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Published in final edited form as: Int J Parasitol. 2020 Nov 29;51(2-3):193–200. doi: 10.1016/j.ijpara.2020.09.005

Development of a CRISPR/Cas9 system in Entamoeba histolytica: proof of concept

Monica Mendes Kangussu-Marcolino a,1, Pedro Morgado a,1, Dipak Manna a,1, Heather Yee a,c, Upinder Singh a,b,*
PMCID: PMC7880892  NIHMSID: NIHMS1652480  PMID: 33264648

Abstract

The protozoan parasite Entamoeba histolytica is an important human pathogen and a leading parasitic cause of death on a global scale. The lack of molecular tools for genome editing hinders the study of important biological functions of this parasite. Due to its versatility, the CRISPR (clustered regularly interspaced short palindromic repeat)-Cas9 system has been successfully used to induce site-specific genomic alterations, including in protozoan parasites. In this study, we optimized CRISPR-Cas9 for use as a genetic tool in E. histolytica. We chose a single plasmid approach containing both guide RNA (gRNA) and Cas9 nuclease expression cassettes. The amebic U6 promoter was used to drive the expression of the gRNA and its expression was confirmed by Northern blot analysis. Stable transfectant cell lines were obtained using a destabilizing domain of dihydrofolate reductase fused to myc-tagged Cas9 (ddCas9). With this system, we were able to induce ddCas9 expression 16 h following treatment with the small molecule ligand trimethoprim (TMP). Stable cell lines expressing ddCas9 and Luc-gRNA or non-specific (NS)-gRNA were transiently transfected with a plasmid containing a mutated luciferase gene (pDeadLuc) targeted by Luc-gRNA and another plasmid with a truncated luciferase gene (pDonorLuc) to restore luciferase expression and consequent activity. We observed that luminescence signal increased for the cell line expressing Luc-gRNA, suggesting that homologous recombination was facilitated by Cas9 activity. This evidence is supported by the presence of chimeric DNA detected by PCR and confirmed by sequencing of the resulting repaired DNA obtained by homologous recombination. We believe this represents the first report of a CRISPR/Cas9 system use in Entamoeba and provides evidence that this genome editing approach can be useful for genetic studies in this early branching eukaryote.

Keywords: Entamoeba histolytica, CRISPR/Cas9, Luciferase, Homologous recombination

1. Introduction

Entamoeba histolytica is a leading parasitic cause of death and an important human pathogen. The parasite infects 500 million people annually and causes colitis and liver abscess in over 50 million people each year with an estimated 100,000 deaths, making it a leading parasitic cause of death (WHO, 1997; Stanley, 2003). Despite its medical importance, the absence of robust molecular tools for genetic manipulation of E. histolytica have held back efforts to interrogate the biological mechanisms of stage conversion, important for transmission, drug target validation, and host-cell interactions. Limited gene knockdown in E. histolytica was achieved following the discovery of a robust endogenous RNA interference (RNAi) pathway and an abundant population of 27 nucleotides (nt) small RNAs in the parasite (Zhang et al., 2013, 2015). The 27 nt small RNAs have nuclear localization and mediate transcriptional gene silencing via histone modifications (Zhang et al., 2011). Taking advantage of the endogenous RNAi pathway led to the development of a novel and robust system for gene silencing, where a gene with abundant small RNAs can “trigger” silencing of other genes fused to it (Morf et al., 2013; Pearson et al., 2013). This has been an important advancement for genetic manipulation in Entamoeba. However, this technology is limited as complementation strategies are challenging due to limited availability of resistance selection markers, and gene knockouts are not yet possible.

Protozoan parasites are early eukaryotes with distinct molecular biology characteristics including features such as multicopy gene families, genome plasticity, and peculiar mechanisms of gene expression (Lanzer et al., 1995; Wickstead et al., 2003). It is particularly important for functional genomics studies in these organisms to implement the use of CRISPR/Cas9 as a versatile tool (Grzybek et al., 2018). Within a relatively short time from its discovery, implementation of a CRISPR (clustered regularly interspaced short palindromic repeat)/Cas9 system as an editing tool has been widely adapted for a range of organisms. The CRISPR/Cas9 system consists of the Streptococcus pyogenes Cas9 endonuclease, that creates a DSB, guided by a user-designed guide RNA (gRNA) to selectively target genetic loci containing a protospacer adjacent motif (PAM) and create a DSB which will be repaired by inserting a point mutation or exogenous DNA (Mali et al., 2013; Doudna and Charpentier, 2014).

