{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T05:16:33Z","timestamp":1788412593212,"version":"build-2803163510"},"reference-count":59,"publisher":"MDPI AG","issue":"9","license":[{"start":{"date-parts":[[2026,8,28]],"date-time":"2026-08-28T00:00:00Z","timestamp":1787875200000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Curr. Issues Mol. Biol.","CIMB"],"abstract":"<jats:p>This review systematically summarizes recent advances in CRISPR-Cas12b-based pathogen nucleic acid detection. Starting with an overview of conventional nucleic acid detection methods and core CRISPR-Cas mechanisms, we highlight the unique properties that distinguish Cas12b from other Cas effectors. We elaborate on the working principles of the CRISPR-Cas12b system and present comparative structural and functional analyses with other Cas variants to underscore its distinctive molecular architecture and enzymatic properties. A comprehensive evaluation of current applications demonstrates the efficacy of CRISPR-Cas12b-based diagnostics across diverse pathogens, including viruses, bacteria, and parasites, with a specific focus on its integration with isothermal amplification techniques. We further examine the translational potential of CRISPR-based diagnostics in clinical settings, while critically analyzing persistent technical challenges including off-target effects, signal amplification limitations, and sample preparation requirements. Targeted strategic recommendations are proposed to optimize detection sensitivity, develop multiplexed detection platforms, and implement point-of-care testing configurations. This review aims to systematically correlate the unique characteristics of Cas12b with its broad diagnostic applications, thereby addressing key research gaps in its progression toward clinical validation and field deployment. It also provides a targeted framework to accelerate the translation of this technology from laboratory platforms to practical diagnostic solutions.<\/jats:p>","DOI":"10.3390\/cimb48090875","type":"journal-article","created":{"date-parts":[[2026,9,1]],"date-time":"2026-09-01T11:33:32Z","timestamp":1788262412000},"page":"875","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":0,"title":["Research Progress in Applications of the CRISPR-Cas12b System in Pathogen Nucleic Acid Detection"],"prefix":"10.3390","volume":"48","author":[{"ORCID":"https:\/\/orcid.org\/0009-0002-2422-2946","authenticated-orcid":false,"given":"Jiangying","family":"Li","sequence":"first","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-5076-9211","authenticated-orcid":false,"given":"Xueyong","family":"Zhang","sequence":"additional","affiliation":[{"name":"The Academy of Animal and Veterinary Sciences, Qinghai University, Xining 810016, China"},{"name":"Qinghai Provincial Key Laboratory of Pathogen Diagnosis for Animal Disease and Green Technical Research for Prevention and Control, Xining 810016, China"}],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"1968","published-online":{"date-parts":[[2026,8,28]]},"reference":[{"key":"ref_1","doi-asserted-by":"crossref","first-page":"118174","DOI":"10.1016\/j.trac.2025.118174","article-title":"CRISPR-Cas technology for rapid detection of pathogens","volume":"185","author":"Daneshgar","year":"2025","journal-title":"Trac-Trends Anal. Chem."},{"key":"ref_2","doi-asserted-by":"crossref","unstructured":"Bhardwaj, P., Kant, R., Behera, S.P., Dwivedi, G.R., and Singh, R. (2022). Next-Generation Diagnostic with CRISPR\/Cas: Beyond Nucleic Acid Detection. