RESULTS
PROOF OF CONCEPT

Evaluation of Selected miRNA Biomarkers for Sepsis Diagnosis

Methodology

This project included patients who were enrolled in the VISION study, formally entitled “Viscoelastic Coagulation Monitor as an Early Index of Sepsis in Patients Admitted with Infection at the Emergency Department: The VISION Project”, a prospective observational investigation registered on ClinicalTrials.gov. The study investigates the potential role of viscoelastic coagulation monitoring (VCM) in the early identification of sepsis progression in patients presenting with infection at the emergency department. The study protocol is approved by the Ethics Committee of ATTIKON University hospital. All procedures were performed in accordance with institutional ethical standards and the Declaration of Helsinki. Informed consent was obtained from all participants or their legal representatives prior to inclusion in the study.

A total of 19 patients presenting with infection were included in the study, of whom 11 fulfilled the Sepsis-3 criteria for sepsis and 8 were classified as non-septic. The mean age of septic patients was 65 ± 6.2 years, while non-septic patients had a mean age of 26.2 ± 4.6 years. Males represented 54.5% of the septic group and 12.5% of the non-septic group. The median NEWS2, qSOFA, and SOFA scores were higher among septic patients 6 (IQR 1–8), 1 (IQR 0–2), and 2 (IQR 2–5), respectively, compared with non-septic patients 0.5 (IQR 0–1.7), 0 (IQR 0–0), and 0 (IQR 0–0). Median procalcitonin (PCT) and mean C-reactive protein (CRP) values were also elevated in the septic group (0.26 ng/ml and 80.9 mg/l, respectively) relative to the non-septic group (0.05 ng/ml and 73.2 mg/l). The most frequent infection sites were lower respiratory tract infections (54.5% in septic, 45% in non-septic patients), followed by intra-abdominal infections (27.3%) and urinary tract infections. One patient in the septic group presented with central nervous system infection, and another with septic shock of unknown primary origin. Clinical and demographic data collected from all participants are presented in detail in the table below.

In order to adjust for the calculation of the relative number of copies, samples from five healthy individuals were included. Peripheral blood samples were obtained from all participants, and serum was separated and stored under standardized conditions for subsequent molecular analysis. Total RNA, including small RNA species, was extracted from serum samples to quantify circulating microRNAs (miRNAs).

The analysis focused on miR-150-5p (hsa-miR-150-5p) and miR-486-5p (hsa- miR-486-5p) with miR-103a-5p (hsa-miR-103a-5p) serving as the endogenous reference gene for normalization. The ΔCT values were calculated for each subject, and clinical correlations were performed between miRNA expression levels and the recorded clinical and biochemical parameters (e.g., CRP, PCT, NEWS2, SOFA). Statistical analyses were conducted using IBM SPSS Statistics software to explore potential associations between miRNA expression profiles and sepsis severity.

Both miR-150-5p and hsa-miR-486-5p were selected based on evidence from the literature suggesting their contribution in immune and inflammatory responses during infection and sepsis. Given their role in immunomodulation and thromboinflammation, these miRNAs have been proposed as potential circulating biomarkers for diagnostic and prognostic strategies in sepsis.

The relative copies of miR-150-5p and of mir are presented in Figures 1 and 2. Data suggest that the copies of mirR are higher in patients with sepsis than patients without sepsis.

Development and Testing of the CRISPR/Cas13a–Catalytic Hairpin Assembly (CHA) Detection System

Polymerase Chain Reaction (PCR) Amplification of gRNA Fragments

To construct the expression plasmids for our CRISPR/Cas13a system, we performed two separate cloning steps. The backbone plasmid contained a variant of the LwCas13a gene, and we inserted:

• gRNA1 cassette targeting hsa-miR-150-5p
• gRNA2 cassette targeting hsa-miR-486-5p

Each gRNA cassette, containing the guide RNA sequence and its regulatory elements, was cloned individually into the Cas13a backbone, generating two distinct plasmids. To prepare the inserts, both gRNA fragments were amplified using gradient PCR (65 °C, 68 °C, and 70 °C) to optimise reaction efficiency. The PCR products were analysed on a 1.5% agarose gel with a 100 bp DNA ladder.

Results

All reactions yielded clear single bands of 590 bp, matching the predicted fragment size. At every temperature the produced bands had identical size and intensity, confirming robust amplification. The PCR products were extracted from the agarose gel and stored for subsequent cloning.

