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Biosensors
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CRISPR/Cas-Based Biosensing Systems: Development and Applications
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CRISPR/Cas-Based Biosensing Systems: Development and Applications

A special issue of Biosensors (ISSN 2079-6374). This special issue belongs to the section "Biosensors and Healthcare".

Deadline for manuscript submissions: closed (31 October 2024) | Viewed by 13544

Special Issue Editor

Dr. Ki Soo Park

E-Mail Website
Guest Editor
Department of Biological Engineering, Konkuk University, Seoul 05029, Republic of Korea
Interests: nucleic acid nanotechnology; molecular diagnostics; exosome analysis platform; biosensing; theranostics
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear Colleagues,

Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) protein, which originated as a bacterial and archaeal defense mechanism, has become an essential tool in various fields, including genome editing, gene expression modulation, cell imaging, and biosensing. Especially, it has shown great promise in biosensing applications due to its easy design, simple operation, and high specificity. Until now, there have been many attempts to develop biosensing platforms for the sensitive detection of target nucleic acids in combination with various signal amplification strategies. Furthermore, the high modularity of CRISPR/Cas protein enables the expansion of detection targets even to small molecules, proteins, exosomes, cells, etc., advancing the area of biosensing.

In this Special Issue, we aim to explore recent advances in CRISPR/Cas-based biosensing systems that can improve the quality of human life. We encourage the submission of papers in the following topics described by the keywords below but also welcome works on related topics.

Dr. Ki Soo Park
Guest Editor

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All submissions that pass pre-check are peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Biosensors is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 2700 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • nucleic acid nanotechnology
  • isothermal amplification
  • molecular diagnostics
  • immunological diagnostics
  • point-of-care testing
  • biosensors
  • in vitro diagnostics
  • clinical applications
  • CRISPR/Cas protein

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Published Papers (5 papers)

