Total genomic DNA (gDNA) from different Trypanosoma parasites (Table 1) was extracted and purified from infected mouse whole blood (at ∼10⁸ Trypanosomes mL⁻¹) using the DNeasy Blood & Tissue Kit (Qiagen, Germany) following the manufacturer’s guidelines. DNA samples were eluted in DNase/RNase-free water and diluted to 1 ng μL⁻¹ before storage at −20°C until further use. The concentration and quality of the purified total gDNA was assessed through gel electrophoresis and spectrophotometric analysis (performed on Nanodrop ND-1000, Thermo Scientific). To determine the analytical sensitivity of the Two-Pot and One-Pot tests with total gDNA, the extraction and purification of T. b. evansi RoTat 1.2 total gDNA was followed by a 1:10 serial dilution from 20 ng μL⁻¹ up to 200 fg μL⁻¹ with DNase/RNase-free water.

Total gDNA of T. b. evansi type A strains was used as a template to amplify by PCR a 615 bp fragment of the Rode Trypanozoon antigenic type 1.2 VSG (RoTat 1.2 VSG) gene (GenBank accession code: AF317914.1). The PCR amplification reaction was as follows: Ten μl of extracted total gDNA (at 1 ng μL⁻¹) were mixed with 15 μL of a PCR-mastermix containing: 2 U GoTaq G2 DNA Polymerase (Promega, United Kingdom), 1x Colorless GoTaq Reaction Buffer (Promega, United Kingdom), 0.4 mM dNTPs (Thermo Fisher Scientific, United States), 0.8 μM TevPCR-Fw primer (Integrated DNA Technologies, United States) and 0.8 μM TevPCR-Rv primer (Integrated DNA Technologies, United States) (See primer sequence in Table 2). Amplifications were performed in a Biometra Trio-block thermocycler at the following cycling conditions: denaturation for 4 min at 94°C, followed by 35 amplification cycles of 1 min, denaturation at 94°C, 1 min primer-template annealing at 55°C, and 1 min polymerization at 72°C. A final elongation step was carried out for 5 min at 72°C. The resulting amplicon DNAs (aDNA) were purified with the GenElute PCR Clean-Up kit (Sigma-Aldrich) following the kit’s guidelines eluting in DNase/RNase-free water and diluted to 100 nM before storage at −20°C until further use. The concentration and quality of the purified aDNA were assessed through gel electrophoresis and spectrophotometric analysis (performed on a Nanodrop ND-1000, Thermo Scientific). To determine the analytical sensitivity of the CRISPR-Cas12b cleavage reactions and the Two-Pot and One-Pot tests with aDNA, T. b. evansi RoTat 1.2 aDNA was 1:10 serially diluted from 100 nM up to 1 aM with DNase/RNase-free water.

The recombinant Alicyclobacillus acidiphilus Cas12b (AapCas12b) protein, selected for the development of the TevRPA-CRISPR tests, was purchased from SignalChem Diagnostics, Canada. The selected suitable sgRNA for this protein includes the Alicyclobacillus acidoterrestris Cas12b (AacCas12b) scaffold sgRNA (Supplementary Table S1), which given the absence of a published native AapCas12b sgRNA, results in a more robust and specific nuclease activity by the AapCas12b protein compared to other sgRNA scaffolds (Joung et al., 2020). All sgRNAs used in this study (Table 2; Supplementary Table S1) were synthesized by Integrated DNA Technologies, United States.

TevCRISPR-Cas12b cis-cleavage assays were performed as follows: 250 nM AapCas12b, 500 nM RoTat1.2sgRNA and 30 nM T. b. evansi RoTat 1.2 aDNA were combined in 1x ThermoPol Reaction Buffer (New England Biolabs, United States) to a final volume of 15 μL. The reaction mix was transferred to a preset thermocycler and incubated for 1 h at different temperatures (50°C, 55°C, 60°C, 65°C, and 70°C). After incubation, the reaction was stopped by adding 2.75 μL of a stop solution (16.9 mM EDTA, 84.5 μg/mL RNAse A, and 67.6 mAU/mL proteinase K) and incubating the mix in a preset thermocycler for 10 min at 56°C. The reaction products were analyzed by electrophoresis on a 1% agarose gel pre-stained with ethidium bromide (EtBr) in TBE buffer (90 mM Tris, 90 mM borate, 2.5 mM EDTA). Electrophoresis was conducted at 100 V for 30 min.