The advances from the use of CRISPR in parasites are remarkable, including the knockout out the gene encoding a rohptry protein kinase, ROP18, not previously achieved with conventional approaches (Shen et al., 2014), a genome-wide screen in Toxoplasma gondii (Sidik et al., 2016, 2018), and a marker-free gene knockout and knockdown system in Plasmodium (Ghorbal et al., 2014; Walker and Lindner, 2019). Other original solutions were developed for Trypanosoma brucei (Beneke et al., 2017; Rico et al., 2018), Cryptosporidium (Vinayak et al., 2015), Trypanosoma cruzi (Lander et al., 2015), Leishmania spp. (Zhang et al., 2017; Beneke et al., 2019), Trichomonas vaginalis (Janssen et al., 2018), and most recently, Eimeria tenella (Hu et al., 2020; Tang et al., 2020).

Here we report the first successful use of CRISPR/Cas9 in Entamoeba. We found that stable expression of Cas9 was only achieved when fused to a dihydrofolate reductase (DHFR) destabilization domain, designed to degrade the protein in the absence of TMP (Liu and Singh, 2014). We demonstrate that Cas9-mediated episomal gene editing is capable of restoring activity of a functionally inactive luciferase gene. This evidence is supported by the PCR detection sequencing confirmation of chimeric DNA result from repair by homologous recombination. This report establishes a CRISPR/Cas9 strategy for gene manipulation in Entamoeba that can be further developed to rigorously interrogate this important and neglected parasite.

2. Materials and methods

2.1. Plasmid construction

The pKT-3M vector originally generated from a pBluescript II KS+ vector (Saito-Nakano et al., 2004), was used as the backbone for all CRISPR related constructs. All primers are listed in Supplementary Table S1. As ampicillin contains a BsaI restriction site that would interfere with subsequent cloning of the gRNA cassette, pKT_Kan was made by replacing the ampicillin resistance gene downstream of the AmpR promoter with kanamycin using primers P1 and P2 to amplify kanamycin and P3 and P4 to amplify pKT-3M without ampicillin. PCR products were assembled using Gibson Assembly (New England Biolabs, USA), following the manufacturer’s instructions.

Subsequently, Streptococcus pyogenes Cas9 was cloned into the pKT-Kan amebic vector under the cystine synthetase promoter and downstream of a putative amebic nuclear localization signal (NLS) (Uribe et al., 2012), the destabilization domain of the Escherichia. coli DHFR (Liu and Singh, 2014), and a 3X myc-tag. The DHFR destabilization domain was fused to Cas9 to generate destabilizing domain containing Cas9 (ddCas9) as it has been successfully used to regulate expression of proteins in Entamoeba. The ddCas9 cassette, consisting of PCR amplified fragments as well as hybridized oligos, was initially assembled in the yeast vector, pRS314. P5 and P6 contained homology to pRS314 and a partial sequence for DHFR was used as hybridized oligos, P7 and P8 were used to PCR amplify DHFR, P9 and P10 were used as hybridized oligos that contained the 3xMyc tag. Cas 9 was amplified using P11, P12, P13, P14, P15, and P16.

Assembly of these fragments was done by taking advantage of homologous recombination in yeast as each fragment contained 20–30 bp of overlap with adjacent fragments or the vector backbone. Briefly, tryptophan auxotroph yeasts were resuspended in 72 μL of transformation buffer (TB; 100mM LiOAc, 1 mM Tris·Cl, pH 8.0, and 0.1 mM EDTA). Yeast was incubated for 30 min on a rotator at room temperature with 500 ng of all hybridized oligos, PCR amplified fragments, and 100 ng of pRS314 (digested with EcoRI and ClaI) together with 8 μg of salmon sperm (New England Biolabs) in 500 μL of polyethelyn glycol solution (PEG; 50% PEG 3300, 100 mM LiOAc, 1 mM Tris·Cl, pH 8.0, and 0.1 mM EDTA). A total of 65 μL of DMSO was added to yeast, followed by incubation at 43°C water bath for 15 min. Yeast was plated on tryptophan depleted agar plates and colonies screened for the presence of a fully assembled ddCas9 cassette. The ddCas9 cassete was digested (XmaI and XhoI) out of pRS314 and into pKT_Kan to generate pKT_Kan-ddCas9 (pddCas9).