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Research

15 pages, 2287 KiB  
Article
A CRISPR/Cas12a-Based System for Sensitive Detection of Antimicrobial-Resistant Genes in Carbapenem-Resistant Enterobacterales
by Jiyong Shin, Sei Rim Kim, Zifan Xie, Yong-Su Jin and Yi-Cheng Wang
Biosensors 2024, 14(4), 194; https://doi.org/10.3390/bios14040194 - 16 Apr 2024
Cited by 2 | Viewed by 2689
Abstract
Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-β-lactamase (NDM) are particularly concerning due to their resistance to most β-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need [...] Read more.
Antimicrobial-resistant (AMR) bacteria pose a significant global health threat, and bacteria that produce New Delhi metallo-β-lactamase (NDM) are particularly concerning due to their resistance to most β-lactam antibiotics, including carbapenems. The emergence and spread of NDM-producing genes in food-producing animals highlight the need for a fast and accurate method for detecting AMR bacteria. We therefore propose a PCR-coupled CRISPR/Cas12a-based fluorescence assay that can detect NDM-producing genes (blaNDM) in bacteria. Thanks to its designed gRNA, this CRISPR/Cas12a system was able to simultaneously cleave PCR amplicons and ssDNA-FQ reporters, generating fluorescence signals. Our method was found to be highly specific when tested against other foodborne pathogens that do not carry blaNDM and also demonstrated an excellent capability to distinguish single-nucleotide polymorphism. In the case of blaNDM-1 carrying E. coli, the assay performed exceptionally well, with a detection limit of 2.7 × 100 CFU/mL: 100 times better than conventional PCR with gel electrophoresis. Moreover, the developed assay detected AMR bacteria in food samples and exhibited enhanced performance compared to previously published real-time PCR assays. Thus, this novel PCR-coupled CRISPR/Cas12a-based fluorescence assay has considerable potential to improve current approaches to AMR gene detection and thereby contribute to mitigating the global threat of AMR. Full article
(This article belongs to the Special Issue CRISPR/Cas-Based Biosensing Systems: Development and Applications)
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10 pages, 1525 KiB  
Article
Aptamer-Based Switching System for Communication of Non-Interacting Proteins
by Younghyeon Kim, Daehan Nam, Eun Sung Lee, Seokjoon Kim, Byung Seok Cha and Ki Soo Park
Biosensors 2024, 14(1), 47; https://doi.org/10.3390/bios14010047 - 16 Jan 2024
Cited by 1 | Viewed by 2108
Abstract
Biological macromolecules, such as DNA, RNA, and proteins in living organisms, form an intricate network that plays a key role in many biological processes. Many attempts have been made to build new networks by connecting non-communicable proteins with network mediators, especially using antibodies. [...] Read more.
Biological macromolecules, such as DNA, RNA, and proteins in living organisms, form an intricate network that plays a key role in many biological processes. Many attempts have been made to build new networks by connecting non-communicable proteins with network mediators, especially using antibodies. In this study, we devised an aptamer-based switching system that enables communication between non-interacting proteins. As a proof of concept, two proteins, Cas13a and T7 RNA polymerase (T7 RNAP), were rationally connected using an aptamer that specifically binds to T7 RNAP. The proposed switching system can be modulated in both signal-on and signal-off manners and its responsiveness to the target activator can be controlled by adjusting the reaction time. This study paves the way for the expansion of biological networks by mediating interactions between proteins using aptamers. Full article
(This article belongs to the Special Issue CRISPR/Cas-Based Biosensing Systems: Development and Applications)
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18 pages, 3788 KiB  
Article
DNA Probes for Cas12a-Based Assay with Fluorescence Anisotropy Enhanced Due to Anchors and Salts
by Irina V. Safenkova, Alexey V. Samokhvalov, Kseniya V. Serebrennikova, Sergei A. Eremin, Anatoly V. Zherdev and Boris B. Dzantiev
Biosensors 2023, 13(12), 1034; https://doi.org/10.3390/bios13121034 - 16 Dec 2023
Cited by 4 | Viewed by 2489
Abstract
CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing [...] Read more.
CRISPR/Cas12a is a potent biosensing tool known for its high specificity in DNA analysis. Cas12a recognizes the target DNA and acquires nuclease activity toward single-stranded DNA (ssDNA) probes. We present a straightforward and versatile approach to transforming common Cas12a-cleavable DNA probes into enhancing tools for fluorescence anisotropy (FA) measurements. Our study involved investigating 13 ssDNA probes with linear and hairpin structures, each featuring fluorescein at one end and a rotation-slowing tool (anchor) at the other. All anchors induced FA changes compared to fluorescein, ranging from 24 to 110 mr. Significant FA increases (up to 180 mr) were obtained by adding divalent metal salts (Mg2+, Ca2+, Ba2+), which influenced the rigidity and compactness of the DNA probes. The specific Cas12a-based recognition of double-stranded DNA (dsDNA) fragments of the bacterial phytopathogen Erwinia amylovora allowed us to determine the optimal set (probe structure, anchor, concentration of divalent ion) for FA-based detection. The best sensitivity was obtained using a hairpin structure with dC10 in the loop and streptavidin located near the fluorescein at the stem in the presence of 100 mM Mg2+. The detection limit of the dsDNA target was equal to 0.8 pM, which was eight times more sensitive compared to the common fluorescence-based method. The enhancing set ensured detection of single cells of E. amylovora per reaction in an analysis based on CRISPR/Cas12a with recombinase polymerase amplification. Our approach is universal and easy to implement. Combining FA with Cas12a offers enhanced sensitivity and signal reliability and could be applied to different DNA and RNA analytes. Full article
(This article belongs to the Special Issue CRISPR/Cas-Based Biosensing Systems: Development and Applications)
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16 pages, 2695 KiB  
Article
CRISPR/Cas12a-Based Detection Platform for Early and Rapid Diagnosis of Scrub Typhus
by Pooja Bhardwaj, Nikita Shrikant Nanaware, Sthita Pragnya Behera, Smita Kulkarni, Hirawati Deval, Rajesh Kumar, Gaurav Raj Dwivedi, Rajni Kant and Rajeev Singh
Biosensors 2023, 13(12), 1021; https://doi.org/10.3390/bios13121021 - 8 Dec 2023
Cited by 2 | Viewed by 3213
Abstract
Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the [...] Read more.
Orientia tsutsugamushi is responsible for causing scrub typhus (ST) and is the leading cause of acute encephalitis syndrome (AES) in AES patients. A rapid and sensitive method to detect scrub typhus on-site is essential for the timely deployment of control measures. In the current study, we developed a rapid, sensitive, and instrument-free lateral flow assay (LFA) detection method based on CRISPR/Cas12a technology for diagnosing ST (named LoCIST). The method is completed in three steps: first, harnessing the ability of recombinase polymerase for isothermal amplification of the target gene; second, CRISPR/Cas12a-based recognition of the target; and third, end-point detection by LFA. The detection limit of LoCIST was found to be one gene copy of ST genomic DNA per reaction, and the process was complete within an hour. In 81 clinical samples, the assay showed no cross-reactivity with other rickettsial DNA and was 100% consistent with PCR detection of ST. LoCIST demonstrated 97.6% sensitivity and 100% specificity. Overall, the LoCIST offers a novel alternative for the portable, simple, sensitive, and specific detection of ST, and it may help prevent and control AES outbreaks due to ST. In conclusion, LoCIST does not require specialized equipment and poses a potential for future applications as a point-of-care diagnostic. Full article
(This article belongs to the Special Issue CRISPR/Cas-Based Biosensing Systems: Development and Applications)
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11 pages, 2134 KiB  
Article
CRISPR/Cas12a Collateral Cleavage Activity for Sensitive 3′–5′ Exonuclease Assay
by Jae Hoon Jeung, Hyogu Han, Chang Yeol Lee and Jun Ki Ahn
Biosensors 2023, 13(11), 963; https://doi.org/10.3390/bios13110963 - 30 Oct 2023
Cited by 1 | Viewed by 2341
Abstract
This study presents a technique for detecting 3′–5′ exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3′–5′ exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility [...] Read more.
This study presents a technique for detecting 3′–5′ exonuclease activity through the use of CRISPR/Cas12a. These enzymes, including 3′–5′ exonuclease (Exo III), perform crucial roles in various cellular processes and are associated with life expectancy. However, imbalances in their expression can increase susceptibility to diseases such as cancer, particularly under prolonged stress. In this study, an activator sequence of CRISPR/Cas12a was constructed on the 5′–end of a hairpin probe (HP), forming a blunt end. When the 3′–end of the HP was hydrolyzed with Exo III activity, the activator sequence of Cas12a was exposed, which led to collateral cleavage of the DNA signal probe and generated a fluorescent signal, allowing sensitive and highly specific Exo III detection. This detection principle relied on the fact that Exo III exclusively cleaves the 3′–end mononucleotide of dsDNA and does not affect ssDNA. Based on this strategy, Exo III activity was successfully assayed at 0.0073 U/mL, demonstrating high sensitivity. In addition, this technique was used to screen candidate inhibitors of Exo III activity. Full article
(This article belongs to the Special Issue CRISPR/Cas-Based Biosensing Systems: Development and Applications)
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