TevCRISPR-Cas12b trans-cleavage reactions were performed as follows: 62.5 nM Cas12b (or 62.5 nM, 120 nM, and 250 nM during optimization assays), 250 nM sgRNA (or 125 nM, 250 nM, 500 nM and 1,000 nM during optimization assays) (six different sgRNAs were assessed, see Table 2; Supplementary Table S1), 250 nM FAM-Q probe (Integrated DNA Technologies, United States) and 30 nM T. b. evansi RoTat 1.2 aDNA (or 5 uL of 100 nM, 10 nM, 1 nM, 100 pM and 10 pM initial concentration, for analytical specificity assessment) were combined in 1x ThermoPol Reaction Buffer (New England Biolabs, United States) to a final volume of 15 μL. The reaction mix was transferred to a preset thermocycler and incubated for 2 h at 50°C (or 50°C, 55°C, 60°C, 65°C and 70°C during optimization assays). The reactions were run using Hard-shell thin wall 96-well PCR Plates (Bio-Rad, United States) on the CFX Connect Real-Time PCR Detection System (Bio-Rad, United States). Fluorescence measurements were read every 30 s at λex: 493 nm, λem: 517 nm.

Two-Pot TevRPA-CRISPR tests include two consecutive reactions, being (i) the specific amplification of the target T. b. evansi DNA through RPA, and (ii) the specific detection of the amplicons through the CRISPR-Cas12b cis- and trans-cleavage activities.

Isothermal RPA amplification was conducted with the TwistAmp Basic kit (TwistDx, Cambridge, United Kingdom) with the protocol suggested by Li et al. (2020) with minor modifications: 10 μL of input aDNA (at 10 fM, 1 fM, 100 aM, 10 aM and 1 aM initial concentration) or total gDNA (at 20 ng μL⁻¹, 2 ng μL⁻¹, 200 pg μL⁻¹, 20 pg μL⁻¹, 2 pg μL⁻¹ and 200 fg μL⁻¹ initial concentration) were incubated with 480 nM of each TevRPA primer, 1x rehydration buffer, 14 mM MgOAc and the lyophilized enzyme pellet of the TwistAmp Basic kit, in a final volume of 50 μL. The reaction mix was transferred to a preset thermocycler and incubated for 30 min at 39°C. The amplified products were first purified using the GenElute PCR Clean-Up kit (Sigma-Aldrich) and visualized by electrophoresis on a 2% agarose gel pre-stained with ethidium bromide (EtBr) in TBE buffer (90 mM Tris, 90 mM borate, 2.5 mM EDTA). Electrophoresis was conducted at 110 V for 40 min.

CRISPR-Cas12b specific detection was performed as follows: 2.5 μL of the previous reaction mix without purification was incubated with 62.5 nM AapCas12b, 250 nM RoTat1.2sgRNA, 250 nM FAM-Q probe (Integrated DNA Technologies, United States) and 1x ThermoPol Reaction Buffer (New England Biolabs, United States) in a final volume of 15 μL. The reaction mix was transferred to a preset thermocycler and incubated for 30–120 min at 50°C. The reactions were run using Hard-shell thin wall 96-well PCR Plates (Bio-Rad, United States) on the CFX Connect Real-Time PCR Detection System (Bio-Rad, United States). Fluorescence measurements were read every 30 s at λex: 493 nm, λem: 517 nm.

One-Pot TevRPA-CRISPR tests combine two reactions in one single tube, being (i) the specific amplification of the target T. b. evansi DNA through RPA, and (ii) the specific detection of the amplicons through the CRISPR-Cas12b cis- and trans-cleavage activities.