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23116052"},{"key":"ref_3","doi-asserted-by":"crossref","unstructured":"Puig-Serra, P., Casado-Rosas, M.C., Martinez-Lage, M., Olalla-Sastre, B., Alonso-Yanez, A., Torres-Ruiz, R., and Rodriguez-Perales, S. (2022). CRISPR Approaches for the Diagnosis of Human Diseases. Int. J. Mol. Sci., 23.","DOI":"10.3390\/ijms23031757"},{"key":"ref_4","doi-asserted-by":"crossref","first-page":"21","DOI":"10.32604\/biocell.2024.056698","article-title":"Advances in CRISPR-based gene editing technology and its application in nucleic acid detection","volume":"49","author":"Chen","year":"2025","journal-title":"Biocell"},{"key":"ref_5","doi-asserted-by":"crossref","first-page":"123388","DOI":"10.1016\/j.talanta.2022.123388","article-title":"Integrating CRISPR\/Cas within isothermal amplification for point-of-Care Assay of nucleic acid","volume":"243","author":"Zhang","year":"2022","journal-title":"Talanta"},{"key":"ref_6","doi-asserted-by":"crossref","first-page":"100471","DOI":"10.1016\/j.cobme.2023.100471","article-title":"Multiplexed CRISPR-based methods for pathogen nucleic acid detection","volume":"27","author":"Lamb","year":"2023","journal-title":"Curr. Opin. Biomed. Eng."},{"key":"ref_7","doi-asserted-by":"crossref","unstructured":"Yuan, B.W., Yuan, C.C., Li, L.L., Long, M., and Chen, Z.L. (2022). Application of the CRISPR\/Cas System in Pathogen Detection: A Review. Molecules, 27.","DOI":"10.3390\/molecules27206999"},{"key":"ref_8","doi-asserted-by":"crossref","first-page":"38989","DOI":"10.1021\/acsomega.4c05489","article-title":"CHAMP: A Centrifugal Microfluidics-Based CRISPR\/Cas12b-Combined Real-Time LAMP One-Pot Method for Mycoplasma pneumoniae Infection Diagnosis","volume":"9","author":"Jing","year":"2024","journal-title":"Acs Omega"},{"key":"ref_9","doi-asserted-by":"crossref","unstructured":"Aman, R., Marsic, T., Rao, G.S., Mahas, A., Ali, Z., Alsanea, M., Al-Qahtani, A., Alhamlan, F., and Mahfouz, M. (2022). ISCAN-V2: A One-Pot RT-RPA-CRISPR\/Cas12b Assay for Point-of-Care SARS-CoV-2 Detection. Front. Bioeng. Biotechnol., 9.","DOI":"10.3389\/fbioe.2021.800104"},{"key":"ref_10","doi-asserted-by":"crossref","first-page":"156","DOI":"10.4161\/rna.27990","article-title":"CRISPR adaptive immune systems of Archaea","volume":"11","author":"Vestergaard","year":"2014","journal-title":"Rna Biol."},{"key":"ref_11","doi-asserted-by":"crossref","unstructured":"Lv, H.L., Wang, J., Zhang, J., Chen, Y.J., Yin, L., Jin, D., Gu, D.Y., Zhao, H.L., Xu, Y., and Wang, J. (2021). Definition of CRISPR Cas12a Trans-Cleavage Units to Facilitate CRISPR Diagnostics. Front. Microbiol., 12.","DOI":"10.3389\/fmicb.2021.766464"},{"key":"ref_12","doi-asserted-by":"crossref","first-page":"3723","DOI":"10.1007\/s11033-023-08240-8","article-title":"Research progress on nucleic acid detection and genome editing of CRISPR\/Cas12 system","volume":"50","author":"Yang","year":"2023","journal-title":"Mol. Biol. Rep."},{"key":"ref_13","doi-asserted-by":"crossref","first-page":"14209","DOI":"10.1021\/acs.analchem.3c01809","article-title":"PNA-Pdx: Versatile Peptide Nucleic Acid-Based Detection of Nucleic Acids and SNPs","volume":"95","author":"Jiang","year":"2023","journal-title":"Anal. Chem."},{"key":"ref_14","doi-asserted-by":"crossref","first-page":"e01271-24","DOI":"10.1128\/spectrum.01271-24","article-title":"One-tube detection of Salmonella Typhimurium using LAMP and CRISPR-Cas12b","volume":"12","author":"Gong","year":"2024","journal-title":"Microbiol. Spectr."