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Microorganisms
Special Issues
CRISPR-Based Diagnostics for Detection of Microorganisms and Beyond
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CRISPR-Based Diagnostics for Detection of Microorganisms and Beyond

A special issue of Microorganisms (ISSN 2076-2607). This special issue belongs to the section "Microbial Biotechnology".

Deadline for manuscript submissions: 31 December 2024 | Viewed by 3873

Special Issue Editor

Prof. Dr. Jin Wang

E-Mail Website
Guest Editor
1. College of Life Sciences, Shanghai Normal University, Shanghai, China
2. Shanghai Tolo Biotechnology Co., Ltd., Shanghai, China
Interests: CRISPR; CRISPR diagnostics; cas engineering; nucleic acid detection; non-nucleic acid detection

Special Issue Information

Dear Colleagues,

Unlike CRISPR Cas9, Cas12 and Cas13 possess trans-cleavage activities against single-stranded nucleic acids, the activities of which have been employed to develop the next-generation CRISPR diagnostic (CRISPR-Dx) systems. So far, dozens of CRISPR-Dx systems have been successfully created, most of which are used in the detection of infectious diseases, non-infectious diseases, SNPs and non-nucleic acid targets. Although CRISPR-Dx technologies have shown merits in accuracy, sensitivity, rapidness and portability, there is still a lot of room for improvement in the detection of microorganisms.

Knowledge in this field will aid in understanding how CRISPR technologies facilitate the development of convenient methods for the sensitive and specific detection of microorganisms, which may include both target nucleic acids and target non-nucleic acids. As the Guest Editor of this Special Issue, I invite you to submit research articles, review articles, and short communications related to CRISPR-based technologies for the detection of microorganisms and beyond.

Prof. Dr. Jin Wang
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. Microorganisms 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

  • CRISPR
  • CRISPR Diagnostics
  • Cas
  • microorganisms
  • next-generation diagnostics
  • molecular diagnostics
  • pathogen detection
  • DNA detection
  • RNA detection
  • amplification-free diagnostics
  • non-nucleic acid detection

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Published Papers (2 papers)

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Research

11 pages, 1504 KiB  
Article
A CRISPR-Cas9-Mediated Large-Fragment Assembly Method for Cloning Genomes and Biosynthetic Gene Cluster
by Yujing Guo, Guang Cai, Huiying Li, Zhenquan Lin, Shuobo Shi, Jin Jin and Zihe Liu
Microorganisms 2024, 12(7), 1462; https://doi.org/10.3390/microorganisms12071462 - 18 Jul 2024
Viewed by 1420
Abstract
The ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient [...] Read more.
The ability to clone large DNA fragments from genomes is valuable for both basic and applied research, such as the construction of synthetic genomes, and the expression of biosynthetic gene clusters (BGCs) for natural product discovery. Here, we report a fast and efficient platform for the direct capture of genome DNAs, by combining CRISPR and Gibson assembly. We demonstrate this method with the ability of cloning large DNA fragments ranging from 30 to 77 kb from various host genomes, achieving a near 100% cloning fidelity for DNA fragments below 50 kb. We next demonstrate this method by the cloning of a 40 kb fragment from Streptomyces ceruleus A3(2), which is rich in BGCs for natural products; and used this method cloning the 40 kb fengycin synthetic gene cluster from B. subtilis 168, encoding for a class of peptides with bioactivity. This method provides efficient and simple opportunities for assembling large DNA constructs from distant sources. Full article
(This article belongs to the Special Issue CRISPR-Based Diagnostics for Detection of Microorganisms and Beyond)
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14 pages, 3326 KiB  
Article
A One-Pot Convenient RPA-CRISPR-Based Assay for Salmonella enterica Serovar Indiana Detection
by Jiansen Gong, Di Zhang, Lixia Fu, Yongyi Dong, Kun Wu, Xinhong Dou and Chengming Wang
Microorganisms 2024, 12(3), 519; https://doi.org/10.3390/microorganisms12030519 - 5 Mar 2024
Viewed by 1605
Abstract
Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and [...] Read more.
Salmonella enterica serovar Indiana (S. Indiana) is among the most prevalent serovars of Salmonella and is closely associated with foodborne diseases worldwide. In this study, we combined a recombinase polymerase amplification (RPA) technique with clustered regularly interspaced short palindromic repeat (CRISPR) and CRISPR-associated (Cas) protein Cas12b (CRISPR/Cas12b)-based biosensing in a one-pot platform to develop a novel one-step identification method for S. Indiana infection diagnosis. The entire RPA-CRISPR/Cas12b reaction can be completed at 41 °C within 1 h without the need for specific instruments. The optimal concentrations of Cas12b and single-guide RNA (sgRNA) for the reaction were the same at 250 nM. The single-stranded DNA (ssDNA) reporter 8C-FQ (5′-/6-FAM/CCCCCCCC/BHQ1/-3′) presented the best performance in the reaction compared with the other reporters. The limit of detection (LoD) of the RPA-CRISPR/Cas12b assay was 14.4 copies per reaction. As for specificity, we successfully identified four S. Indiana strains among twenty-two Salmonella strains without any false-positive results, presenting 100% accuracy for S. Indiana, and no cross-reactions were observed in eight other pathogens. Moreover, a total of 109 chicken carcasses were classified by the S. Indiana RPA-CRISPR assay and PCR methods from three processing points, including 43 post-shedding, 35 post-evisceration, and 31 post-chilling. There were 17 S. Indiana-positive samples identified during the whole processing step, consisting of nine post-shedding, five post-evisceration, and three post-chilling. The corresponding S. Indiana-positive rates of post-shedding, post-evisceration, and post-chilling were 20.93% (9/43), 14.29% (5/35), and 9.68% (3/31), respectively. Results from the S. Indiana one-step RPA-CRISPR/Cas12b assay were totally in agreement with those obtained using a traditional culture method, demonstrating 100% agreement with no false-positive or false-negative results observed. Altogether, the RPA-CRISPR/Cas12b assay developed in this study represents a promising, accurate, and simple diagnostic tool for S. Indiana detection. Full article
(This article belongs to the Special Issue CRISPR-Based Diagnostics for Detection of Microorganisms and Beyond)
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