The gRNA cassette, containing a 20 bp non-specific (NS) target sequence flanked by BsaI restrictions sites and located downstream of the E. histolytica U6 promoter, was synthesized by GenScript (Piscataway, NJ, USA) and cloned into the pddCas9 vector to generate pKT_Kan_ddCas9_NS-gRNA (pCas9-gNS). Target-specific gRNA oligos against firefly luciferase (FLuc) or E. histolytica Myb1 (EHI_197980) were designed using the Eukaryotic Pathogen CRISPR gRNA Design Tool (Peng and Tarleton, 2015) and cloned into pKT_Kan_ddCas9_NS-gRNA to generate pKT_Kan_ddCas9_FLuc-gRNA (pCas9-gLuc) or pKT_Kan_ddCas9_Myb-gRNA (pCas9-gMyb) constructs. The final constructs containing Cas9 and gRNA expression cassetes are 11,161 bp plasmids.

Expression of the renilla luciferase reporter gene was driven by the cysteine synthetase promoter in the pKT_CS Renilla (pRLuc) vector (Morf et al., 2013). The enolase promoter drives the expression of firefly luciferase in the pKT_firefly luciferase (pFLuc) expression vector (Ehrenkaufer and Singh, 2012).

To test for CRISPR/Cas9-mediated repair, we modified the pFLuc to generate the dead luciferase (pDeadLuc) vector. Using site-directed mutagenesis with QuikChange Lightning (Agilent, Santa Clara, CA, USA) we introduced a nonsense mutation (G58T), conferring early translation termination, adjacent to a gRNA target sequence near the 5’ end of the gene. The donor luciferase construct (pDonorLuc), which encodes for the first 435 amino acids of firefly luciferase that restores G58 and a G54A mutation that ablates the PAM sequence, was also generated using the pFLuc with QuickChage Lightning.

A construct DonorLuc with linker was generated by cloning the 32 bp linker before the starting of Luciferase at HindIII site. All constructs were checked by sequencing. All the primers used for making constructs were listed in Supplementary Table S1.

2.2. Parasite culture and transfections

Entamoeba histolytica (strain HM-1:IMSS ) was grown axenically in TYI-S-33 medium under standard culture conditions at 36.5 °C (Diamond et al., 1978; MacFarlane and Singh, 2006).

Parasites were transfected using two different methods. For obtaining stably transfected cell lines we used the Lipofectamine 3000-based method (Olvera et al., 1997); 5×105 trophozoites were seeded into 25 mm Petri dishes and allowed to grow for 24 h before transfection following the manufacturer’s instructions. On the day of transfection, 7.5 μL of Lipofectamine 3000 reagent were mixed with 150 μL of Opti-MEM while 5 μg of each plasmid DNA were incubated with 7.5 μL of P3000 reagent (Thermo Fisher Scientific, USA) in 150 μL of Opti-MEM medium (Gibco – Thermo Fisher Scientific). Diluted DNA (with P3000 Reagent) was then mixed with diluted Lipofectamine 3000 reagent and incubated for 10 min at room temperature. Cells were washed once with 1x PBS followed by addition of 2 ml of serum-free Opti-MEM. The Lipofectamine 3000-DNA mixture was added in drops across the Petri dish, and the dishes were covered by parafilm to minimize oxygen exposure. Parasites were incubated at 37 °C for 3 h, iced for 5 min to release parasites from the dish, and transferred to a 15 mL glass tube containing fresh TYI medium. For generation of stable transfectants, parasites were allowed to grow for 48 h after transfection before addition of drug selection. Parasites were selected at 3 μg/mL of neomycin and increased to a final concentration of 12 and 24 μg/mL of neomycin. We generated the following cell lines: Eh_Cas9-gNS, Eh_Cas9-gLuc and Eh_Cas9-gMyb from the transfections with pCas9-gNS, pCas9-gLuc and pCas9-gMyb, respectively.