},{"key":"ref_15","doi-asserted-by":"crossref","first-page":"1814","DOI":"10.1016\/j.cell.2016.11.053","article-title":"PAM-Dependent Target DNA Recognition and Cleavage by C2c1 CRISPR-Cas Endonuclease","volume":"167","author":"Yang","year":"2016","journal-title":"Cell"},{"key":"ref_16","doi-asserted-by":"crossref","first-page":"63","DOI":"10.1038\/s41421-018-0069-3","article-title":"Repurposing CRISPR-Cas12b for mammalian genome engineering","volume":"4","author":"Teng","year":"2018","journal-title":"Cell Discov."},{"key":"ref_17","doi-asserted-by":"crossref","first-page":"385","DOI":"10.1016\/j.molcel.2015.10.008","article-title":"Discovery and Functional Characterization of Diverse Class 2 CRISPR-Cas Systems","volume":"60","author":"Shmakov","year":"2015","journal-title":"Mol. Cell"},{"key":"ref_18","doi-asserted-by":"crossref","first-page":"gkaf519","DOI":"10.1093\/nar\/gkaf519","article-title":"Catalytic-state structure of Candidatus Hydrogenedentes Cas12b revealed by cryo-EM studies","volume":"53","author":"Li","year":"2025","journal-title":"Nucleic Acids Res."},{"key":"ref_19","doi-asserted-by":"crossref","first-page":"310","DOI":"10.1016\/j.molcel.2016.11.040","article-title":"C2c1-sgRNA Complex Structure Reveals RNA-Guided DNA Cleavage Mechanism","volume":"65","author":"Liu","year":"2017","journal-title":"Mol. Cell"},{"key":"ref_20","doi-asserted-by":"crossref","first-page":"1172","DOI":"10.1038\/s41594-023-01042-3","article-title":"Mechanistic and evolutionary insights into a type V-M CRISPR\u2013Cas effector enzyme","volume":"30","author":"Omura","year":"2023","journal-title":"Nat. Struct. Mol. Biol."},{"key":"ref_21","doi-asserted-by":"crossref","first-page":"195","DOI":"10.1039\/D5ME00140D","article-title":"Exploring the temperature stability of CRISPR-Cas12b using molecular dynamics simulations","volume":"11","author":"Jia","year":"2026","journal-title":"Mol. Syst. Des. Eng."},{"key":"ref_22","doi-asserted-by":"crossref","first-page":"117","DOI":"10.1016\/j.sbi.2016.12.007","article-title":"Physical and molecular bases of protein thermal stability and cold adaptation","volume":"42","author":"Pucci","year":"2017","journal-title":"Curr. Opin. Struct. Biol."},{"key":"ref_23","doi-asserted-by":"crossref","first-page":"108719","DOI":"10.1016\/j.biotechadv.2025.108719","article-title":"A comprehensive review of methodological and technological advancement in PCR during the last 15 years","volume":"85","author":"Li","year":"2025","journal-title":"Biotechnol. Adv."},{"key":"ref_24","doi-asserted-by":"crossref","first-page":"986","DOI":"10.1101\/gr.6.10.986","article-title":"Real time quantitative PCR","volume":"6","author":"Heid","year":"1996","journal-title":"Genome Res."},{"key":"ref_25","doi-asserted-by":"crossref","first-page":"165","DOI":"10.1128\/CMR.19.1.165-256.2006","article-title":"Real-time PCR in clinical microbiology: Applications for routine laboratory testing","volume":"19","author":"Espy","year":"2006","journal-title":"Clin. Microbiol. Rev."},{"key":"ref_26","doi-asserted-by":"crossref","first-page":"e704","DOI":"10.1016\/S2666-5247(21)00214-7","article-title":"Diagnostic performances of common nucleic acid tests for SARS-CoV-2 in hospitals and clinics: A systematic review and meta-analysis","volume":"2","author":"Au","year":"2021","journal-title":"Lancet Microbe"},{"key":"ref_27","doi-asserted-by":"crossref","first-page":"132","DOI":"10.1186\/s13059-019-1742-z","article-title":"CDetection: CRISPR-Cas12b-based DNA detection with sub-attomolar sensitivity and single-base specificity","volume":"20","author":"Teng","year":"2019","journal-title":"Genome Biol."