and intracellular amastigotes14, the ability to maintain the axenic culture nonetheless provides valuable experimental tools to study the basic biology of the clinically relevant stage of Leishmania15,16. In the case of T. cruzi, literatures regarding the presence of naturally occurring extracellular amastigotes (EA)17 and in vitro production of EA17,18,19 date back to decades ago. In addition, EA is known to have an infectious capability20, albeit less than that of trypomastigote, and the mechanism of amastigote host invasion has been elucidated in recent years (reviewed by Bonfim-Melo et al.21). However, unlike Leishmania, EA had not been utilized as an experimental tool in T. cruzi, primarily because EA had been regarded as an obligate intracellular parasite, and thus had not been considered as “replicative form” in a practical sense.
Recently, our group proposed to utilize EA of T. cruzi as a temporal axenic culture22. Amastigotes of T. cruzi Tulahuen strain can replicate free of host cells in LIT medium at 37 °C for up to 10 days without major deterioration or loss of amastigote-like properties. During the host-free growth period, EA was successfully utilized for exogenous gene expression by conventional electroporation, drug titration assay with trypanocidal compounds, and CRISPR/Cas9-mediated knockout followed by growth phenotype monitoring. In this report, we describe the detailed protocol to produce in vitro derived EA and to utilize the axenic amastigote in knockout experiments.
NOTE: An overview of the entire experimental flow is depicted in Figure 1.
Figure 1: Overview of the knockout experiment using EA. Tissue culture-derived trypomastigotes are harvested and differentiated into EA. gRNA is transfected into Cas9-expressing amastigotes by electroporation, and growth phenotype of the knockout amastigote is evaluated either by axenic replication or by intracellular replication after host cell invasion. Please click here to view a larger version of this figure.
1. Parasite Culture Preparations
2. Differentiation of Trypomastigotes into EA
3. Electroporation of EA
4. Monitoring the Growth of Knockout Cells as Axenic Amastigotes
5. Monitoring the Growth of Knockout Cells as Intracellular Amastigotes
Isolation of trypomastigotes by the swim-out procedure
To harvest fresh trypomastigotes from contaminating old EAs by swim-out procedure, cell pellets need to be incubated at least for 1 h. Incubating the pellets for more than 2 h does not significantly increase the number of trypomastigotes swimming in the solution (Figure 2B). In this particular experiment, the percentage of trypomastigote in the initial mixture was 38%, and the percentage after the swim-out was above 98% at any given time points. From two T-75 flasks of confluent co-culture, we routinely obtain 3-4 x 107 cells of pure trypomastigote by this swim-out protocol.
Growth monitoring of knockout amastigote as axenic culture
Unlike other developmental stages of T. cruzi, flagellar-less amastigotes are practically static. Thus, staining with PI helps to distinguish live amastigotes from dead ones. PI is impermeable to the intact cell membrane but easily penetrates dead cells (Figure 3A). Amastigotes transfected with gRNA against essential gene, TcCGM1 (TcCLB.511807.80), showed a significant growth defect comparing to the control group that received gRNA not homologous to the T. cruzi genome (Figure 3B).
Growth monitoring of knockout parasites as intracellular amastigotes
The essentiality of the target gene in the amastigote stage can also be demonstrated by evaluation of the growth phenotype of knockout T. cruzi as intracellular amastigote (Figure 4). The fraction of host nuclei that are associated with smaller T. cruzi nuclei was significantly lower in TcCGM1-KO group comparing to the control group.
Knockout of a stage-specific gene causes phenotypic difference in amastigote and trypomastigote stages
Paraflagellar rod component PAR1 is highly expressed in trypomastigote stage, but is downregulated in the amastigote stage of T. cruzi22,31. Indeed, transfection of gRNA against TcPAR1 (TcCLB.506755.20) did not significantly affect the growth of EA after 4 days of axenic culturing (Figure 5B). gRNA transfection followed by host cell invasion also showed that TcPAR1-KO does not inhibit the growth of intracellular amastigotes, in terms of the fraction of infected host cells at 4 days post infection (Figure 5C). The number of parasites within individual host cell did not seem to be affected by the knockout, either (Supplemental Figure 2). These results indicate that TcPAR1 is not essential in the amastigote stage of T. cruzi.