For the transient transfections we used the Attractene-based method (Ralston et al., 2014); parasites were grown to confluency in T25 flasks and 8×105 trophozoites were used per transfection condition. On the day of transfection, a total of 15–20 μg of plasmid DNA was incubated for 10 min with 37.5 μL Attractene (Qiagen, DE) in a total volume of 200 μL of M199 medium (Gibco) in a 2 mL cryovial. Cells were washed once with 1x PBS and resuspended at 4.4×105 cells/mL with supplemented M199 (M199 containing 7.4 mM L-cysteine, 23 mM Hepes Na, 0.5 mM ascorbic acid, pH adjusted to 6.8 and then sterile filtered followed by supplementation with 15% heat-inactivated bovine serum. Parasites (1.8 mL) were added to Attractene-DNA mixture and then incubated at 37 °C for 3 h, iced for 10 min to release parasites from the dish, and transferred to a 15 mL glass tube containing fresh TYI medium. Parasites transiently transfected were collected 16–20 h post-transfection.

2.3. Western blot analysis

The stable parasite cell lines Eh_Cas9-gNS, Eh_Cas9-gLuc and Eh_Cas9-gMyb were maintained at 12 and 24 μg/ml of G418. Parasites were collected 16 h post-treatment with 10 μM of the stabilizing ligand TMP or vehicle control (DMSO) and 20 μg of protein lysate were resolved by SDS-PAGE and transferred to polyvinylidene difluoride (PVDF) for immunoblotting. The membranes were blotted with antibodies against Myc or actin. Horseradish peroxidase (HRP)-conjugated secondary antibodies were used. All Western blotting antibodies were obtained from Cell Signaling (Danvers, MA, USA).

2.4. Northern blot analysis

Total RNA was isolated from stable parasite cell lines Eh_Cas9-gNS and Eh_Cas9-gMyb using Trizol. 32P-radiolabeled probes used to detect the Myb target-specific gRNA region or the non-specific generic gRNA region were generated by using the mirVana micro (mi)RNA probe construction kit (Ambion, USA) following the manufacturer’s protocol. Northern blot analysis was done using a previously published protocol (Zhang et al., 2008). Briefly, 50 μg of RNA were loaded onto a denaturing 12% polyacrylamide gel, transferred to a membrane, probed with end-labeled 32P-labeled oligonucleotides in perfectHyb buffer (Sigma-Aldrich, USA) at 37 °C and washed using low (2X SSC, 0.1% SDS at room temperature for 15 min) and medium (1X SSC, 0.1% SDS at 37 °C for 15 min) stringency conditions.

2.5. Luciferase CRISPR-dependent episomal DNA repair assay

The cell lines Eh_Cas9-gNS and Eh_Cas9-gLuc were transiently transfected, as described above, with 5 μg of each expression construct listed for each transient transfection condition. All samples received 10 μg of TMP upon transfer to glass tubes with fresh TYI medium following 3 h transfection incubation. Parasites were collected 16–20 h post-transfection and lysed in passive lysis buffer with the addition of protease inhibitors (E1300 lysis buffer (Promega Madison, WI, USA), 1x HALT (Thermo Fisher Scientific)). A total of 50 μg of cell lysate was assayed for firefly and renilla luciferase activity using the Dual-Luciferase® Reporter Assay System (Promega,) following the manufacturer’s instructions. Firefly luciferase relative intensity units (RLU) were normalized to renilla luciferase RLU. One-way ANOVA and a multiple comparison Turkey test were used to determine statistically significant differences (P < 0.001) between samples (GraphPad Prism version 8.0.0).

3. Results

3.1. Expression and stabilization of Cas9

Expression of Cas9 has been previously shown to be toxic in other organisms, necessitating different strategies such as nuclear localization and conditional expression to develop a functional CRISPR/Cas9 system (Serpeloni et al., 2016; Janssen et al., 2018; Rico et al., 2018). Accordingly, our initial attempts to establish stably expressing Streptococcus pyogenes Cas9 (SpCas9) cell lines were unsuccessful. To circumvent this issue, we cloned SpCas9 into the pKT-3M amebic vector downstream of a putative amebic nuclear localization signal (Uribe et al., 2012), the destabilization domain of the E. coli dihydrofolate reductase (ddDHFR), previously demonstrated to effectively regulate protein levels in Entamoeba (Liu and Singh, 2014), and a myc-tag (Fig. 1A). With this strategy, we generated parasites stably transfected with pCas9 plasmids. We demonstrated that the expression of myc-tagged ddCas9 is stabilized after a 16 h period in the presence of TMP (Fig. 1B).