For this assay, 5 μL of input aDNA (at 1 pM, 100 fM, 10 fM, 1 fM, 100 aM and 10 aM initial concentration) or total gDNA (at 20 ng μL⁻¹, 2 ng μL⁻¹, 200 pg μL⁻¹, 20 pg μL⁻¹ and 2 pg μL⁻¹) was incubated with 480 nM of each TevRPA primer, 1x rehydration buffer, 14 mM MgOAc, the lyophilized enzyme pellet of the TwistAmp Basic kit, 62.5 nM AapCas12b, 250 nM RoTat1.2sgRNA and 250 nM FAM-Q probe (Integrated DNA Technologies, United States) in a final volume of 15 μL. The reaction mix was transferred to a preset thermocycler and incubated for 60–120 at 39°C. The reactions were run using Hard-shell thin wall 96-well PCR Plates (Bio-Rad, United States) on the CFX Connect Real-Time PCR Detection System (Bio-Rad, United States). Fluorescence measurements were read every 30 s at λex: 493 nm, λem: 517 nm.

Eight-week-old male C57BL/6 mice (purchased from Janvier, France) were divided into two groups of six individuals. In each group, five mice were inoculated intraperitoneally with 2000 T. b. evansi STIB 816 parasites in 200 μL of HBSS buffer (140 mM NaCl, 5 mM KCl, 1 mM CaCl₂, 0.4 mM MgSO₄ 7H₂O, 0.5 mM MgCl₂ 6H₂O, 0.3 mM Na₂HPO₄ 2H₂O, 0.4 nM KH₂PO₄, 6 mM D-Glucose, 4 mM Sodium bicarbonate; Thermo Fisher Scientific, United States). Of note, bloodstream trypanosome parasites were stored at −80°C as blood aliquots containing 50% Alsever’s solution (Sigma–Aldrich) and 10% glycerol (final V/V). One mouse in each group was used as a negative control and was not infected. The mice were tail bled at different times post-infection. The mice in Group 1 were bled at days 1, 3, 5 and 7 post-infection. The animals in Group 2 were bled at days 0, 2, 4, 6, 8 and 10 post-infection. All individuals from Group 2 were treated with Berenil (40 mg per kg), administered intraperitoneally at day 5 post-infection. Blood samples were collected from the tail and mixed with heparinized saline (10-fold at 10 units/mL; Sigma-Aldrich, United States) to prevent coagulation. Then, 2.5 μL of the collected blood was used to follow-up mice parasitemia by diluting the sample 200-fold in HBSS buffer and assessing parasitemia under the VisiScope IT415 PH light microscope (VWR, United States). The rest of the collected blood was used to extract and purify the total gDNA using the DNeasy Blood & Tissue Kit (Qiagen, Germany) following the manufacturer’s guidelines. DNA samples were eluted in DNase/RNase-free water on equal volumes to the initial sample (i.e., no sample concentration). The resulting gDNA was used to evaluate the samples using the TevPCR (used as a gold standard to assess positivity), Two-Pot TevRPA-CRISPR and the One-Pot TevRPA-CRISPR tests. The TevPCR was performed as described in Claes et al. (2004). TevRPA-CRISPR Two-Pot and the TevRPA-CRISPR One-Pot tests were performed as described previously in this study on optimized conditions. Fluorescence values from positive and negative samples from the Two-Pot TevRPA-CRISPR and the One-Pot TevRPA-CRISPR tests were evaluated by a Receiver Operating Characteristic (ROC) curve analysis for determining test’s positivity thresholds, as well as sensitivity and specificity scores (Supplementary Table S2; Supplementary Figure S9).

All experiments, maintenance and care of the mice complied with the European Convention for the Protection of Vertebrate Animals (ECPVA) used for Experimental and Other Scientific Purposes guidelines (CETS No 123) and were approved by the Ethical Committee for Animal Experiments (ECAE) at the Vrije Universiteit Brussel (Permit Number: 17-220-02). Mice were monitored daily. Humane endpoints were used during the study, based on weight loss, whereby animals with >25% weight loss were sacrificed using carbon dioxide treatment. The study was conducted in accordance with the local legislation and institutional requirements.