},{"key":"ref_28","doi-asserted-by":"crossref","first-page":"2228","DOI":"10.1021\/acssynbio.9b00209","article-title":"HOLMESv2: A CRISPR-Cas12b-Assisted Platform for Nucleic Acid Detection and DNA Methylation Quantitation","volume":"8","author":"Li","year":"2019","journal-title":"ACS Synth. Biol."},{"key":"ref_29","doi-asserted-by":"crossref","unstructured":"Chen, X., Tan, Y., Wang, S., Wu, X., Liu, R., Yang, X., Wang, Y., Tai, J., and Li, S. (2021). A CRISPR-Cas12b\u2013Based Platform for Ultrasensitive, Rapid, and Highly Specific Detection of Hepatitis B Virus Genotypes B and C in Clinical Application. Front. Bioeng. Biotechnol., 9.","DOI":"10.3389\/fbioe.2021.743322"},{"key":"ref_30","doi-asserted-by":"crossref","first-page":"e06716","DOI":"10.1002\/advs.202506716","article-title":"One-Pot Isothermal Nucleic Acid Amplification Assisted CRISPR\/Cas Detection Technology: Challenges, Strategies, and Perspectives","volume":"12","author":"Liu","year":"2025","journal-title":"Adv. Sci."},{"key":"ref_31","doi-asserted-by":"crossref","first-page":"125616","DOI":"10.1016\/j.talanta.2023.125616","article-title":"Rapid and sensitive detection of nucleic acids using an RAA-CRISPR\/ Cas12b one-pot detection assay (Rcod)","volume":"271","author":"Lin","year":"2024","journal-title":"Talanta"},{"key":"ref_32","doi-asserted-by":"crossref","first-page":"5519","DOI":"10.1039\/D4AY00977K","article-title":"A LAMP-CRISPR\/Cas12b rapid detection platform for canine parvovirus detection","volume":"16","author":"Chen","year":"2024","journal-title":"Anal. Methods"},{"key":"ref_33","doi-asserted-by":"crossref","first-page":"103926","DOI":"10.1016\/j.ebiom.2022.103926","article-title":"A thermostable Cas12b from Brevibacillus leverages one-pot discrimination of SARS-CoV-2 variants of concern","volume":"77","author":"Nguyen","year":"2022","journal-title":"eBioMedicine"},{"key":"ref_34","doi-asserted-by":"crossref","first-page":"339494","DOI":"10.1016\/j.aca.2022.339494","article-title":"A warm-start digital CRISPR\/Cas-based method for the quantitative detection of nucleic acids","volume":"1196","author":"Wu","year":"2022","journal-title":"Anal. Chim. Acta"},{"key":"ref_35","doi-asserted-by":"crossref","first-page":"101037","DOI":"10.1016\/j.xcrm.2023.101037","article-title":"Engineering highly thermostable Cas12b via de novo structural analyses for one-pot detection of nucleic acids","volume":"4","author":"Nguyen","year":"2023","journal-title":"Cell Rep. Med."},{"key":"ref_36","doi-asserted-by":"crossref","first-page":"107838","DOI":"10.1016\/j.jsb.2022.107838","article-title":"Structural principles of CRISPR-Cas enzymes used in nucleic acid detection","volume":"214","author":"Das","year":"2022","journal-title":"J. Struct. Biol."},{"key":"ref_37","doi-asserted-by":"crossref","first-page":"113541","DOI":"10.1016\/j.bios.2021.113541","article-title":"CRISPR-Cas based virus detection: Recent advances and perspectives","volume":"193","author":"Yin","year":"2021","journal-title":"Biosens. Bioelectron."