Fig. 1.

Fig. 1.

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Destabilization domain-containing Cas9 (ddCas9) and guide RNA (gRNA) expression in Entamoeba. (A) Schematic representation of the single vector construct which expresses both Cas9 and gRNA. Streptococcus pyogenes Cas9 was cloned into the pKT-3M amebic vector, under the regulation of cysteine synthetase promoters (5’ and 3’EhCS), downstream of a putative amebic nuclear localization signal (NLS), the destabilization domain of the E. coli dihydrofolate reductase (DHFR), and a Myc-tag. The gRNA expression cassette is included, containing a 20 bp target sequence (either the non-specific RNA (NS-gRNA) shown here, luciferase, or a Myb DNA binding gene (Myb1 - EHI_197980) ) and the gRNA scaffold cloned downstream of the Entamoeba histolytica (Eh) U6 promoter. Arrows denote transcription direction. (B) Expression of Myc-tagged ddCas9 in E. histolytica. Immunoblot of total protein (20 μg) from Eh_Cas9-gNS cell line after 16 h in the presence or absence of 10 mM of trimethoprim (TMP), indicated by ‘+’ and ‘−’ signs. Primary immunoblotting antibody (IB) against Myc was used and the top band pointed with an arrow indicates the expected stable Cas9 in the presence of the small molecule ligand trimethoprim (TMP). Anti-actin antibody was used as a loading control. (C-D) gRNA oligonucleotides expressed in E. histolytica. Total RNA (50 μg) was isolated from Eh_Cas9-gNS and Eh_Cas9-gMyb cell lines, resolved onto a denaturing polyacrylamide gel and transferred to a membrane. Radiolabeled probes were used to detect the common tracerRNA portion of the gRNA (C) and the Myb target-specific gRNA region (D). nt, nucleotide.

The gRNA expression cassette was included in the same vector, containing the gRNA scaffold cloned downstream of the E. histolytica U6 promoter and a 20 bp non-specific (NS), luciferase-specific, or Myb-specific target sequence, targeting the amebic Myb gene, EHI_197980 (Miranda et al., 1996), to generate pCas9-gNS, pCas9-gLuc and pCas9-gMyb expression vectors, respectively. We transfected these plasmids into E. histolytica HM-I:IMSS, using the Lipofectamine method, to generate the stable parasite cell lines Eh_Cas9-gNS, Eh_Cas9-gLuc and Eh_Cas9-gMyb. The expression of gRNAs was confirmed by Northern blot analyses using the same membrane incubated with radiolabeled probes to detect the generic tracerRNA region, which should be present in all cell lines (Fig. 1C) and Myb target-specific gRNA region, which should only be present in the Myb-gRNA expressing cell line (Fig. 1D). Non-transfected cells are not labeled with the probe to detect the generic tracerRNA region, as shown in Supplementary Fig. S1.

3.2. Cas9-dependent luciferase repair system

To demonstrate that the CRISPR/Cas9 system functions in Entamoeba, we designed a Cas9-dependent DNA repair assay for the restoration of luciferase activity based on an approach previously used in Cryptosporidium parvum (Vinayak et al., 2015). As E. histolytica possesses some of the machinery for non-homologous end-joining but lacks key components of the machinery, a characteristic observed in other eukaryotic parasite systems (Kelso et al., 2017), our CRISPR system is designed to facilitate Cas9-mediated DNA DSB repair through directed homologous recombination which can be achieved by introducing a transgene containing flanking regions to the DSB site.

