CRISPR-Cas9-based electrochemical biosensor for the detection of gene 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 the and genes that confer resistance to rifampicin and isoniazid. While many molecular diagnostic tools focus on , the gene 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 of mutation.

EXPERIMENTAL APPROACH

A guide RNA (gRNA) specific to the mutation site on gene was designed using the Benchling CRISPR tool, considering on-target and off-target scores, specificity, and cleavage sites within the genome. 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 a mutation. 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 10 to 10 aM.

CONCLUSION

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