},{"key":"ref_38","doi-asserted-by":"crossref","first-page":"816","DOI":"10.1126\/science.1225829","article-title":"A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity","volume":"337","author":"Jinek","year":"2012","journal-title":"Science"},{"key":"ref_39","doi-asserted-by":"crossref","first-page":"436","DOI":"10.1126\/science.aar6245","article-title":"CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity","volume":"360","author":"Chen","year":"2018","journal-title":"Science"},{"key":"ref_40","doi-asserted-by":"crossref","first-page":"212","DOI":"10.1038\/s41467-018-08224-4","article-title":"Engineering of CRISPR-Cas12b for human genome editing","volume":"10","author":"Strecker","year":"2019","journal-title":"Nat. Commun."},{"key":"ref_41","doi-asserted-by":"crossref","first-page":"438","DOI":"10.1126\/science.aam9321","article-title":"Nucleic acid detection with CRISPR-Cas13a\/C2c2","volume":"356","author":"Gootenberg","year":"2017","journal-title":"Science"},{"key":"ref_42","doi-asserted-by":"crossref","first-page":"839","DOI":"10.1126\/science.aav4294","article-title":"Programmed DNA destruction by miniature CRISPR-Cas14 enzymes","volume":"362","author":"Harrington","year":"2018","journal-title":"Science"},{"key":"ref_43","doi-asserted-by":"crossref","first-page":"1430","DOI":"10.1021\/acssensors.0c02312","article-title":"CRISPR Systems for COVID-19 Diagnosis","volume":"6","author":"Rahimi","year":"2021","journal-title":"ACS Sens."},{"key":"ref_44","doi-asserted-by":"crossref","first-page":"173","DOI":"10.1146\/annurev-virology-110615-035747","article-title":"Genomic Analysis of Viral Outbreaks","volume":"3","author":"Wohl","year":"2016","journal-title":"Annu. Rev. Virol."},{"key":"ref_45","doi-asserted-by":"crossref","first-page":"031409","DOI":"10.1063\/5.0142494","article-title":"Rapid and ultrasensitive detection of mpox virus using CRISPR\/Cas12b-empowered graphene field-effect transistors","volume":"10","author":"Wang","year":"2023","journal-title":"Appl. Phys. Rev."},{"key":"ref_46","doi-asserted-by":"crossref","first-page":"418","DOI":"10.1007\/s43657-025-00240-x","article-title":"SIMPLE: A One-Pot RPA-Cas12b Method for Single Nucleotide Variations Identification with an Expanded Scope and Clinical Applications","volume":"5","author":"Guo","year":"2025","journal-title":"Phenomics"},{"key":"ref_47","doi-asserted-by":"crossref","first-page":"3260","DOI":"10.1016\/j.scib.2024.06.012","article-title":"Discovery and engineering of ChCas12b for precise genome editing","volume":"69","author":"Wei","year":"2024","journal-title":"Sci. Bull."},{"key":"ref_48","doi-asserted-by":"crossref","unstructured":"Gong, J.S., Zhang, D., Fu, L.X., Dong, Y.Y., Wu, K., Dou, X.H., and Wang, C.M. (2024). A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection. Microorganisms, 12.","DOI":"10.3390\/microorganisms12030519"},{"key":"ref_49","doi-asserted-by":"crossref","first-page":"110534","DOI":"10.1016\/j.foodcont.2024.110534","article-title":"Rapid and accurate detection of Salmonella enterica serovar Enteritidis using a one-step LAMP-CRISPR\/Cas12b method","volume":"164","author":"Gong","year":"2024","journal-title":"Food Control"},{"key":"ref_50","doi-asserted-by":"crossref","first-page":"136912","DOI":"10.1016\/j.ijbiomac.2024.136912","article-title":"Rapid and multiple visual detection of Fasciola hepatica in feces via recombinase polymerase amplification integrated with CRISPR\/Cas12a technology","volume":"282","author":"Yang","year":"2024","journal-title":"Int. J. Biol. Macromol."