Advancing CRISPR/Cas Biosensing with Integrated Devices

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Learn More CiteCitationCitation and abstractCitation and referencesMore citation options ShareShare onFacebookX (Twitter)WeChatLinkedInRedditEmailJump toExpandCollapse EditorialFebruary 28, 2025Advancing CRISPR/Cas Biosensing with Integrated DevicesClick to copy article linkArticle link copied!Guozhen Liu*Guozhen LiuIntegrated Devices and Intelligent Diagnosis (ID2) Laboratory, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, China*Email: [email protected]More by Guozhen Liuhttps://orcid.org/0000-0002-0556-6404Open PDFACS SensorsCite this: ACS Sens. 2025, 10, 2, 575–576Click to copy citationCitation copied!https://pubs.acs.org/doi/10.1021/acssensors.5c00330https://doi.org/10.1021/acssensors.5c00330Published February 28, 2025 Publication History Received 27 January 2025Published online 28 February 2025Published in issue 28 February 2025editorialCopyright © 2025 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissionsThis publication is licensed for personal use by The American Chemical Society. ACS PublicationsCopyright © 2025 American Chemical SocietySubjectswhat are subjectsArticle subjects are automatically applied from the ACS Subject Taxonomy and describe the scientific concepts and themes of the article.AssaysBiosensingBiotechnologyGeneticsSensorsRather than being famous only in the gene editing field, by revealing the collateral cleavage activity of Cas12a, Cas13a, and Cas14 effectors, Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/CRISPR associated (Cas) systems (i.e., CRISPR/Cas) have received significant credit in modern analytical science with the capability of detecting versatile analytes with superior sensitivity and specificity. (1,2) A variety of exciting CRISPR/Cas biosensing systems have now been developed successfully for detection of different analytes varying from nucleic acids to non-nucleic acids (such as metabolites, proteins, exosomes, and metal ions). Although the most popular signal output in CRISPR/Cas biosensors is fluorescence, various signal output modalities such as colorimetric, electrochemiluminescence, electrochemical, and electrical have been applied in CRISPR/Cas biosensing systems. Furthermore, the potential of CRISPR/Cas has been demonstrated in multiplex detection by integration with microfluidics or other devices enabling identification of the presence of multiple targets. However, despite extensive efforts and success to develop CRISPR/Cas diagnostic tools based on trans-cleavage enzymatic activity, these systems encounter unavoidable challenges, including inadequate detection limit (near the picomole level) for detecting clinically relevant biomarkers at subpicomolar levels and limited catalytic efficiency for DNA cleavage. These limitations significantly hinder the widespread adoption of CRISPR/Cas diagnostic tools in clinical diagnostics and point-of-care testing.To further enhance detection sensitivity and avoid the necessity for sophisticated and costly equipment, nucleic acids-based preamplification techniques, including thermal-dependent amplification, such as polymerase chain reaction (PCR), and thermal-independent amplification, rolling circle amplification (RCA), recombinase polymerase amplification (RPA), or loop-mediated isothermal amplification (LAMP), are frequently integrated with CRISPR/Cas based assays. Although preamplification techniques significantly increase the sensitivity, they inevitably overshadow Cas effectors and neglect the intrinsic detection capability of Cas effectors. Preamplification also extends detection time and reduces the efficiency of subsequent detection due to nonspecific amplification and primer interference, while substantially increasing the risk of aerosol contamination. The most sensitive nucleic acid amplification strategies employ exponential amplification formats in which amplicons (amplification products) are recycled as primers or templates. However, because of the exponential format, nonspecific background products that lead to false-positive results are inevitable after long reaction times and can be caused by, for example, contaminants, off-template polymerase products, and secondary structures of primers or templates. Therefore, the reaction time of exponential amplification has to be evaluated and controlled in practice to avoid the generation of unwanted signals. Consequently, the sensitivity of exponential amplification-based diagnostics is always determined by the resolution between true- and false-positive signals generated by diluted standard samples and blank/negative controls, respectively.Development of preamplification-free strategies aims to achieve rapid one-pot detection with high detection limit (attomolar or single-molecule detection levels) and high adaptability. This can be achieved by optimizing key components of CRISPR/Cas biosensors (such as reporters), employing cascade amplification techniques, adopting microfluidic droplet analysis, and integrating with other signal readout patterns. Conventional single-strand DNA reporters have been replaced by a range of nanoparticle-based reporters, (3) such as gold nanoparticle (AuNP) reporters, quantum dot reporters, platinum nanoparticles, and