Thus, in our assay strategy we have three components: the stable cell lines expressing Cas9 and gRNA, a mutated luciferase gene (DeadLuc) that lacks luciferase activity, and the repair DNA (DonorLuc) consisting of a truncated version of the FLuc gene containing the first 435 amino acids of the correct sequence the luciferase gene (Fig. 2A). Both DeadLuc and DonorLuc are provided as transient transfected plasmids (pDeadLuc and pDonorLuc), using the Attractene method of transfection. To generate pDeadLuc, we introduced a stop codon by inserting a G58T mutation in the full-length FLuc gene to ablate luciferase activity. The cell line Eh_Cas9-gLuc expresses a gRNA that we designed to target a PAM site a few nucleotides upstream from the stop codon in pDeadLuc. The plasmid pDonorLuc, containing the coding region of just the first 435 amino acids (aa) of the luciferase gene, is designed to restore read-through translation of the luciferase gene and confer resistance to further Cas9-mediated cleavage due to a point mutation that removes the PAM site targeted by gLuc-RNA (Fig. 2B). To confirm that no luminescence signal was detected from each component alone, all plasmids were independently evaluated using a dual luciferase assay to determine that they were functionally inactive and gave the expected results (Fig. 2C).

Fig. 2.

Fig. 2.

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Expression vectors used in Cas9-dependent DNA repair assay. (A) Schematic of all plasmids used in this study: (a) Plasmids for ddCas9 expression in Entamoeba histolytica (pddCas9) containing the expression cassete for the guide RNA (gRNA)targeting Luciferase (pCas9-gLuc) or a non-specific gRNA (pCas9-gNS). Both plasmids were used to generate stable cell lines. (b) The dead luciferase expression construct (pDeadLuc), the donor luciferase construct encoding the first 435 amino acids (pDonorLuc) and the donor luciferase construct containing a 20nt foreign sequence upstream to the luciferase ORF (pDonorLinker). The three plasmids were used in transient assays to evaluate Cas9 activity. (c) Full-length renilla (pRLuc) and firefly luciferase (pFLuc) expression vectors with respective amebic regulatory regions. Both plasmids were used as control in the transient assays. (B) Comparing sequences from the original and mutated Luciferase genes used in this study. The dead luciferase construct contains a nonsense mutation (G58T) (pDeadLuc), conferring early translation termination. The donor luciferase construct contains a G54A mutation to ablate the protospacer adjacent motif (PAM) sequence (pDonorLuc). (C) Measuring luciferase activity of transiently expressed vectors. Entamoeba histolytica were transfected with 10 μg of either pCas9-gLuc, pCas9-gNS, pDeadLuc, pDonorLuc, or pFLuc (positive control); 5 μg of pRLuc were added to each sample as a transfection control. Parasites were collected 16–20 h post-transfection and 50 μg of cell lysate were assayed for firefly and renilla luciferase activity. Data shown is representative of two independent experiments.

In order to test whether our CRISPR/Cas9 strategy worked to repair luciferase function, we transiently transfected Eh_Cas9-gLuc cells with plasmids pDeadLuc and pDonorLuc, treated with TMP and assayed for luminescence signal after 16 h. As a control cell line, we used Eh_Cas9-gNS, designed to express a non-specific gRNA, which should not be able to mediate a Cas9-dependent DSB within the DeadLuc gene. Consistent with our assay strategy, co-transfection of Eh_Cas9-gLuc cells with pDeadLuc and pDonorLuc constructs consistently restored luciferase activity well above the levels observed under control conditions (Fig. 3). Eh_Cas9-gNS did not present a significant increase in luminescence signal for either co-transfection or single transfection (Fig. 3), and neither did transfections in the absence of donor DNA.

Fig. 3.

Fig. 3.

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Luciferase activity derived from CRISPR/Cas9 mediated repair of episomal DNA. Entamoeba histolytica stable cell lines Eh_Cas9-gNS and Eh_Cas9-gLuc (Cas9-gNS and Cas9-Luc for short) were transiently transfected with a mixture of 5 μg of the dead luciferase expression construct (pDeadLuc) combined with 5 μg of either the donor luciferase construct encoding the first 435 amino acids (pDonorLuc) or a non-homologous construct (pEhGFP). All cell lines received 5 μg of renilla expressing plasmid as internal control. After transfection, all samples received 10 μM of small molecule ligand trimethoprim (TMP). Parasites were collected 16–20 h post-transfection and 50 μg of cell lysate were assayed for firefly and renilla luciferase activity. Firefly luciferase Relative Light Units (RLU) were normalized to renilla luciferase RLU. Data shown is from seven biological replicates; results obtained from Eh_Cas9-gLuc plus pDeadLuc and pDonorLuc were significantly different from all other samples (P < 0.001).