},{"key":"ref_51","doi-asserted-by":"crossref","first-page":"e00923-23","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":"ref_52","doi-asserted-by":"crossref","first-page":"94","DOI":"10.1186\/s40249-024-01266-5","article-title":"A novel single-tube LAMP-CRISPR\/Cas12b method for rapid and visual detection of zoonotic Toxoplasma gondii in the environment","volume":"13","author":"Liang","year":"2024","journal-title":"Infect. Dis. Poverty"},{"key":"ref_53","doi-asserted-by":"crossref","unstructured":"Alvarez-Rodr\u00edguez, A., Li, Z., Jin, B.K., Stijlemans, B., Geldhof, P., and Magez, S. (2025). A CRISPR-Cas-based recombinase polymerase amplification assay for ultra-sensitive detection of active Trypanosoma brucei evansi infections. Front. Mol. Biosci., 12.","DOI":"10.3389\/fmolb.2025.1512970"},{"key":"ref_54","doi-asserted-by":"crossref","unstructured":"Li, X., Dang, Z., Tang, W., Zhang, H., Shao, J., Jiang, R., Zhang, X., and Huang, F. (2024). Detection of Parasites in the Field: The Ever-Innovating CRISPR\/Cas12a. Biosensors, 14.","DOI":"10.3390\/bios14030145"},{"key":"ref_55","doi-asserted-by":"crossref","unstructured":"Ulloa, D., N\u00fa\u00f1ez, C., Matamala, R., San Mart\u00edn, A., P\u00e1ez-De \u00c1vila, D., Mercado-Vides, J., Narv\u00e1ez, J., Aguirre, J., Effer, B., and Iturrieta-Gonz\u00e1lez, I. (2025). CRISPR-Cas12 Application for the Detection of Pneumocystis jirovecii in Immunodepression Patients Through Fluorescent and Lateral Flow Colorimetric Assay. Int. J. Mol. Sci., 26.","DOI":"10.3390\/ijms26178732"},{"key":"ref_56","doi-asserted-by":"crossref","first-page":"6392","DOI":"10.1038\/s41467-025-61748-4","article-title":"Regulating cleavage activity and enabling microRNA detection with split sgRNA in Cas12b","volume":"16","author":"Wang","year":"2025","journal-title":"Nat. Commun."},{"key":"ref_57","doi-asserted-by":"crossref","first-page":"2824","DOI":"10.1021\/acssynbio.1c00401","article-title":"Advances in Field Detection Based on CRISPR\/Cas System","volume":"10","author":"Yuan","year":"2021","journal-title":"ACS Synth. Biol."},{"key":"ref_58","doi-asserted-by":"crossref","first-page":"e03475-22","DOI":"10.1128\/spectrum.03475-22","article-title":"Development of CRISPR\/Cas12b-Based Multiple Cross Displacement Amplification Technique for the Detection of Mycobacterium tuberculosis Complex in Clinical Settings","volume":"11","author":"Yang","year":"2023","journal-title":"Microbiol. Spectr."},{"key":"ref_59","first-page":"3145","article-title":"Ultraspecific One-Pot CRISPR-Based \u201cGreen-Yellow-Red\u201d Multiplex Detection Strategy Integrated with Portable Cartridge for Point-of-Care Diagnosis","volume":"96","author":"Chen","year":"2024","journal-title":"Anal. Chem."}],"container-title":["Current Issues in Molecular Biology"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.mdpi.com\/1467-3045\/48\/9\/875\/pdf","content-type":"unspecified","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2026,9,3]],"date-time":"2026-09-03T04:33:16Z","timestamp":1788409996000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.mdpi.com\/1467-3045\/48\/9\/875"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2026,8,28]]},"references-count":59,"journal-issue":{"issue":"9","published-online":{"date-parts":[[2026,9]]}},"alternative-id":["cimb48090875"],"URL":"https:\/\/doi.org\/10.3390\/cimb48090875","relation":{},"ISSN":["1467-3045"],"issn-type":[{"value":"1467-3045","type":"electronic"}],"subject":[],"published":{"date-parts":[[2026,8,28]]}}}