aggregation-induced emission agent reporters. AuNPs were used in a CRISPR/Cas12a biosensor integrated with surface enhanced Raman spectroscopy (SERS) signal readout to achieve a detection limit of detection as low as 10 aM. (4) By utilizing autocatalytic nucleic acid circuits, a CRISPR/Cas biosensor enabled DNA detection with the attomolar detection limit without the need for preamplification. (5) Although these systems demonstrated the ability to detect nucleic acids, integration of immunoassays into the CRISPR/Cas system can achieve the detection of other analytes in low abundance. (6)CRISPR/Cas technologies have demonstrated promise in biosensing beyond a simple assay. (7) The integration of CRISPR technology with engineering tools has led to significant advances in molecular biology and healthcare from in vitro diagnosis to in vivo monitoring, from tube assays to integrated devices. (8−10) Integrated with a lateral flow assay, a face mask incorporated with a lyophilized CRISPR/Cas sensor was developed for the noninvasive detection of SARS-CoV-2 at room temperature in 90 min that requires no user intervention other than the press of a button. (11) Microfluidic paper-based analytical devices were also integrated with CRISPR/Cas12a biosensors to realize the supersensitive detection of pathogenic bacteria in foods. (10) A wearable microneedle patch that uses CRISPR-activated graphene biointerfaces was reported for the extraction and long-term monitoring of universal cell-free DNA. It enables the real-time detection of nucleic acid biomarkers over 10 days in vivo, highlighting its potential for early disease screening and prognosis. (12) A single-step CRISPR detection of monkeypox virus in 15 min was developed with a vest-pocket diagnostic device with a sensitivity of 0.5 copies μL–1 and 100% concordance with real-time PCR in clinical validation, (13) which is adaptable to resource-limited settings. Multiplex detection is challenging due to the collateral cleavage activity of certain Cas enzymes, resulting in interference due to possible cross-reactivity among multiple analytes. (14) Solutions, such as engineering high-fidelity Cas variants, coupling with optimal crRNA designs, using orthogonal Cas effectors, and integrating with microfluidic technologies, allow simultaneous detection of multiple targets without interference. We recently integrated CRISPR-Cas12a immunosensing on glass fiber with a portable fluorescence reader to achieve sensitive detection of various proteins with a low pM detection limit, including cytokines in synovial joint fluids in a point-of-care scenario. (6) By eliminating the expensive instrument and tedious sample preparation, CRISPR/Cas-mediated devices that provide a simple sample-to-answer will continue to bring about a breakthrough in point-of-care diagnosis. (10,11)CRISPR/Cas-based biosensors are much further away from maturation. This editorial encourages submissions on CRISPR/Cas-mediated biosensing devices that demonstrate significant advancements in existing biosensors to address the growing need for quick, cost-effective, sensitive, and accurate field-deployable detection of multiple analytes from in vitro to in vivo. (11) With the integration with nanotechnologies, microfluidics, Cas enzyme-based orthogonal systems, and artificial intelligence, our aim is to achieve preamplification free CRISPR/Cas biosensors to facilitate comprehensive and reliable multiple analyte detection. (15) And it will lead to a breakthrough in high throughput biomarker discovery (16) by designing "all-in-one" devices that combine sample processing, amplification, and readout. Real-time monitoring of target analytes or in vivo bioimaging will be another highlight of the research that CRISPR/Cas biosensors can bring to the biomedical engineering field if novel reporters are discovered to integrate with devices that provide continuous signals corresponding to the target analytes. CRISPR/Cas biosensors typically involve multiple steps, including target extraction, amplification, and signal readout, which can become time-consuming and prone to contamination. In addition to device integration, CRISPR/Cas-mediated detection will respond with innovations in the Cas enzyme and reagent discovery to realize a one-pot assay with desirable detection limit and reduced assay time. Realizing the full potential of these CRISPR/Cas biosensing platforms requires sustained interdisciplinary collaboration between scientists, biologists, engineers, clinicians, and policy makers. Continued innovation in the engineering of CRISPR/Cas components, device integration, and regulatory standardization is essential to translate these cutting-edge technologies from the laboratory to the bedside and beyond, although there are challenges. We are confident that CRISPR/Cas biosensing systems continue to match proudly as diagnostic tools that are not only rapid and accurate but also affordable and globally accessible, driving significant improvements in precision medicine and sustainability in modern analytical science.Author InformationClick to copy section linkSection link copied!Corresponding AuthorGuozhen Liu, Integrated Devices and Intelligent Diagnosis (ID2) Laboratory, School of Medicine, The Chinese University of Hong Kong, Shenzhen 518172, China, https://orcid.org/0000-0002-0556-6404, Email: [email protected]NotesViews expressed in this editorial are those of the author and not necessarily the views of the ACS.ReferencesClick to copy section linkSection link copied! This article references 16 other publications. 