After observing that Cas9 can restore luciferase activity in Entamoeba, we wished to determine the specific recombination results between pDeadLuc and pDonorLuc constructs at the DNA level. For that, we inserted a 20 bp foreign sequence (linker) at the donor DNA to allow differential amplification of the recombined DNA (Fig. 4A). We took advantage of a HindIII restriction site just upstream of the Luciferase435 open reading frame (ORF) on pDonorLuc to insert the linker sequence to generate the plasmid pDonorLinker (Fig. 2A). In order to test the recombination, Eh_Cas9-gLuc was transiently transfected with pDeadLuc and pDonorLinker, and treated with TMP overnight. The genomic DNA of the transfection was extracted and PCR-amplified using the primer for the 20 bp linker sequence (P17) and a primer annealing at the 3′-end of the Luciferase gene (P20), which should not hybridize to the donor DNA. Genomic DNA from a pDeadLuc only transfection, done in parallel and treated with TMP overnight, was our recombination negative control. As loading control, we used a different set of primers (P21/P20) with both genomic DNA samples. P21 anneals at Eno5′-region, present in pDeadLuc and, therefore, this set of primers amplifies the two samples (recombination test (RT) and control (C)).

Fig. 4.

Fig. 4.

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Episomal homologous recombination mediated by CRISPR/Cas9. (A) Schematic representation of plasmids for dead luciferase expression (pDeadLuc) and for donor luciferase construct encoding the first 435 amino acids containing a 20-nucleotide foreign sequence upstream to the luciferase ORF (pDonorLinker)and the result recombined DNA. P17 represents a primer specific to the foreign linker inserted at the donor plasmid to detect recombination. P20 represents the reverse primer, aligning at 3’ portion of the luciferase gene. P21 represents the primer at 5’ Eno promoter, present in both plasmids (B) PCR product obtained from genomic DNA extracted from Eh_Cas9-gLuc transfected either with both plasmids (RT – test) or with only pDeadLuc (C – control). PCR using primers P17/P20 generates a product only when there is recombination, the product generated by PCR with P21/P20 is a loading control as it aligns with pDeadLuc plasmid. (C) Alignment of plasmid sequences pDeadLuc and pDonorLinker with the sequencing obtained from recombined DNA. For sequencing, the PCR product obtained from the amplification using primers P17-P20 genomic DNA extract from the test sample as template was cloned into TOPO vector and sequenced using M13 primers. The protospacer adjacent motif (PAM) site (AGG) is highlighted in green (light grey) with bold characters and the stop codon (TGA) in red (dark grey) with italicized characters and the single point mutation nucleotides in these sites are with clear background. The underlined and italicized sequence of characters represents the portion of DNA present in the whole luciferase gene and not in the DonorLuc gene.

Fig. 4B shows that only the transfection with both plasmids (RT) yields a PCR product in the correct predicted size (1431 bp). We confirmed by sequencing that this PCR product matches the expected sequence predicted for recombination between pDeadLuc and pDonorLinker, as shown by the chimeric sequence (Fig. 4C). We identified that, as expected, the PAM site is absent and the stop codon has been removed in the recombined PCR product. This indicates that the Cas9 recombination occurred exactly as one would have predicted.

4. Discussion

Despite the great impact of amebiasis in global public health, E. histolytica is understudied and its molecular toolbox is limited. A few options for studying functional genomics are available such as trigger-mediated gene silencing, expression of recombinant protein, and destabilization domain for regulated protein expression (Morf et al., 2013; Liu and Singh, 2014; Suresh et al., 2016). However, due to a variable number of nuclei and polyploidy (Lopez-Revilla and Gomez, 1978; Marquez-Monter et al., 1990; Willhoeft and Tannich, 1999), gene knockout tools have not been developed to date.

