{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,14]],"date-time":"2026-08-14T19:35:08Z","timestamp":1786736108569,"version":"build-2736575974"},"reference-count":22,"publisher":"Frontiers Media SA","license":[{"start":{"date-parts":[[2025,5,21]],"date-time":"2025-05-21T00:00:00Z","timestamp":1747785600000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":["frontiersin.org"],"crossmark-restriction":true},"short-container-title":["Front. Immunol."],"abstract":"<jats:p><jats:italic>Mycobacterium tuberculosis<\/jats:italic> (<jats:italic>Mtb<\/jats:italic>) invades and survives inside macrophages, evading detection and resisting antibiotic treatment, which results in severe clinical consequences such as fatal respiratory failure and systemic inflammation. Rapid and specific detection of intracellular <jats:italic>Mtb<\/jats:italic> is crucial for accurate diagnosis and optimizing treatment strategies. In this study, we developed a one-step CRISPR\/Cas12a assay targeting the <jats:italic>IS6110<\/jats:italic> gene for the specific and rapid detection of intracellular <jats:italic>Mtb<\/jats:italic>. Upon efficient delivery into RAW264.7 macrophages, the assay enabled direct visualization of <jats:italic>Mtb IS6110<\/jats:italic> nucleic acid, generating detectable fluorescence signals. The diagnostic performance was further validated using bronchoalveolar lavage fluid (BALF) samples from clinical participants, achieving a sensitivity of 94%, which surpassed conventional methods such as culture (67%) and Xpert (78%), while maintaining a specificity of 100%. This CRISPR\/Cas12a-based assay offers a highly sensitive, rapid, and innovative approach for intracellular <jats:italic>Mtb<\/jats:italic> detection, with significant potential to enhance tuberculosis diagnostic methodologies and improve clinical outcomes.<\/jats:p>","DOI":"10.3389\/fimmu.2025.1597654","type":"journal-article","created":{"date-parts":[[2025,5,21]],"date-time":"2025-05-21T05:26:07Z","timestamp":1747805167000},"update-policy":"https:\/\/doi.org\/10.3389\/crossmark-policy","source":"Crossref","is-referenced-by-count":4,"title":["Ultra-sensitive in situ detection of intracellular Mycobacterium tuberculosis with CRISPR\/Cas12a"],"prefix":"10.3389","volume":"16","author":[{"given":"Zhiqiang","family":"Lin","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhe","family":"Song","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hong","family":"Yu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yang","family":"Zhou","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Deliang","family":"Liu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Peize","family":"Zhang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Li","family":"Wei","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guiqin","family":"Dai","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Guangyan","family":"Liang","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Zhuojun","family":"He","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Xiaorong","family":"Hu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Yuying","family":"Chen","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Pengfei","family":"Zhao","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Hongzhou","family":"Lu","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mingbin","family":"Zheng","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1965","published-online":{"date-parts":[[2025,5,21]]},"reference":[{"key":"B1","doi-asserted-by":"publisher","first-page":"685","DOI":"10.1038\/s41579-022-00731-y","article-title":"Anti-tuberculosis treatment strategies and drug development: challenges and priorities","volume":"20","author":"Dartois","year":"2022","journal-title":"Nat Rev Microbiol"},{"key":"B2","doi-asserted-by":"publisher","DOI":"10.1038\/s41579-022-00763-4","article-title":"Immune evasion and provocation by Mycobacterium tuberculosis","volume":"20","author":"Chandra","year":"2022","journal-title":"Nat Rev Microbiol"},{"key":"B3","doi-asserted-by":"publisher","first-page":"370","DOI":"10.1038\/s41577-024-01124-3","article-title":"How macrophage heterogeneity affects tuberculosis disease and therapy","volume":"25","author":"Russell","year":"2025","journal-title":"Nat Rev Immunol"},{"key":"B4","doi-asserted-by":"publisher","DOI":"10.1016\/j.autrev.2025.103758","article-title":"Assessment and management for latent tuberculosis before advanced therapies for immune-mediated inflammatory diseases: a comprehensive review","volume":"24","author":"Jha","year":"2025","journal-title":"Autoimmun Rev"},{"key":"B5","doi-asserted-by":"publisher","DOI":"10.1016\/S2666-5247(23)00367-1","article-title":"Accuracy of the tuberculosis molecular bacterial load assay to diagnose and monitor response to anti-tuberculosis therapy: a longitudinal comparative study with standard-of-care smear microscopy, Xpert Mtb\/Rif Ultra, and culture in Uganda","volume":"5","author":"Musisi","year":"2024","journal-title":"Lancet Microbe"},{"key":"B6","doi-asserted-by":"publisher","DOI":"10.1128\/JCM.01582-19","article-title":"Advances in molecular diagnosis of Tuberculosis","volume":"58","author":"MacLean","year":"2020","journal-title":"J Clin