However, the number of trypomastigotes emerged out of host cells at 5 days post infection was significantly lower in TcPAR1-KO co-culture, comparing to the control co-culture (Figure 5D). Also, differentiated trypomastigotes within host 3T3 cell before egression appeared to be quite active in control group (Supplemental Movie 1) but seemed sluggish in TcPAR1-KO group (Supplemental Movies 2). These results suggest that TcPAR1 plays an important role in the trypomastigote stage of T. cruzi, presumably by providing motility to help the egression, despite its non-essentiality in amastigote stage.
Figure 2: Isolation of trypomastigote by swim-out procedure. (A) Schematic representation of the protocol. Red = presence of trypomastigote. (B) The number of trypomastigotes that have swum out of the pellet at indicated time points. Initial pellets contained total of 3 x 107 parasites, 38% of which were trypomastigotes and the rest of which were amastigotes. Experiments were performed in triplicate and mean values (±SD) are plotted. The purity of trypomastigotes in solution was above 98% at any given time points. (C) Microscopy images of parasites before swim-out procedure (left), trypomastigotes that have swum out of a pellet (middle), and parasites remaining in a pellet (right) after 1 h of incubation. Scale bars = 10 µm. Please click here to view a larger version of this figure.
Figure 3: Growth monitoring of axenic amastigote. (A) Propidium iodide (PI) exclusion assay. Microscopy images of EA on a hemocytometer in bright field (left), fluorescence channel (middle), and overlaid image (right). Yellow arrows indicate PI-stained dead cells. Scale bars = 20 µm. (B) Cell count of Cas9-expressing amastigotes after transfection with gRNAs against TcCGM1 (open circle) and control gRNA with no homology to T. cruzi genome (closed circle). Electroporations were performed in triplicates, and mean values (±SD) are plotted. (**p < 0.01 by Welch’s t-test). This figure has been modified from Takagi et al.22 Please click here to view a larger version of this figure.
Figure 4: Growth of knockout amastigotes after host invasion. (A) Nuclear staining of the host-parasite co-culture after intracellular replication of amastigotes transfected with gRNAs against TcCGM1 and control gRNA. gRNA-transfected EAs were applied onto 3T3 cells 1 day after electroporation and allowed to infect host cells for 2 days. Amastigotes remaining outside of the host cells were washed away, and the host-parasite co-culture was incubated for an additional 2 days. Scale bars = 20 µm. This figure has been modified from Takagi et al.22 (B) Percentage of host 3T3 cells infected by control amastigotes (black bar) and TcCGM1-KO amastigotes (gray bar). The mean (±SD) of three infection experiments are plotted (**p < 0.01, Welch’s t-test). This figure has been modified from Takagi et al.22 Please click here to view a larger version of this figure.
Figure 5: Phenotypic difference between knockout amastigote and differentiated trypomastigote. (A) Schematic representation of experimental time line. (B) Cell counts of axenic amastigote at 4 days post transfection for control amastigotes (black bar) and TcPAR1-KO amastigotes (gray bar). Mean values (±SD) of three electroporation experiments are plotted (n.s.: not significant, Mann-Whitney U test). (C) Percentage of infected host 3T3 cells at 4 days post infection by control amastigote (black bar) and TcPAR1-KO amastigote (gray bar). Mean values (±SD) of three culture wells are plotted (n.s.: not significant, Mann-Whitney U test). (D) Number of trypomastigote released into culture medium at 5 days post infection for control co-culture (black bar) and TcPAR1-KO co-culture (gray bar). Mean values (±SD) of three culture wells are plotted (*p < 0.05, Mann-Whitney U test). Please click here to view a larger version of this figure.
Supplemental Table 1: Composition of LIT medium. LIT medium was prepared according to ATCC 1029, except for the amount of disodium hydrogen phosphate. Please click here to download the table.
