W36 - Development of a Gold Electrochemical Sensor for High-Sensitivity and Specificity Detection of CRISPR-Cas9 Interactions with Target and Off-Target DNA

Introduction: : CRISPR-Cas9 gene editing holds significant promise in medicine, enabling therapies for diseases like cancer, HIV, and sickle cell disease. The essential parts of a CRISPR complex include the Cas enzyme and guide RNA (gRNA). On-target specificity of gRNA is crucial for safe and accurate editing. Even when the gRNA almost matches the target DNA, except for just three or fewer nucleotide mismatches, the Cas9 enzyme can still mistakenly cut the DNA at that site. This off-target cleavage can create insertions or deletions during the cell’s repair process, leading to unintended genetic mutations. This is a challenge in CRISPR applications, as high sequence similarity alone isn’t enough to ensure safety, and minor mismatches may not be sufficient to prevent off-target effects. Off-target effects may also occur through DNA bulges or mismatched alignments, potentially impairing gene function or triggering adverse outcomes. Testing gRNA specificity in relevant biological models and minimizing Cas9 exposure time can reduce off-target risks. Strategies such as truncated gRNAs, dual gRNA systems, and limited exposure duration have shown up to 5000-fold improvements in specificity but may hinder the detection of single-point mutations. Existing detection techniques are often limited by sequencing depth, high cost, false positives, or complex workflows. Thus, there is a need for faster, more accurate, and simplified methods to evaluate gRNA specificity and CRISPR activity. This study proposes a gold electrochemical sensor combined with electrochemical impedance spectroscopy (EIS) to distinguish on- vs. off-target DNA binding, cleavage, and dissociation of CRISPR-Cas9, offering a label-free, rapid, and high-sensitivity approach.

Materials and

Methods: : To prepare the electrochemical sensors, an acrylic substrate (0.7 mm thick) was used to fabricate a three-electrode gold electrochemical sensor via laser cutting and sputtering of 10 nm Cr and 100 nm Au. Custom double-stranded DNA (dsDNA) oligonucleotides, representing healthy and sickle cell disease (SCD) sequences with 5′-thiol modifications, were immobilized on the gold working electrode. gRNA targeting the SCD sequence was purchased commercially. A DNA immobilization buffer containing Tris, 1M KH₂PO₄, and TCEP was used to reduce the disulfide bond and enable surface conjugation. The Cas reaction buffer (Tris-HCl and MgCl₂) was used for subsequent enzyme reactions. After soaking and stabilizing the sensor in ultrapure water, EIS was recorded at each step: baseline, DNA immobilization, Cas9 reaction, and washing. EIS measurements were performed continuously at 10 mV amplitude over 100 Hz to 200 kHz. DNA concentrations (1 µM, 10 µM, and 20 µM) and Cas9-gRNA complex concentrations (25nM, 50nM, and 100nM) were optimized based on sensor response, measured as capacitance change and charge transfer resistance (Rct) fitted to a Randles circuit using a custom Python script. The Cas9-gRNA complex was pre-assembled before reaction with immobilized DNA. All assays were conducted at 37 °C and performed in triplicate to ensure reproducibility. Control experiments lacking DNA or the Cas complex were also run. Capacitance changes indicated DNA immobilization efficiency, while Rct changes after Cas9 incubation reflected DNA cleavage. Reaction rates were extracted from Nyquist plots to compare on-target (healthy) versus off-target (SCD) responses.

Results, Conclusions, and Discussions:: The developed electrochemical biosensor demonstrated high reproducibility, with triplicate measurements under all experimental conditions yielding a maximum standard deviation of 0.56. DNA immobilization efficiency was tested with 1 µM, 10 µM, and 20 µM concentrations. The 10 µM dsDNA concentration produced the highest percentage change in capacitance, indicating optimal surface conjugation, while 20 µM showed reduced response, likely due to steric hindrance limiting DNA attachment. CRISPR-Cas9 cleavage activity was quantified by measuring changes in Rct using EIS. The Cas9-DNA reaction buffer generated semi-circular Nyquist plots, suitable for modeling with a Randles circuit. Among Cas9-gRNA concentrations tested (25, 50, and 100nM), 100nM yielded the largest Rct change and clearest differentiation between healthy (on-target) and sickle cell disease (off-target) DNA, establishing it as the optimal concentration. Sensor sensitivity, calculated as the slope of the Rct response, was 0.044 nM⁻¹ for healthy DNA and 0.039 nM⁻¹ for SCD DNA, confirming the platform’s ability to resolve single-nucleotide mismatches with high analytical precision.
This study presents a label-free, non-Faradaic EIS biosensor capable of distinguishing between on-target and off-target Cas9-DNA interactions with single-base resolution. Unlike genome-wide sequencing-based approaches such as GUIDE-seq and DISCOVER-seq, this system enables continuous, time-resolved detection without requiring DNA amplification, labeling, or post-processing. Its low material cost, simplified fabrication, and rapid signal acquisition make it a practical tool for pre-screening guide RNA specificity in CRISPR workflows, particularly in early-stage assay development or quality control.
While the platform offers significant advantages in sensitivity, specificity, and simplicity, its current reliance on low-salt buffers and gold electrodes may limit application under physiological conditions or in complex biological fluids. Future improvements could include evaluating alternative conductive materials, such as carbon-based or polymer electrodes, or integrating the sensor into multiplexed microfluidic systems for higher throughput and broader biological compatibility. Additionally, validating the platform in cell lysates or clinical samples could expand its translational relevance. Overall, this micro-biosensor provides a scalable, accessible, and analytically robust method for CRISPR-Cas9 evaluation, with promising applications in precision medicine, therapeutic development, diagnostic screening, and agricultural biotechnology.

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