Biophysical characterization of spCas9 binding and cleavage using real-time electronic biosensors

CRISPR-Cas9 enables curative genome editing but requires precise control of target recognition, particularly when single-nucleotide polymorphisms (SNPs) influence specificity. Conventional biochemical and optical assays often rely on endpoint or ensemble-averaged measurements and therefore fail to resolve the real-time binding dynamics underlying off-target interactions. Here, we report a label-free, non-faradaic electrochemical impedance spectroscopy (nfEIS) platform that directly monitors spCas9-gRNA interactions on gold microelectrodes with single-base resolution at the sickle cell disease (SCD) locus. A guide RNA was designed to perfectly match the SCD mutation (A to T) while introducing a single PAM-proximal mismatch with the wild-type DNA (WD) sequence. Using 63-nucleotide synthetic DNA substrates representing SCD and WD targets, concentration-dependent binding assays were performed to extract equilibrium parameters. Hill-model analysis revealed higher affinity for the SCD target (k _D = 0.09 nM) relative to WD (k _D = 0.3 nM), confirming strong on-target binding and weakened interaction at the mismatch site. Magnesium dependence evaluation showed that 5 mM Mg²⁺ enhanced discrimination by stabilizing on-target complexes while destabilizing mismatched binding, whereas at 1 mM Mg²⁺ this selectivity was lost. Time-resolved kinetic measurements using 1 nM spCas9 and exponential fitting of the curve revealed rapid association (t _1/2 = 1.85 min) and dissociation rates (t _1/2 = 5.24 min) for SCD, consistent with efficient R-loop formation. In contrast, the WD target exhibited slower association (t _1/2 = 2.68 min) and recurring transient binding with delayed dissociation (t _1/2 = 34.38 min), corroborated by endpoint gel assays. Cas9 lacking gRNA showed only weak, unstable interactions. Overall, these results demonstrate that Cas9 specificity arises from both affinity differences and binding-residence dynamics. nfEIS thus provides a real-time, label-free platform for probing Cas9 fidelity, Mg²⁺-dependent activation, and gRNA design for therapeutic genome editing and diagnostics.