Microbiol"},{"key":"B7","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2024.1454076","article-title":"Era-Crispr\/Cas12a System: A Rapid, Highly sensitive and specific assay for","volume":"14","author":"Gan","year":"2024","journal-title":"Mycobacterium tuberculosis. Front Cell Infect Microbiol"},{"key":"B8","doi-asserted-by":"publisher","DOI":"10.1038\/s41587-019-0249-1","article-title":"Visualizing RNA dynamics in live cells with bright and stable fluorescent RNAs","volume":"37","author":"Chen","year":"2019","journal-title":"Nat Biotechnol"},{"key":"B9","doi-asserted-by":"publisher","DOI":"10.1038\/s41422-022-00712-z","article-title":"CRISPR-Fisher enables high-sensitivity imaging of nonrepetitive DNA in living cells through phase separation-mediated signal amplification","volume":"32","author":"Lyu","year":"2022","journal-title":"Cell Res"},{"key":"B10","doi-asserted-by":"publisher","first-page":"1166","DOI":"10.1038\/s41467-025-56516-3","article-title":"Tunable control of Cas12 activity promotes universal and fast one-pot nucleic acid detection","volume":"16","author":"Cheng","year":"2025","journal-title":"Nat Commun"},{"key":"B11","doi-asserted-by":"publisher","first-page":"gkaf017","DOI":"10.1093\/nar\/gkaf017","article-title":"Proximity-activated guide RNA of CRISPR-Cas12a for programmable diagnostic detection and gene regulation","volume":"53","author":"Hu","year":"2025","journal-title":"Nucleic Acids Res"},{"key":"B12","doi-asserted-by":"publisher","first-page":"54","DOI":"10.1016\/j.jinf.2021.04.032","article-title":"Tb-Quick: CRISPR-Cas12b-assisted rapid and sensitive detection of","volume":"83","author":"Sam","year":"2021","journal-title":"Mycobacterium tuberculosis. J Infect"},{"key":"B13","doi-asserted-by":"publisher","DOI":"10.3389\/fbioe.2023.1233353","article-title":"Development and preliminary assessment of a CRISPR-Cas12a-based multiplex detection of Mycobacterium tuberculosis complex","volume":"11","author":"Xiao","year":"2023","journal-title":"Front Bioeng Biotechnol"},{"key":"B14","doi-asserted-by":"publisher","first-page":"e0092323","DOI":"10.1128\/jcm.00923-23","article-title":"Rapid detection of Mycobacterium tuberculosis in sputum using CRISPR-Cas12b combined with cross-priming amplification in a single reaction","volume":"62","author":"Peng","year":"2024","journal-title":"J Clin Microbiol"},{"key":"B15","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2019.1664939","article-title":"CRISPR-based rapid and ultra-sensitive diagnostic test for Mycobacterium tuberculosis","volume":"8","author":"Ai","year":"2019","journal-title":"Emerg Microbes Infect"},{"key":"B16","doi-asserted-by":"publisher","DOI":"10.1080\/09273948.2020.1787462","article-title":"IS1081-based multi-targeted LAMP: an opportunity to detect tubercular uveitis","volume":"30","author":"Sharma","year":"2022","journal-title":"Ocul Immunol Inflammation"},{"key":"B17","doi-asserted-by":"publisher","DOI":"10.1080\/22221751.2023.2239940","article-title":"The interaction of macrophages and CD8 T cells in bronchoalveolar lavage fluid is associated with latent tuberculosis infection","volume":"12","author":"Yang","year":"2023","journal-title":"Emerg Microbes Infect"},{"key":"B18","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2015.05.005","article-title":"Cyclic GMP-AMP synthase is an innate immune DNA sensor for","volume":"17","author":"Collins","year":"2015","journal-title":"Mycobacterium tuberculosis. Cell Host Microbe"},{"key":"B19","doi-asserted-by":"publisher","DOI":"10.1016\/j.chom.2012.03.007","article-title":"Mycobacterium tuberculosis activates the DNA-dependent cytosolic surveillance pathway within macrophages","volume":"11","author":"Manzanillo","year":"2012","journal-title":"Cell Host Microbe"},{"key":"B20","doi-asserted-by":"publisher","first-page":"e2461","DOI":"10.1371\/journal.pone.0002461","article-title":"Exosomes derived from M. bovis BCG-infected macrophages activate antigen-specific CD4+ and CD8+ T cells in vitro and in vivo","volume":"3","author":"Giri","year":"2008","journal-title":"PloS One"},{"key":"B21","doi-asserted-by":"publisher","DOI":"10.3389\/fcimb.2022.912831","article-title":"Extracellular vesicles in mycobacteria and tuberculosis","volume":"12","author":"Mehaffy","year":"2022","journal-title":"Front Cell Infect Microbiol"},{"key":"B22","doi-asserted-by":"publisher","DOI":"10.3389\/fmicb.2022.1039040","article-title":"Extracellular vesicle-mediated regulation of macrophage polarization in bacterial infections","volume":"13","author":"Qu","year":"2022","journal-title":"Front Microbiol"}],"container-title":["Frontiers in Immunology"],"original-title":[],"link":[{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1597654\/full","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2025,5,21]],"date-time":"2025-05-21T05:26:08Z","timestamp":1747805168000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.frontiersin.org\/articles\/10.3389\/fimmu.2025.1597654\/full"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2025,5,21]]},"references-count":22,"alternative-id":["10.3389\/fimmu.2025.1597654"],"URL":"https:\/\/doi.org\/10.3389\/fimmu.2025.1597654","relation":{},"ISSN":["1664-3224"],"issn-type":[{"value":"1664-3224","type":"electronic"}],"subject":[],"published":{"date-parts":[[2025,5,21]]},"article-number":"1597654"}}