Supplemental Figure 1: Effect of electroporation buffer on amastigote cell viability. EA was electroporated either in electroporation buffer or in EM buffer. Amastigote was stained with PI after 24 h to count the number of live and dead amastigotes. Percentages of dead cells in each group and in the no pulse group are plotted. The mean (±SD) of three electroporation experiments are shown (*p < 0.05, n.s.: not significant, Kruskal-Wallis test). Please click here to download the figure.
Supplemental Figure 2: Microscopy images of nuclear-stained TcPAR1-KO and control intracellular amastigotes. Co-culture of host 3T3 cells and transfected amastigotes were fixed and stained at 4 days post infection. Scale bars: 20 µm. Please click here to download the figure.
Supplemental Figure 3: Growth phenotype of Cas9-expressing epimastigote. Cell counts of wildtype epimastigotes (WT), epimastigotes that constitutively expresses Cas9-EGFP (Cas9-EGFP), and epimastigotes that expresses Cas9-EGFP under the regulation of amastigote-specific 3’-UTR (Cas9-EGFP-AmaUTR) are plotted in log scale. Epimastigotes were cultivated in LIT medium (10% FCS) at 28˚C. Parasite cell density was adjusted to 1 x 106 cells/mL on Day 0. Cumulative cell counts are calculated as cell density multiplied by the total dilution factor. The mean (±SD) of three culture flasks are shown (*p<0.05, n.s.: not significant, Kruskal-Wallis test). Please click here to download the figure.
Supplemental Movie 1: Microscopy video image of control co-culture. Number and activeness of trypomastigotes that have differentiated from control amastigotes. Video image at 5 days post infection is shown. Please click here to download the movie.
Supplemental Movie 2: Microscopy video image of TcPAR1-KO co-culture. Number and activeness of trypomastigotes that have differentiated from TcPAR1-KO amastigotes. Video image at 5 days post infection is shown. Please click here to download the movie.
We demonstrated that the axenic culture of T. cruzi amastigotes can be utilized in CRISPR/Cas9-mediated gene knockout, by electroporating gRNA directly into Cas9-expressing EA. This way, the essentiality of the target gene specifically in amastigote stage can be evaluated without going through other developmental stages.
Another beneficial aspect of amastigote transfection is the convenience in testing for a large number of target genes. Once the co-culture of Cas9-expressing T. cruzi and host mammalian cell is established, it takes only few days to transform the tissue-derived trypomastigotes into EA and transfect gRNA to obtain knockout amastigotes. gRNA is the only material that needs to be tailored to each target gene, but synthetic gRNA is available from number of manufacturers, so it can simply be purchased. An alternative method to analyze the stage specific essentiality of target genes would be to establish an inducible knockout system32. In that case, plasmids must be constructed for each target gene and transfected to the parasite in the epimastigote stage to allow drug selection of the transfectants, since transfection efficiency of the plasmid is much lower than that of gRNA (<15% for plasmid26 as supposed to >96% for gRNA22). Selected transfectants must be differentiated into metacyclic trypomastigotes to infect host cells to finally induce a knockout in intracellular amastigote. This whole process would take 1-2 months.
In this report, we used PI to distinguish dead cells in axenic amastigote culture to quantitate the cell density with a hemocytometer. Alternatively, metabolic assays such as the resazurin viability assay can be employed to estimate the number of live amastigotes, which is more suited for a high throughput format. In our hands, 1 x 105 amastigotes per well yield sufficient redox activity to be detected by a resazurin assay after 5 h of incubation.
One drawback of establishing a Cas9-expressing cell line is the potential cellular burden and non-specific cleavage due to Cas9 overexpression5,33. There are several reports indicating that constitutive expression of Cas9 has no effect on the growth rate of T. cruzi4,6,7,8. However, in one instance5 and also in our hands, Cas9-expressing parasite showed slow growth comparing to the wildtype. In order to overcome this issue, we employed the amastigote-specific 3’-UTR to restrict the expression of Cas9 to amastigote stage22. Conjugation of amastin 3’-UTR to the Cas9 open reading frame restored the replication rate of transgenic parasite (Supplemental Figure 3), thereby producing robust host-parasite co-culture to continuously supply trypomastigotes for in vitro amastigogenesis. Recently, other groups have demonstrated that introduction of gRNA/Cas9 RNP complex to wildtype parasite can cause genome editing in T. cruzi7,9. This method should be explored as a less stressful approach to the parasite9, since study in mammalian cells suggests that the use of RNP reduces unwanted off-target genome editing, owing to the short half-life of Cas9 protein34.