CRISPR/Cas9 is a versatile tool which is changing the landscape of molecular biology for a great variety of organisms. In this study, we provide a proof of concept of its function on episomal DNA in E. histolytica. CRISPR/Cas9 is especially useful for organisms known to be polyploid or have multicopy gene families, common features among protozoan parasites. Leishmania is an example of protozoa with multicopy gene families and a remarkable genome plasticity including variable ploidy, that makes gene knockout challenging (Bryant et al., 2019). With the CRISPR/Cas9 system optimized, it is now possible to knockout multicopy genes in Leishmania and even perform high throughput knockout screening, which allowed the generation of 100 mutants to evaluate flagellar defects (Zhang et al., 2017; Beneke et al., 2019; Bryant et al., 2019).

Cas9 expression has been reported to be cytotoxic in several systems including Toxoplasma gondii, Trichomonas vaginalis and Trypanosoma. brucei (Serpeloni et al., 2016; Janssen et al., 2018; Rico et al., 2018). After unsuccessful attempts to constitutively express Cas9, we obtained stable transfected cell lines using a destabilization domain previously used in E. histolytica (Liu and Singh, 2014) to regulate Cas9 expression (ddCas9) suggesting that Cas9 expression is toxic for E. histolytica. A similar strategy was previously used for other parasites, T. gondii and T. vaginalis (Serpeloni et al., 2016; Janssen et al., 2018).

Entamoeba is known to repair DSB by homologous recombination (Lopez-Casamichana et al., 2008; Singh et al., 2013; Kelso et al., 2016) and to increase expression of meiosis related genes during encystation (Ehrenkaufer et al., 2013; Singh et al., 2013). Recombination in Entamoeba has been demonstrated in episomal DNA, causing flipping of an intervening gene sequence, and is increased by UV exposure, encystation conditions, and growth stress such as heat shock, oxygen stress, and serum starvation (Singh et al., 2013). In our study, we demonstrate CRISPR/Cas9 mediated recombination by restoration of luciferase activity only in the presence of all necessary components (Cas9, luciferase guide RNA and donor luciferase DNA). Additionally, our sequencing confirms that the recombination occurred as expected, with no mismatch near the recombination site.

In conclusion, this study is to our knowledge the first to demonstrate activity of CRISPR/Cas9 in Entamoeba. Development of a CRISPR/Cas9 system for genomic editing is ongoing and we are investing in the complete optimization, looking at efficiency, specificity and off-target effects. We expect that demonstration of genomic editing, a phenomenon never reported in Entamoeba, will transform and bring a fruitful future to E. histolytica functional genomics. We will be better suited to address important biological questions, validate potential drug targets and develop other elegant tools for studying this impactful parasite.

Supplementary Material

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Supplementary Fig. S1. Northern Blot. Guide RNA (gRNA) oligonucleotides expressed in Entamoeba [in full] histolytica. Total RNA (50 μg) was isolated from non-transfected E. histolytica (strain HM-1:IMSS) and Eh_Cas9-gNS, stably transfected with plasmid for ddCas9 and non-specific guide RNA expression resolved onto a denaturing polyacrylamide gel and transferred to a membrane. Radiolabeled probes were used to detect the common tracerRNA portion of the gRNA (gRNA scaffold). nt, nucleotides.

Acknowledgments

We thank all members of the Singh laboratory for helpful discussions and critical reading of the manuscript. We also thank the members of Andrew Fire’s laboratory for insightful discussions. This work was supported by The National Institute of Health, USA, R21 AI149268-01, R21-AI102277-01 to Upinder Singh, the Institutional Research and Academic Career Development Award (IRACDA), USA Grant –5K12GM088033 to Pedro Morgado and Stanford Maternal and Child Health Research Institute (MCHRI, USA) Postdoctoral Support grant to Monica Kangussu-Marcolino.

Footnotes

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

1
NIHMS1652480-supplement-1.xlsx (12.1KB, xlsx)
2

Supplementary Fig. S1. Northern Blot. Guide RNA (gRNA) oligonucleotides expressed in Entamoeba [in full] histolytica. Total RNA (50 μg) was isolated from non-transfected E. histolytica (strain HM-1:IMSS) and Eh_Cas9-gNS, stably transfected with plasmid for ddCas9 and non-specific guide RNA expression resolved onto a denaturing polyacrylamide gel and transferred to a membrane. Radiolabeled probes were used to detect the common tracerRNA portion of the gRNA (gRNA scaffold). nt, nucleotides.

NIHMS1652480-supplement-2.png (472.4KB, png)

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