CRISPR-Cas9-based electrochemical biosensor for the detection of katG 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 rpoB and katG genes that confer resistance to rifampicin and isoniazid. While many molecular diagnostic tools focus on rpoB, the...
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International Association of Physical Chemists (IAPC)
2025-06-01
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| Series: | ADMET and DMPK |
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| Online Access: | https://pub.iapchem.org/ojs/index.php/admet/article/view/2766 |
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| author | Dika Apriliana Wulandari Muhammad Ihda Hamlu Liwaissunati Zein Salma Nur Zakiyyah Safri Ishmayana Mehmet Ozsoz Yeni Wahyuni Hartati Irkham |
| author_facet | Dika Apriliana Wulandari Muhammad Ihda Hamlu Liwaissunati Zein Salma Nur Zakiyyah Safri Ishmayana Mehmet Ozsoz Yeni Wahyuni Hartati Irkham |
| author_sort | Dika Apriliana Wulandari |
| collection | DOAJ |
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Background and purpose: Multidrug-resistant tuberculosis (MDR-TB) remains a significant challenge in tuberculosis (TB) treatment, driven by simultaneous mutations in the rpoB and katG genes that confer resistance to rifampicin and isoniazid. While many molecular diagnostic tools focus on rpoB, the katG 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 katG mutation. Experimental approach: A guide RNA (gRNA) specific to the mutation site on katG gene was designed using the Benchling CRISPR tool, considering on-target and off-target scores, specificity, and cleavage sites within the Mycobacterium tuberculosis 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(CN6)]3-/4- 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 katG 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 101 to 106 aM. Conclusion: The CRISPR/Cas9-based electrochemical biosensor exhibits high sensitivity and specificity for the detection katG mutation, offering a promising platform for rapid MDR-TB diagnostics.
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| format | Article |
| id | doaj-art-220d5e98d50546efae404f8e7a237193 |
| institution | OA Journals |
| issn | 1848-7718 |
| language | English |
| publishDate | 2025-06-01 |
| publisher | International Association of Physical Chemists (IAPC) |
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| series | ADMET and DMPK |
| spelling | doaj-art-220d5e98d50546efae404f8e7a2371932025-08-20T02:21:14ZengInternational Association of Physical Chemists (IAPC)ADMET and DMPK1848-77182025-06-0113310.5599/admet.2766CRISPR-Cas9-based electrochemical biosensor for the detection of katG gene mutations in isoniazid-resistant tuberculosisDika Apriliana Wulandari0https://orcid.org/0009-0003-8361-2116Muhammad Ihda Hamlu Liwaissunati Zein1https://orcid.org/0000-0001-6656-7315Salma Nur Zakiyyah2https://orcid.org/0000-0003-0985-5980Safri Ishmayana3https://orcid.org/0000-0002-9825-4425Mehmet Ozsoz4https://orcid.org/0000-0003-4037-3445Yeni Wahyuni Hartati5https://orcid.org/0000-0003-1463-6352Irkham6https://orcid.org/0000-0001-9938-2931Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang 45363, IndonesiaDepartment of Chemistry “Giacomo Ciamician”, Alma Mater Studiorum – University of Bologna, Bologna 40126, ItalyDepartment of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang 45363, IndonesiaDepartmeent of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang 45363, IndonesiaDepartment of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang 45363, Indonesia and Department of Biomedical Engineering, Near East University, Mersin 99138, Turkey Department of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang 45363, Indonesia and Study Center of Sensor and Green Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Bandung 40132, IndonesiaDepartment of Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Sumedang 45363, Indonesia and Study Center of Sensor and Green Chemistry, Faculty of Mathematics and Natural Science, Universitas Padjadjaran, Bandung 40132, Indonesia Background and purpose: Multidrug-resistant tuberculosis (MDR-TB) remains a significant challenge in tuberculosis (TB) treatment, driven by simultaneous mutations in the rpoB and katG genes that confer resistance to rifampicin and isoniazid. While many molecular diagnostic tools focus on rpoB, the katG 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 katG mutation. Experimental approach: A guide RNA (gRNA) specific to the mutation site on katG gene was designed using the Benchling CRISPR tool, considering on-target and off-target scores, specificity, and cleavage sites within the Mycobacterium tuberculosis 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(CN6)]3-/4- 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 katG 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 101 to 106 aM. Conclusion: The CRISPR/Cas9-based electrochemical biosensor exhibits high sensitivity and specificity for the detection katG mutation, offering a promising platform for rapid MDR-TB diagnostics. https://pub.iapchem.org/ojs/index.php/admet/article/view/2766Electrochemistryguide RNAferrocene signaldrug-resistant tuberculosis |
| spellingShingle | Dika Apriliana Wulandari Muhammad Ihda Hamlu Liwaissunati Zein Salma Nur Zakiyyah Safri Ishmayana Mehmet Ozsoz Yeni Wahyuni Hartati Irkham CRISPR-Cas9-based electrochemical biosensor for the detection of katG gene mutations in isoniazid-resistant tuberculosis ADMET and DMPK Electrochemistry guide RNA ferrocene signal drug-resistant tuberculosis |
| title | CRISPR-Cas9-based electrochemical biosensor for the detection of katG gene mutations in isoniazid-resistant tuberculosis |
| title_full | CRISPR-Cas9-based electrochemical biosensor for the detection of katG gene mutations in isoniazid-resistant tuberculosis |
| title_fullStr | CRISPR-Cas9-based electrochemical biosensor for the detection of katG gene mutations in isoniazid-resistant tuberculosis |
| title_full_unstemmed | CRISPR-Cas9-based electrochemical biosensor for the detection of katG gene mutations in isoniazid-resistant tuberculosis |
| title_short | CRISPR-Cas9-based electrochemical biosensor for the detection of katG gene mutations in isoniazid-resistant tuberculosis |
| title_sort | crispr cas9 based electrochemical biosensor for the detection of katg gene mutations in isoniazid resistant tuberculosis |
| topic | Electrochemistry guide RNA ferrocene signal drug-resistant tuberculosis |
| url | https://pub.iapchem.org/ojs/index.php/admet/article/view/2766 |
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