Another optional modification to the protocol would be the co-transfection of donor DNA as a template for homologous recombination. It has been reported that the donor DNA containing homologous sequences to upstream and downstream of the cleavage site, in a form of either double-stranded DNA4,5,8,35 or single-stranded oligonucleotide7,9, facilitates the repair process of double-stranded break caused by Cas9 nuclease. Although some reports indicate that microhomology mediated end joining without donor template can repair the cleavage and introduce a deletion mutation5, introduction of the defined sequence to the mutation site nonetheless helps to confirm successful genome editing, because it is difficult to show the DNA evidence of gene knockout in some cases, even though the target protein reduction and phenotypic outcome suggest the target gene was mutated4.
Cas9/gRNA RNP transfection and co-transfection of a donor DNA are not demonstrated in this report to simplify the description of the protocol and to focus on the preparation of EA and the use of axenic culture thereafter. If one wishes to perform those experiments, it can be done by simply replacing the components of electroporation.
Our method of utilizing EA of T. cruzi as an experimental tool potentially enables a variety of stage-specific studies, including transient gene expression by plasmid transfection and drug efficacy test22. However, the effectiveness of this approach has been tested only in the Tulahuen strain thus far. Since strains of T. cruzi are quite diverse, applicability of this protocol to other strains must be investigated.
The authors have nothing to disclose.
This work was supported in part by JSPS KAKENHI Grant Number 18K15141 to Y.T.
20% formalin solution | FUJIFILM Wako Pure Chemical | 068-03863 | fixing cells |
25 cm2 double seal cap culture flask | AGC Techno Glass | 3100-025 | |
75 cm2 double seal cap culture flask | AGC Techno Glass | 3110-075 | |
All-in One Fluorescence Microscope | Keyence | BZ-X710 | |
Alt-R CRISPR-Cas9 crRNA (for Control) | IDT | custom made | target sequence = GGACGGCACCTTCATCTACAAGG |
Alt-R CRISPR-Cas9 crRNA (for TcCGM1) | IDT | custom made | target sequence = TAGCCGCGATGGAGAGTTTATGG |
Alt-R CRISPR-Cas9 crRNA (for TcPAR1) | IDT | custom made | target sequence = CGTGGAGAACGCCATTGCCACGG |
Alt-R CRISPR-Cas9 tracrRNA | IDT | 1072532 | to anneal with crRNA |
Amaxa Nucleofector device | LONZA | AAN-1001 | electroporation |
Basic Parasite Nucleofector Kit 2 | LONZA | VMI-1021 | electroporation |
BSA | Sigma-Aldrich | A3294 | component of the medium for in-vitro amastigogenesis |
Burker-Turk disposable hemocytometer | Watson | 177-212C | cell counting |
Coster 12-well Clear TC-Treated Multiple Well Plates | Corning | 3513 | |
DMEM | FUJIFILM Wako Pure Chemical | 044-29765 | culture medium |
Fetal bovine serum, Defined | Hyclone | SH30070.03 | heat-inactivate before use |
G-418 Sulfate Solution | FUJIFILM Wako Pure Chemical | 077-06433 | selection of transformant |
Hemin chloride | Sigma-Aldrich | H-5533 | component of LIT medium |
Hoechst 33342 | Thermo Fisher Scientific | H3570 | staining of nuclei |
Liver infusion broth, Difco | Becton Dickinson | 226920 | component of LIT medium |
MES | FUJIFILM Wako Pure Chemical | 349-01623 | component of the medium for in-vitro amastigogenesis |
PBS (–) | FUJIFILM Wako Pure Chemical | 166-23555 | |
Propidium Iodide | Sigma-Aldrich | P4864-10ML | staining of dead cells |
RPMI 1646 | Sigma-Aldrich | R8758 | medium for metacyclogenesis |