1Chertow, D. S. Next-generation diagnostics with CRISPR. Science 2018, 360, 381– 382, DOI: 10.1126/science.aat4982 Google Scholar1Next-generation diagnostics with CRISPRChertow Daniel S.Science (Washington, DC, United States) (2018), 360 (6387), 381-382CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science) There is no expanded citation for this reference. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpt1Witbs%253D&md5=b83cc13b01ff95f5b9dba8b21ee480c02Li, Y.; Li, S.; Wang, J.; Liu, G. CRISPR/Cas systems towards next-generation biosensing. Trends Biotechnol. 2019, 37, 730– 743, DOI: 10.1016/j.tibtech.2018.12.005 Google Scholar2CRISPR/Cas Systems towards Next-Generation BiosensingLi, Yi; Li, Shiyuan; Wang, Jin; Liu, GuozhenTrends in Biotechnology (2019), 37 (7), 730-743CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.) A review. Beyond its remarkable genome editing ability, the CRISPR/Cas9 effector has also been utilized in biosensing applications. The recent discovery of the collateral RNA cleavage activity of the Cas13a effector has sparked even greater interest in developing novel biosensing technologies for nucleic acid detection and promised significant advances in CRISPR diagnostics. Now, along with the discovery of Cas12 collateral cleavage activities on single-stranded DNA (ssDNA), several CRISPR/Cas systems have been established for detecting various targets, including bacteria, viruses, cancer mutations, and others. Based on key Cas effectors, we provide a detailed classification of CRISPR/Cas biosensing systems and propose their future utility. As the field continues to mature, CRISPR/Cas systems have the potential to become promising candidates for next-generation diagnostic biosensing platforms. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXksVyntQ%253D%253D&md5=99d745d312b053b1c9582bcbd832ab923Ki, J. CRISPR/Cas-assisted colorimetric biosensor for point-of-use testing for African swine fever virus. ACS Sens. 2022, 7, 3940– 3946, DOI: 10.1021/acssensors.2c02007 Google Scholar3CRISPR/Cas-Assisted Colorimetric Biosensor for Point-of-Use Testing for African Swine Fever VirusKi, Jisun; Na, Hee-Kyung; Yoon, Sun Woo; Le, Van Phan; Lee, Tae Geol; Lim, Eun-KyungACS Sensors (2022), 7 (12), 3940-3946CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society) African swine fever virus (ASFV) causes a highly contagious and fatal disease affecting both domesticated and wild pigs. Substandard therapies and inadequate vaccinations cause severe economic damages from pig culling and removal of infected carcasses. Therefore, there is an urgent need to develop a rapid point-of-use approach that assists in avoiding the spread of ASFV and reducing economic loss. In this study, we developed a colorimetric sensing platform based on dual enzymic amplification that combined the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-assocd. protein 12a (Cas12a) system and the enzyme urease for accurate and sensitive detection of ASFV. The mechanism of the sensing platform involves a magnetic bead-anchored urease-conjugated single-stranded oligodeoxynucleotide (MB@urODN), which in the presence of ASFV dsDNA is cleaved by activated CRISPR/Cas12a. After magnetically sepg. the free urease, the presence of virus can be confirmed by measuring the colorimetric change in the soln. The advantage of this method is that it can detect the presence of virus without undergoing a complex target gene duplication process. The established method detected ASFV from three clin. specimens collected from porcine clin. tissue samples. The proposed platform is designed to provide an adequate, simple, robust, highly sensitive and selective anal. technique for rapid zoonotic disease diagnosis while eliminating the need for vast or specialized tools. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XivFSgsrbJ&md5=192b3a94a2eeda9308c22970296517d24Yin, B. A CRISPR-Cas12a integrated SERS nanoplatform with chimeric DNA/RNA hairpin guide for ultrasensitive nucleic acid detection. Theranostics 2022, 12, 5914, DOI: 10.7150/thno.75816 Google Scholar4A CRISPR-Cas12a integrated SERS nanoplatform with chimeric DNA/RNA hairpin guide for ultrasensitive nucleic acid detectionYin, Bohan; Zhang, Qin; Xia, Xinyue; Li, Chuanqi; Ho, Willis Kwun Hei; Yan, Jiaxiang; Huang, Yingying; Wu, Honglian; Wang, Pui; Yi, Changqing; Hao, Jianhua; Wang, Jianfang; Chen, Honglin; Wong, Siu Hong Dexter; Yang, MoTheranostics (2022), 12 (13), 5914-5930CODEN: THERDS; ISSN:1838-7640. (Ivyspring International Publisher) CRISPR-Cas12a has been integrated with nanomaterial-based optical techniques, such as surface-enhanced Raman scattering (SERS), to formulate a powerful amplification-free nucleic acid detection system. However, nanomaterials impose steric hindrance to limit the accessibility of CRISPR-Cas12a to the narrow gaps (SERS hot spots) among nanoparticles (NPs) for producing a significant change in signals after nucleic acid detection. To overcome this restriction, we specifically design chimeric