The GraphPad Prism 10 software was used for statistical analyses. Analytical sensitivity, and analytical specificity analyses were conducted using three technical replicates. The results presented were chosen as the most representative from two independent experiments. All statistical analyses were conducted using a one-way ANOVA, followed by Dunnett’s multiple comparison test. Values are expressed as mean ± standard deviation (SD) and p-values are shown. Specificity and sensitivity were evaluated through Receiver Operating Characteristic (ROC) curve analysis, which included a 95% confidence interval (CI) for both metrics, based on a sample size of n = 66.

Development of a TevRPA-CRISPR test requires the pre-amplification of a double-stranded DNA (dsDNA) target by RPA, followed by a highly specific cleavage and detection of the resulting amplicon through the CRISPR-Cas12b machinery (Figure 1A). For this, it is critical to choose a proper target region for the Cas12b-sgRNA complex, which must contain a PAM sequence (5′-TTN-3′) followed by a protospacer sequence (Teng et al., 2018). The target sequence was chosen based on: (i) the presence within the TevRPA amplicon, outside the primers or primer binding sites; (ii) the occurrence of a PAM sequence; (iii) the degree of nucleotide sequence identity between different T. b. evansi strains from different origins; and (iv) the absence of single nucleotide polymorphisms (SNPs).

First, the specificity of the RoTat 1.2 VSG gene to T. b. evansi type A was verified by PCR amplification (Supplementary Figure S1). Then, the TevRPA targeted sequence included within the aDNAs collected from 13 T. b. evansi type A parasites was analyzed (Table 1). A 99.65% nucleotide sequence identity was observed, including the presence of a unique SNP in two geographically closely related T. b. evansi type A strains, being T. b. evansi KAZAKHSTAN and T. b. evansi STIB 816, from Kazakhstan and P.R. of China, respectively (Supplementary Figure S2). As a result, and in accordance with the above-mentioned criteria, six sgRNAs targeting the TevRPA amplicon were designed (Supplementary Table S1). The efficacy of all sgRNAs was analyzed in conjunction with AapCas12b for cleaving (through cis-cleavage) and detecting (through trans-cleavage) T. b. evansi aDNA. Among the sgRNAs, sgRNA_6, designated as RoTat1.2sgRNA, exhibited the most rapid and robust fluorescence signals throughout the reaction (Supplementary Figure S3). Consequently, RoTat1.2sgRNA was selected for subsequent development stages of the TevRPA-CRISPR diagnostic test. Next, the optimal AapCas12b:RoTat1.2sgRNA molar ratio to target T. b. evansi aDNA was evaluated, indicating a 4:1 ratio (i.e., 62.5 nM AapCas12b and 250 nM RoTat1.2sgRNA) as the best combination to reduce assay costs while maximizing the cleavage (Supplementary Figure S4). Previous studies have reported an optimal AapCas12b temperature range between 31°C and 59°C for the cis-cleavage and up to 50°C–60°C for the trans-cleavage (Joung et al., 2020; Teng et al., 2018; Huyke et al., 2022). To corroborate if those results apply to our CRISPR-AapCas12b- RoTat1.2sgRNA design, a temperature range analysis was performed for both cis- and trans-cleavage reactions. A nearly total cis-cleavage of the target T. b. evansi aDNA was observed up to 55°C, while at 60ºC–70°C the cleavage was reduced but still visible (Figure 1B). Nevertheless, the trans-cleavage of the fluorescent probes was optimal from 40ºC to 60°C, and progressively decreased but measurable when increasing the reaction temperature at 65ºC–70°C (Figure 1C). As such, 50°C was selected as the optimal reaction temperature. Finally, the analytical sensitivity of the TevCRISPR-Cas12b assay was determined at the optimized reaction conditions. To achieve this, the TevCRISPR-Cas12b assays were conducted on 1:10 serially diluted T. b. evansi aDNA samples, using a negative control sample in which aDNA was absent. Figure 1D shows that the TevCRISPR-Cas12b assay detects target T. b. evansi aDNA up to a pM concentration.