Transformation of the vector with LwCas13a gene into NEB Stable cells

Before starting with the vector transformation, we assessed the competency of our NEB Stable cells. Assessing cell competency is important for troubleshooting transformations. To test competency, NEB Stable cells were transformed with the plasmid pUC019 (resistance to Ampicillin) and plated on LB agar with Ampicillin. After approximately 24 hours, colonies appeared, indicating that the cells were sufficiently competent for subsequent experiments. In order to engineer the two plasmids consisting of the desired fragments and a backbone containing the LwCas13a gene, the backbone first had to also be amplified. For amplification of the plasmid backbone, it was transformed into Escherichia coli (NEB Stable strain) using a standard transformation protocol. Transformed colonies were cultured in LB medium supplemented with chloramphenicol, and plasmid DNA was isolated using a miniprep protocol.

Results of vector transformation

The purified plasmid DNA was analyzed by 1% w/v agarose gel electrophoresis to confirm amplification and plasmid size (approximately 5.5 kb). To verify plasmid identity, restriction digestion was performed using the EcoRV-HF enzyme, which was predicted to cut the plasmid at three sites, producing fragments of approximately 3393 bp, 1521 bp, and 521 bp. These results confirmed the successful isolation of the plasmid that would be used as vector in our subsequent experiments.

Gibson Assembly

To clone our two gRNA fragments into the vector, we performed Gibson Assembly. First, we prepared vector/backbone overhangs by PCR and confirmed the correct product on agarose gel (5360 bp band). The vector was then extracted from the agarose gel, since other non desirable products were also observed in the electrophoresis. After the isolation of the vector with overhangs, we performed a Gibson Assembly with a 7:1 insert-to-vector ratio, to account for the lower backbone concentration after extraction.

Results of assembly

To verify correct assembly, we performed a restriction digest using EcoRV-HF. Both the overhang vector and the Gibson product yielded three bands. However, the second band differed in size: 1521 bp in the overhang vector vs. 1926 bp in the Gibson product. The agarose gel electrophoresis confirmed successful Gibson Assembly, since the right band pattern was observed after digest with EcoRV-HF.

While downstream expression analysis presented challenges on SDS-PAGE, the assembly step itself demonstrated correct integration. For functional assays, we continued with purified commercial Cas13a and our designed crRNAs.

CRISPR/Cas13a & Catalytic Hairpin Assembly (CHA)

Results of CRISPR/Cas13a and Catalytic Hairpin Assembly Reaction

We tested our CRISPR/Cas13a system coupled with the CHA (Catalyzed Hairpin Assembly) reaction using two miRNA biomarkers, hsa-miR-150-5p and hsa-miR-486-5p, to evaluate:

1. Whether the system can successfully detect miRNAs.
2. Whether the concentration of H1–H2 duplex increases with increasing miRNA concentration, enabling potential biomarker quantification.
3. Whether any optimization of hairpins is required to improve system performance.

A total of 9 reaction tubes were used:

• 4 tubes for hsa-miR-150-5p (2 for 100 nM, 2 for 200 nM)
• 4 tubes for hsa-miR-486-5p (2 for 100 nM, 2 for 200 nM)
• 1 tube as a negative control

After incubating all reagents in the tubes at 37°C for 1 hour, reactions were analyzed by 3% agarose gel electrophoresis.

Observations

• In the absence of Hairpin 0 and miRNA (Lanes 2–3), the H1–H2 duplex was formed but at a low concentration compared to the reactions containing both Hairpin 0 and miRNA. This indicates that a spontaneous reaction between Hairpins 1 and 2 occurs, which we aimed to minimize to improve system performance. READ MORE
• The optimized Hairpin 0 (H0) and Hairpin 2 (H2) further enhanced duplex formation compared to the unoptimized version, demonstrating that hairpin design significantly affects reaction efficiency. READ MORE
• Both hsa-miR-150-5p and hsa-miR-486-5p were successfully detected using this biological system.
• The percentage increase in H1–H2 duplex concentration reflects the efficiency of our combined CRISPR/Cas13a–CHA system. The newly designed hairpins showed an overall improvement in duplex formation. Additionally, duplex concentration increased with higher concentrations of miRNA targets. Using the optimized DNA hairpins, a 51.1% increase in H1–H2 duplex concentration was observed for hsa-miR-150-5p when the miRNA concentration was raised from 100 nM to 200 nM, suggesting that doubling the biomarker concentration approximately doubled duplex formation. In contrast, for hsa-miR-486-5p, a 1.8% increase in duplex concentration was observed under the same conditions. In both cases, duplex concentration showed a positive correlation with increasing miRNA biomarker concentration.
• The moderate increase observed for hsa-miR-486-5p is considered more representative of the system’s performance, as the electrophoresis results indicated that the reaction at 100 nM with modified hairpins did not run properly.

Future Steps

Additional experiments should be conducted for both miRNA biomarkers to establish a clear correlation between biomarker concentration, duplex concentration, and subsequent signal production. These results will support the integration of the CRISPR/Cas13a–CHA system into an electrochemical biosensor for quantitative detection of miRNA biomarkers.

Abbreviations