DNA/RNA hairpins (displacers) that can be destabilized by activated CRISPR-Cas12a in the presence of target DNA, liberating excessive RNA that can disintegrate a core-satellite nanocluster via toehold-mediated strand displacement for orchestrating a promising "on-off" nucleic acid biosensor. The core-satellite nanocluster comprises a large gold nanoparticle (AuNP) core surrounded by small AuNPs with Raman tags via DNA hybridization as an ultrabright Raman reporter, and its disassembly leads to a drastic decrease of SERS intensity as signal readouts. We further introduce a magnetic core to the large AuNPs that can facilitate their sepn. from the disassembled nanostructures to suppress the background for improving detection sensitivity. As a proof-of-concept study, our findings showed that the application of displacers was more effective in decreasing the SERS intensity of the system and attained a better limit of detection (LOD, 10 aM) than that by directly using activated CRISPR-Cas12a, with high selectivity and stability for nucleic acid detection. Introducing magnetic-responsive functionality to our system further improves the LOD to 1 aM. Our work not only offers a platform to sensitively and selectively probe nucleic acids without pre-amplification but also provides new insights into the design of the CRISPR-Cas12a/SERS integrated system to resolve the steric hindrance of nanomaterials for constructing biosensors. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xit12nurzK&md5=33e4d73cf233e629bd2c56718062532c5Shi, K. A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Sci. Adv. 2021, 7, eabc7802 DOI: 10.1126/sciadv.abc7802 Google ScholarThere is no corresponding record for this reference.6Zou, S. CRISPR-Cas12a immunosensing on glass fiber for point-of-care quantification of multiple inflammation biomarkers in osteoarthritis. Device 2024, 2, 100319, DOI: 10.1016/j.device.2024.100319 Google ScholarThere is no corresponding record for this reference.7Shi, R.; Zhong, L.; Liu, G.; Mak, W. C. CRISPR/Cas Biosensing Technology: From Lab Assays to Integrated Portable Devices towards Wearables. TrAC Trends Anal. Chem. 2024, 177, 117796, DOI: 10.1016/j.trac.2024.117796 Google ScholarThere is no corresponding record for this reference.8Broughton, J. P. CRISPR–Cas12-based detection of SARS-CoV-2. Nat. Biotechnol. 2020, 38, 870– 874, DOI: 10.1038/s41587-020-0513-4 Google Scholar8CRISPR-Cas12-based detection of SARS-CoV-2Broughton, James P.; Deng, Xianding; Yu, Guixia; Fasching, Clare L.; Servellita, Venice; Singh, Jasmeet; Miao, Xin; Streithorst, Jessica A.; Granados, Andrea; Sotomayor-Gonzalez, Alicia; Zorn, Kelsey; Gopez, Allan; Hsu, Elaine; Gu, Wei; Miller, Steve; Pan, Chao-Yang; Guevara, Hugo; Wadford, Debra A.; Chen, Janice S.; Chiu, Charles Y.Nature Biotechnology (2020), 38 (7), 870-874CODEN: NABIF9; ISSN:1087-0156. (Nature Research) Abstr.: An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in Dec. 2019. COVID-19, the disease assocd. with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA exts. We validated our method using contrived ref. samples and clin. samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% pos. predictive agreement and 100% neg. predictive agreement. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntlejt7w%253D&md5=a1ccd699e2945474307f349518da37e59Lee, I.; Kwon, S.-J.; Heeger, P.; Dordick, J. S. Ultrasensitive ImmunoMag-CRISPR Lateral Flow Assay for Point-of-Care Testing of Urinary Biomarkers. ACS Sens. 2024, 9, 92– 100, DOI: 10.1021/acssensors.3c01694 Google ScholarThere is no corresponding record for this reference.10Nguyen, P. Q. Wearable materials with embedded synthetic biology sensors for biomolecule detection. Nat. Biotechnol. 2021, 39, 1366– 1374, DOI: 10.1038/s41587-021-00950-3 Google Scholar10Wearable materials with embedded synthetic biology sensors for biomolecule detectionNguyen, Peter Q.; Soenksen, Luis R.; Donghia, Nina M.; Angenent-Mari, Nicolaas M.; de Puig, Helena; Huang, Ally; Lee, Rose; Slomovic, Shimyn; Galbersanini, Tommaso; Lansberry, Geoffrey; Sallum, Hani M.; Zhao, Evan M.; Niemi, James B.; Collins, James J.Nature Biotechnology (2021), 39 (11), 1366-1374CODEN: NABIF9; ISSN:1087-0156. (Nature Portfolio) Integrating synthetic biol. into wearables could expand opportunities for noninvasive monitoring of physiol. status, disease states and exposure to pathogens or toxins. However, the operation of synthetic circuits generally requires the presence of living, engineered bacteria, which has limited their application in wearables. Here we report lightwt., flexible substrates and textiles functionalized with freeze-dried, cell-free synthetic circuits, including CRISPR-based tools, that detect metabolites, chems. and pathogen nucleic acid signatures. The wearable devices are activated upon rehydration from aq. exposure events and report the presence of specific mol. targets by colorimetric changes or via an optical fiber network that detects fluorescent and luminescent outputs. The detection limits for nucleic acids rival current lab. methods such as quant. PCR. We demonstrate the development of a face mask with a lyophilized CRISPR sensor for wearable, noninvasive detection of SARS-CoV-2 at room temp. within 90 min, requiring no user intervention other than the press of a button. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVenurnK&md5=70c78eaf02d067096407bd97d4ceb1ea11Dai, Y.; Wu, Y.; Liu, G.; Gooding, J. J. CRISPR mediated biosensing toward understanding cellular biology and point-of-care diagnosis. Angew. Chem., Int. Ed. 2020, 59, 20754– 20766, DOI: 10.1002/anie.202005398 Google Scholar11CRISPR Mediated Biosensing Toward Understanding Cellular Biology and Point-of-Care DiagnosisDai, Yifan; Wu, Yanfang; Liu, Guozhen; Gooding, J. JustinAngewandte Chemie, International Edition (2020), 59 (47), 20754-20766CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA) A review. Recent advances in CRISPR based biotechnologies have greatly expanded the authors' capabilities to repurpose CRISPR for the development of biomol. sensors for diagnosing diseases and understanding cellular pathways. The key attribute that allows CRISPR to be widely used is the programmable and highly selective mechanism. In this Minireview, the authors first illustrate the mol. principle of CRISPR functioning process from sensing to actuating. Next, the CRISPR based biosensing strategies for nucleic acids, proteins and small mols. are summarized. The authors highlight some of recent advances in applications for in vitro detection of biomols. and in vivo imaging of cellular networks. Finally, the challenges with, and exciting prospects of, CRISPR based biosensing developments are discussed. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Oks7nP&md5=0119652222aa5d39b8f64ccb8aceb96f12Yang, B.; Kong, J.; Fang, X. Programmable CRISPR-Cas9 microneedle patch for long-term capture and real-time monitoring of universal cell-free DNA. Nat. Commun. 2022, 13, 3999, DOI: 10.1038/s41467-022-31740-3 Google Scholar12Programmable CRISPR-Cas9 microneedle patch for long-term capture and real-time monitoring of universal cell-free DNAYang, Bin; Kong, Jilie; Fang, XueenNature Communications (2022), 13 (1), 3999CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio) Recent advances in biointerfaces have led to the development of wearable devices that can provide insights into personal health. As wearable modules, microneedles can ext. analytes of interest from interstitial fluid in a minimally invasive fashion. However, some microneedles are limited by their ability to perform highly effective extn. and real-time monitoring for macromol. biomarkers simultaneously. Here we show the synergetic effect of CRISPR-activated graphene biointerfaces, and report an online wearable microneedle patch for extn. and in vivo long-term monitoring of universal cell-free DNA. In this study, this wearable system enables real-time monitoring of Epstein-Barr virus, sepsis, and kidney transplantation cell-free DNA, with anti-interference ability of 60% fetal bovine serum, and has satisfactory stable sensitivity for 10 days in vivo. The exptl. results of immunodeficient mouse models shows the feasibility and practicability of this proposed method. This wearable patch holds great promise for long-term in vivo monitoring of cell-free DNA and could potentially be used for early disease screening and prognosis. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhvVChtr3N&md5=1e4c4fdfe726801050f38a8fda03c78d13Wang, Y. Ultrasensitive single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device. Nat. Commun. 2024, 15, 3279, DOI: 10.1038/s41467-024-47518-8 Google ScholarThere is no corresponding record for this reference.14Li, Y.; Liu, L.; Liu, G. CRISPR/Cas multiplexed biosensing: a challenge or an insurmountable obstacle?. Trends Biotechnol. 2019, 37, 792– 795, DOI: 10.1016/j.tibtech.2019.04.012 Google Scholar14CRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable Obstacle?Li, Yi; Liu, Linyang; Liu, GuozhenTrends in Biotechnology (2019), 37 (8), 792-795CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.) A review. Performing multiplex detection is still an elusive goal for mol. diagnostics. CRISPR/Cas-based biosensing has demonstrated potential for multiplex detection. Instead of being an insurmountable obstacle, CRISPR/Cas multiplexed biosensing is a realistic challenge with some recent successful applications. Strategic considerations are required to fully explore its potential in multiplex diagnostics. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpsVOrsL4%253D&md5=414b5e06e7ed5c55a528e930054e0b8815Song, J. Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE. Nat. Biotechnol. 2024, DOI: 10.1038/s41587-024-02426-6 Google ScholarThere is no corresponding record for this reference.16Rahimi, S.; Balusamy, S. R.; Perumalsamy, H.; Ståhlberg, A.; Mijakovic, I. CRISPR-Cas target recognition for sensing viral and cancer biomarkers. Nucleic Acids Res. 2024, 52, 10040– 10067, DOI: 10.1093/nar/gkae736 Google ScholarThere is no corresponding record for this reference.Cited By Click to copy section linkSection link copied!This article has not yet been cited by other publications.Download PDFFiguresReferences Get e-AlertsGet e-AlertsACS SensorsCite this: ACS Sens. 2025, 10, 2, 575–576Click to copy citationCitation copied!https://doi.org/10.1021/acssensors.5c00330Published February 28, 2025 Publication History Received 27 January 2025Published online 28 February 2025Published in issue 28 February 2025Copyright © 2025 American Chemical Society. This publication is available under these Terms of Use. Request reuse permissionsArticle Views-Altmetric-Citations-Learn about these metrics closeArticle Views are the COUNTER-compliant sum of full text article downloads since November 2008 (both PDF and HTML) across all institutions and individuals. 