The AapCas12b-RoTat1.2sgRNA complex was designed with the aim of adapting this technology to the TevRPA. Hence, first a Two-Pot test was developed, in which the RPA amplification is directly followed by the cleavage of the resulting amplicon through the CRISPR-Cas12b machinery, and the cleavage and detection of a fluorescent probe (Figure 2A). This test was performed following the optimal conditions for both reactions, in two separate tubes, with a total reaction time of 1 h. Using this approach, the analytical sensitivities of both TevRPA and Two-Pot TevRPA-CRISPR tests were evaluated. While TevRPA allows to detect up to 100 aM of the target T. b. evansi aDNA, the Two-Pot TevRPA-CRISPR test improved this detection limit by 10-fold, detecting up to 10 aM of T. b. evansi aDNA after 30 min of reaction (Figures 2B, C, F), or by 100-fold, detecting up to 1 aM of T. b. evansi aDNA after 60–120 min of reaction (Supplementary Figure S5). When using T. b. evansi gDNA instead, the TevRPA allowed to detect up to 200 pg of the target gDNA, whereas the Two-Pot TevRPA-CRISPR test improved this detection limit by 10-fold, detecting up to 20 pg of T. b. evansi gDNA after 30 min of reaction (Figures 2D, E, H), or by 100-fold, detecting up to 2 pg of T. b. evansi gDNA after 60–120 min of reaction (Supplementary Figure S6). Although the CRISPR-Cas12b technology enhances sensitivity, its primary advantage lies in ensuring test specificity by serving as a “second verification” of amplicon accuracy following the initial amplification step (Joung et al., 2020). To probe the test’s analytical specificity, the Two-Pot TevRPA-CRISPR was performed on different Trypanosoma spp. gDNA samples (listed in Table 1) including those that can be found coexisting in the same territories as T. b. evansi. As expected, the TevRPA-CRISPR test resulted in a positive fluorescence signal only when T. b. evansi type A gDNA was present (Figures 2G, I).

Having demonstrated the feasibility and adaptability of CRISPR-Cas12b within the TevRPA Two-Pot system, combining both steps into a single-pot reaction was addressed, with the goal of creating an efficient and reliable diagnostic test for POC use. The One-Pot TevRPA-CRISPR assay integrates RPA amplification with CRISPR-Cas12b-mediated cleavage and detection within a single reaction mixture, enabling the detection of T. b. evansi type A gDNA within 1 h. The amplification rate and efficiency of these RPA-CRISPR assays are significantly affected by primer concentration and magnesium acetate (MgOAc) levels. Therefore, the optimized concentration for both reagents was determined, being 14 mM MgOAc and 480 mM TevRPA primers, respectively (Supplementary Figure S7). Finally, the analytical sensitivities of the One-Pot TevRPA-CRISPR test were evaluated, detecting up to 100–10 aM of T. b. evansi aDNA (Figures 3B, C; Supplementary Figure S8), and 20 pg of T. b. evansi gDNA (Figures 3D, E; Supplementary Figure S8).

After developing the Two-Pot and One-Pot TevRPA-CRISPR designs, test efficacy in diagnosing both active and cured infections of T. b. evansi was validated. Ten C57BL/6 mice were infected with T. b. evansi STIB 816, divided into two groups. The presence of parasites was assessed by microscopy, TevPCR (Claes et al., 2004), Two-pot TevRPA-CRISPR, and One-Pot TevRPA-CRISPR at various time points of infection. Group 1 was left untreated, while Group 2 was treated with Berenil 5 days post-infection (Figures 4A, B). Both Two-Pot and One-Pot TevRPA-CRISPR assays accurately detected all infected samples in both untreated and treated groups, matching the performance of the gold standard TevPCR (Kappa value = 1) (Figures 5A, B). All infected mice in Group 1 were euthanized by day 8 post-infection, as they started to show signs of infection-associated pathology. In contrast, all mice in Group 2 survived, indicating successful parasite clearance following Berenil treatment. Both Two-Pot and One-Pot TevRPA-CRISPR yielded negative results in post-treatment non-infected samples, as corroborated by TevPCR, validating their effectiveness as “test-of-cure” assays (Figure 5B). Finally, the preliminary sensitivity and specificity of both TevRPA-CRISPR tests were evaluated, achieving 100% for both metrics (Supplementary Table S2).