CRISPR-Cas9-based electrochemical biosensor for the detection ofgene mutations in isoniazid-resistant tuberculosis.

BACKGROUND AND PURPOSE: Multidrug-resistant tuberculosis (MDR-TB) remains a significant challenge in tuberculosis (TB) treatment, driven by simultaneous mutations in theandgenes that confer resistance to rifampicin and isoniazid. While many molecular diagnostic tools focus on, thegene is often overlooked despite its critical role in confirming MDR-TB. This study aims to develop a CRISPR/Cas9-based electrochemical biosensor for the rapid and selective detection ofmutation.

EXPERIMENTAL APPROACH: A guide RNA (gRNA) specific to the mutation site ongene was designed using the Benchling CRISPR tool, considering on-target and off-target scores, specificity, and cleavage sites within thegenome. The selected gRNA achieved the highest on-target score of 61.2 and an off-target score of 49.0 at cut position 2928, with a PAM sequence of AGG. Its cleavage efficiency was validated experimentally using an electrochemical biosensing platform incorporating a gold-modified screen-printed carbon electrode (SPCE/Au). Redox response enhancement by [Fe(CN)]confirmed the improved performance of the electrode.

KEY RESULTS: The biosensor system detects the target DNA through hybridization with DNA probe-Fc, forming double-stranded DNA (dsDNA) that is recognized and cleaved by the Cas9/gRNA complex. This cleavage significantly reduces the ferrocene oxidation signal, indicating the presence of amutation. Non-mutated target DNA produces a nondetectable ferrocene signal, suggesting that the Cas9 enzyme may remain bound to the electrode without cleavage. The CRISPR/Cas9 electrochemical biosensor demonstrated a low detection limit of 7.5530 aM and a detection range of 10to 10aM.

CONCLUSION: The CRISPR/Cas9-based electrochemical biosensor exhibits high sensitivity and specificity for the detectionmutation, offering a promising platform for rapid MDR-TB diagnostics.