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Science 2018, 360, 381– 382, DOI: 10.1126/science.aat4982 1Next-generation diagnostics with CRISPRChertow Daniel S.Science (Washington, DC, United States) (2018), 360 (6387), 381-382CODEN: SCIEAS; ISSN:0036-8075. (American Association for the Advancement of Science) There is no expanded citation for this reference. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1cXpt1Witbs%253D&md5=b83cc13b01ff95f5b9dba8b21ee480c02Li, Y.; Li, S.; Wang, J.; Liu, G. CRISPR/Cas systems towards next-generation biosensing. Trends Biotechnol. 2019, 37, 730– 743, DOI: 10.1016/j.tibtech.2018.12.005 2CRISPR/Cas Systems towards Next-Generation BiosensingLi, Yi; Li, Shiyuan; Wang, Jin; Liu, GuozhenTrends in Biotechnology (2019), 37 (7), 730-743CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.) A review. Beyond its remarkable genome editing ability, the CRISPR/Cas9 effector has also been utilized in biosensing applications. The recent discovery of the collateral RNA cleavage activity of the Cas13a effector has sparked even greater interest in developing novel biosensing technologies for nucleic acid detection and promised significant advances in CRISPR diagnostics. Now, along with the discovery of Cas12 collateral cleavage activities on single-stranded DNA (ssDNA), several CRISPR/Cas systems have been established for detecting various targets, including bacteria, viruses, cancer mutations, and others. Based on key Cas effectors, we provide a detailed classification of CRISPR/Cas biosensing systems and propose their future utility. As the field continues to mature, CRISPR/Cas systems have the potential to become promising candidates for next-generation diagnostic biosensing platforms. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXksVyntQ%253D%253D&md5=99d745d312b053b1c9582bcbd832ab923Ki, J. CRISPR/Cas-assisted colorimetric biosensor for point-of-use testing for African swine fever virus. ACS Sens. 2022, 7, 3940– 3946, DOI: 10.1021/acssensors.2c02007 3CRISPR/Cas-Assisted Colorimetric Biosensor for Point-of-Use Testing for African Swine Fever VirusKi, Jisun; Na, Hee-Kyung; Yoon, Sun Woo; Le, Van Phan; Lee, Tae Geol; Lim, Eun-KyungACS Sensors (2022), 7 (12), 3940-3946CODEN: ASCEFJ; ISSN:2379-3694. (American Chemical Society) African swine fever virus (ASFV) causes a highly contagious and fatal disease affecting both domesticated and wild pigs. Substandard therapies and inadequate vaccinations cause severe economic damages from pig culling and removal of infected carcasses. Therefore, there is an urgent need to develop a rapid point-of-use approach that assists in avoiding the spread of ASFV and reducing economic loss. In this study, we developed a colorimetric sensing platform based on dual enzymic amplification that combined the clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-assocd. protein 12a (Cas12a) system and the enzyme urease for accurate and sensitive detection of ASFV. The mechanism of the sensing platform involves a magnetic bead-anchored urease-conjugated single-stranded oligodeoxynucleotide (MB@urODN), which in the presence of ASFV dsDNA is cleaved by activated CRISPR/Cas12a. After magnetically sepg. the free urease, the presence of virus can be confirmed by measuring the colorimetric change in the soln. The advantage of this method is that it can detect the presence of virus without undergoing a complex target gene duplication process. The established method detected ASFV from three clin. specimens collected from porcine clin. tissue samples. The proposed platform is designed to provide an adequate, simple, robust, highly sensitive and selective anal. technique for rapid zoonotic disease diagnosis while eliminating the need for vast or specialized tools. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XivFSgsrbJ&md5=192b3a94a2eeda9308c22970296517d24Yin, B. A CRISPR-Cas12a integrated SERS nanoplatform with chimeric DNA/RNA hairpin guide for ultrasensitive nucleic acid detection. Theranostics 2022, 12, 5914, DOI: 10.7150/thno.75816 4A CRISPR-Cas12a integrated SERS nanoplatform with chimeric DNA/RNA hairpin guide for ultrasensitive nucleic acid detectionYin, Bohan; Zhang, Qin; Xia, Xinyue; Li, Chuanqi; Ho, Willis Kwun Hei; Yan, Jiaxiang; Huang, Yingying; Wu, Honglian; Wang, Pui; Yi, Changqing; Hao, Jianhua; Wang, Jianfang; Chen, Honglin; Wong, Siu Hong Dexter; Yang, MoTheranostics (2022), 12 (13), 5914-5930CODEN: THERDS; ISSN:1838-7640. (Ivyspring International Publisher) CRISPR-Cas12a has been integrated with nanomaterial-based optical techniques, such as surface-enhanced Raman scattering (SERS), to formulate a powerful amplification-free nucleic acid detection system. However, nanomaterials impose steric hindrance to limit the accessibility of CRISPR-Cas12a to the narrow gaps (SERS hot spots) among nanoparticles (NPs) for producing a significant change in signals after nucleic acid detection. To overcome this restriction, we specifically design chimeric DNA/RNA hairpins (displacers) that can be destabilized by activated CRISPR-Cas12a in the presence of target DNA, liberating excessive RNA that can disintegrate a core-satellite nanocluster via toehold-mediated strand displacement for orchestrating a promising "on-off" nucleic acid biosensor. The core-satellite nanocluster comprises a large gold nanoparticle (AuNP) core surrounded by small AuNPs with Raman tags via DNA hybridization as an ultrabright Raman reporter, and its disassembly leads to a drastic decrease of SERS intensity as signal readouts. We further introduce a magnetic core to the large AuNPs that can facilitate their sepn. from the disassembled nanostructures to suppress the background for improving detection sensitivity. As a proof-of-concept study, our findings showed that the application of displacers was more effective in decreasing the SERS intensity of the system and attained a better limit of detection (LOD, 10 aM) than that by directly using activated CRISPR-Cas12a, with high selectivity and stability for nucleic acid detection. Introducing magnetic-responsive functionality to our system further improves the LOD to 1 aM. Our work not only offers a platform to sensitively and selectively probe nucleic acids without pre-amplification but also provides new insights into the design of the CRISPR-Cas12a/SERS integrated system to resolve the steric hindrance of nanomaterials for constructing biosensors. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38Xit12nurzK&md5=33e4d73cf233e629bd2c56718062532c5Shi, K. A CRISPR-Cas autocatalysis-driven feedback amplification network for supersensitive DNA diagnostics. Sci. Adv. 2021, 7, eabc7802 DOI: 10.1126/sciadv.abc7802 There is no corresponding record for this reference.6Zou, S. CRISPR-Cas12a immunosensing on glass fiber for point-of-care quantification of multiple inflammation biomarkers in osteoarthritis. Device 2024, 2, 100319, DOI: 10.1016/j.device.2024.100319 There is no corresponding record for this reference.7Shi, R.; Zhong, L.; Liu, G.; Mak, W. C. CRISPR/Cas Biosensing Technology: From Lab Assays to Integrated Portable Devices towards Wearables. TrAC Trends Anal. Chem. 2024, 177, 117796, DOI: 10.1016/j.trac.2024.117796 There is no corresponding record for this reference.8Broughton, J. P. CRISPR–Cas12-based detection of SARS-CoV-2. Nat. Biotechnol. 2020, 38, 870– 874, DOI: 10.1038/s41587-020-0513-4 8CRISPR-Cas12-based detection of SARS-CoV-2Broughton, James P.; Deng, Xianding; Yu, Guixia; Fasching, Clare L.; Servellita, Venice; Singh, Jasmeet; Miao, Xin; Streithorst, Jessica A.; Granados, Andrea; Sotomayor-Gonzalez, Alicia; Zorn, Kelsey; Gopez, Allan; Hsu, Elaine; Gu, Wei; Miller, Steve; Pan, Chao-Yang; Guevara, Hugo; Wadford, Debra A.; Chen, Janice S.; Chiu, Charles Y.Nature Biotechnology (2020), 38 (7), 870-874CODEN: NABIF9; ISSN:1087-0156. (Nature Research) Abstr.: An outbreak of betacoronavirus severe acute respiratory syndrome (SARS)-CoV-2 began in Wuhan, China in Dec. 2019. COVID-19, the disease assocd. with SARS-CoV-2 infection, rapidly spread to produce a global pandemic. We report development of a rapid (<40 min), easy-to-implement and accurate CRISPR-Cas12-based lateral flow assay for detection of SARS-CoV-2 from respiratory swab RNA exts. We validated our method using contrived ref. samples and clin. samples from patients in the United States, including 36 patients with COVID-19 infection and 42 patients with other viral respiratory infections. Our CRISPR-based DETECTR assay provides a visual and faster alternative to the US Centers for Disease Control and Prevention SARS-CoV-2 real-time RT-PCR assay, with 95% pos. predictive agreement and 100% neg. predictive agreement. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXntlejt7w%253D&md5=a1ccd699e2945474307f349518da37e59Lee, I.; Kwon, S.-J.; Heeger, P.; Dordick, J. S. Ultrasensitive ImmunoMag-CRISPR Lateral Flow Assay for Point-of-Care Testing of Urinary Biomarkers. ACS Sens. 2024, 9, 92– 100, DOI: 10.1021/acssensors.3c01694 There is no corresponding record for this reference.10Nguyen, P. Q. Wearable materials with embedded synthetic biology sensors for biomolecule detection. Nat. Biotechnol. 2021, 39, 1366– 1374, DOI: 10.1038/s41587-021-00950-3 10Wearable materials with embedded synthetic biology sensors for biomolecule detectionNguyen, Peter Q.; Soenksen, Luis R.; Donghia, Nina M.; Angenent-Mari, Nicolaas M.; de Puig, Helena; Huang, Ally; Lee, Rose; Slomovic, Shimyn; Galbersanini, Tommaso; Lansberry, Geoffrey; Sallum, Hani M.; Zhao, Evan M.; Niemi, James B.; Collins, James J.Nature Biotechnology (2021), 39 (11), 1366-1374CODEN: NABIF9; ISSN:1087-0156. (Nature Portfolio) Integrating synthetic biol. into wearables could expand opportunities for noninvasive monitoring of physiol. status, disease states and exposure to pathogens or toxins. However, the operation of synthetic circuits generally requires the presence of living, engineered bacteria, which has limited their application in wearables. Here we report lightwt., flexible substrates and textiles functionalized with freeze-dried, cell-free synthetic circuits, including CRISPR-based tools, that detect metabolites, chems. and pathogen nucleic acid signatures. The wearable devices are activated upon rehydration from aq. exposure events and report the presence of specific mol. targets by colorimetric changes or via an optical fiber network that detects fluorescent and luminescent outputs. The detection limits for nucleic acids rival current lab. methods such as quant. PCR. We demonstrate the development of a face mask with a lyophilized CRISPR sensor for wearable, noninvasive detection of SARS-CoV-2 at room temp. within 90 min, requiring no user intervention other than the press of a button. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3MXhsVenurnK&md5=70c78eaf02d067096407bd97d4ceb1ea11Dai, Y.; Wu, Y.; Liu, G.; Gooding, J. J. CRISPR mediated biosensing toward understanding cellular biology and point-of-care diagnosis. Angew. Chem., Int. Ed. 2020, 59, 20754– 20766, DOI: 10.1002/anie.202005398 11CRISPR Mediated Biosensing Toward Understanding Cellular Biology and Point-of-Care DiagnosisDai, Yifan; Wu, Yanfang; Liu, Guozhen; Gooding, J. JustinAngewandte Chemie, International Edition (2020), 59 (47), 20754-20766CODEN: ACIEF5; ISSN:1433-7851. (Wiley-VCH Verlag GmbH & Co. KGaA) A review. Recent advances in CRISPR based biotechnologies have greatly expanded the authors' capabilities to repurpose CRISPR for the development of biomol. sensors for diagnosing diseases and understanding cellular pathways. The key attribute that allows CRISPR to be widely used is the programmable and highly selective mechanism. In this Minireview, the authors first illustrate the mol. principle of CRISPR functioning process from sensing to actuating. Next, the CRISPR based biosensing strategies for nucleic acids, proteins and small mols. are summarized. The authors highlight some of recent advances in applications for in vitro detection of biomols. and in vivo imaging of cellular networks. Finally, the challenges with, and exciting prospects of, CRISPR based biosensing developments are discussed. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB3cXhs1Oks7nP&md5=0119652222aa5d39b8f64ccb8aceb96f12Yang, B.; Kong, J.; Fang, X. Programmable CRISPR-Cas9 microneedle patch for long-term capture and real-time monitoring of universal cell-free DNA. Nat. Commun. 2022, 13, 3999, DOI: 10.1038/s41467-022-31740-3 12Programmable CRISPR-Cas9 microneedle patch for long-term capture and real-time monitoring of universal cell-free DNAYang, Bin; Kong, Jilie; Fang, XueenNature Communications (2022), 13 (1), 3999CODEN: NCAOBW; ISSN:2041-1723. (Nature Portfolio) Recent advances in biointerfaces have led to the development of wearable devices that can provide insights into personal health. As wearable modules, microneedles can ext. analytes of interest from interstitial fluid in a minimally invasive fashion. However, some microneedles are limited by their ability to perform highly effective extn. and real-time monitoring for macromol. biomarkers simultaneously. Here we show the synergetic effect of CRISPR-activated graphene biointerfaces, and report an online wearable microneedle patch for extn. and in vivo long-term monitoring of universal cell-free DNA. In this study, this wearable system enables real-time monitoring of Epstein-Barr virus, sepsis, and kidney transplantation cell-free DNA, with anti-interference ability of 60% fetal bovine serum, and has satisfactory stable sensitivity for 10 days in vivo. The exptl. results of immunodeficient mouse models shows the feasibility and practicability of this proposed method. This wearable patch holds great promise for long-term in vivo monitoring of cell-free DNA and could potentially be used for early disease screening and prognosis. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BB38XhvVChtr3N&md5=1e4c4fdfe726801050f38a8fda03c78d13Wang, Y. Ultrasensitive single-step CRISPR detection of monkeypox virus in minutes with a vest-pocket diagnostic device. Nat. Commun. 2024, 15, 3279, DOI: 10.1038/s41467-024-47518-8 There is no corresponding record for this reference.14Li, Y.; Liu, L.; Liu, G. CRISPR/Cas multiplexed biosensing: a challenge or an insurmountable obstacle?. Trends Biotechnol. 2019, 37, 792– 795, DOI: 10.1016/j.tibtech.2019.04.012 14CRISPR/Cas Multiplexed Biosensing: A Challenge or an Insurmountable Obstacle?Li, Yi; Liu, Linyang; Liu, GuozhenTrends in Biotechnology (2019), 37 (8), 792-795CODEN: TRBIDM; ISSN:0167-7799. (Elsevier Ltd.) A review. Performing multiplex detection is still an elusive goal for mol. diagnostics. CRISPR/Cas-based biosensing has demonstrated potential for multiplex detection. Instead of being an insurmountable obstacle, CRISPR/Cas multiplexed biosensing is a realistic challenge with some recent successful applications. Strategic considerations are required to fully explore its potential in multiplex diagnostics. >> More from SciFinder ®https://chemport.cas.org/services/resolver?origin=ACS&resolution=options&coi=1%3ACAS%3A528%3ADC%252BC1MXpsVOrsL4%253D&md5=414b5e06e7ed5c55a528e930054e0b8815Song, J. Amplifying mutational profiling of extracellular vesicle mRNA with SCOPE. Nat. Biotechnol. 2024, DOI: 10.1038/s41587-024-02426-6 There is no corresponding record for this reference.16Rahimi, S.; Balusamy, S. R.; Perumalsamy, H.; Ståhlberg, A.; Mijakovic, I. CRISPR-Cas target recognition for sensing viral and cancer biomarkers. Nucleic Acids Res. 2024, 52, 10040– 10067, DOI: 10.1093/nar/gkae736 There is no corresponding record for this reference.