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      Advances in the application of CRISPR-Cas technology in rapid detection of pathogen nucleic acid

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          Abstract

          Clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated proteins (Cas) are widely used as gene editing tools in biology, microbiology, and other fields. CRISPR is composed of highly conserved repetitive sequences and spacer sequences in tandem. The spacer sequence has homology with foreign nucleic acids such as viruses and plasmids; Cas effector proteins have endonucleases, and become a hotspot in the field of molecular diagnosis because they recognize and cut specific DNA or RNA sequences. Researchers have developed many diagnostic platforms with high sensitivity, high specificity, and low cost by using Cas proteins (Cas9, Cas12, Cas13, Cas14, etc.) in combination with signal amplification and transformation technologies (fluorescence method, lateral flow technology, etc.), providing a new way for rapid detection of pathogen nucleic acid. This paper introduces the biological mechanism and classification of CRISPR-Cas technology, summarizes the existing rapid detection technology for pathogen nucleic acid based on the trans cleavage activity of Cas, describes its characteristics, functions, and application scenarios, and prospects the future application of this technology.

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          A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity.

          Martin Jinek, Krzysztof Chylinski, Ines Fonfara … (2012)
          Clustered regularly interspaced short palindromic repeats (CRISPR)/CRISPR-associated (Cas) systems provide bacteria and archaea with adaptive immunity against viruses and plasmids by using CRISPR RNAs (crRNAs) to guide the silencing of invading nucleic acids. We show here that in a subset of these systems, the mature crRNA that is base-paired to trans-activating crRNA (tracrRNA) forms a two-RNA structure that directs the CRISPR-associated protein Cas9 to introduce double-stranded (ds) breaks in target DNA. At sites complementary to the crRNA-guide sequence, the Cas9 HNH nuclease domain cleaves the complementary strand, whereas the Cas9 RuvC-like domain cleaves the noncomplementary strand. The dual-tracrRNA:crRNA, when engineered as a single RNA chimera, also directs sequence-specific Cas9 dsDNA cleavage. Our study reveals a family of endonucleases that use dual-RNAs for site-specific DNA cleavage and highlights the potential to exploit the system for RNA-programmable genome editing.
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            Multiplex genome engineering using CRISPR/Cas systems.

            Le Cong, F. Ran, David T. Cox … (2013)
            Functional elucidation of causal genetic variants and elements requires precise genome editing technologies. The type II prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats)/Cas adaptive immune system has been shown to facilitate RNA-guided site-specific DNA cleavage. We engineered two different type II CRISPR/Cas systems and demonstrate that Cas9 nucleases can be directed by short RNAs to induce precise cleavage at endogenous genomic loci in human and mouse cells. Cas9 can also be converted into a nicking enzyme to facilitate homology-directed repair with minimal mutagenic activity. Lastly, multiple guide sequences can be encoded into a single CRISPR array to enable simultaneous editing of several sites within the mammalian genome, demonstrating easy programmability and wide applicability of the RNA-guided nuclease technology.
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              CRISPR-Cas12–based detection of SARS-CoV-2

              James Broughton, Xianding Deng, Guixia Yu … (2020)
              An outbreak of betacoronavirus SARS-CoV-2 began in Wuhan, China in December 2019. COVID-19, the disease associated with 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 extracts. We validated our method using contrived reference samples and clinical samples from US patients, 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 CDC SARS-CoV-2 real-time RT-PCR assay, with 95% positive predictive agreement and 100% negative predictive agreement.. SARS-CoV-2 in patient samples is detected in under an hour using a CRISPR-based lateral flow assay.
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                Author and article information

                Contributors
                Journal
                Journal ID (nlm-ta): Front Mol Biosci
                Journal ID (iso-abbrev): Front Mol Biosci
                Journal ID (publisher-id): Front. Mol. Biosci.
                Title: Frontiers in Molecular Biosciences
                Publisher: Frontiers Media S.A.
                ISSN (Electronic): 2296-889X
                Publication date (Electronic): 21 September 2023
                Publication date Collection: 2023
                Volume: 10
                Electronic Location Identifier: 1260883
                Affiliations
                [1] 1 Key Laboratory of Pollution Exposure and Health Intervention of Zhejiang Province , Shulan International Medical College , Zhejiang Shuren University , Hangzhou, China
                [2] 2 Faculty of Medicine , Macau University of Science and Technology , Avenida Wai Long Taipa , Macau, China
                [3] 3 Department of Hepatobiliary and Pancreatic Surgery , Department of Liver Transplantation , Shulan (Hangzhou) Hospital , Zhejiang Shuren University School of Medicine , Hangzhou, China
                [4] 4 NHC Key Laboratory of Combined Multi-Organ Transplantation , Hangzhou, China
                [5] 5 The Organ Repair and Regeneration Medicine Institute of Hangzhou , Hangzhou, China
                [6] 6 Jinan Microecological Biomedicine Shandong Laboratory , Jinan, China
                [7] 7 Zhejiang Chinese Medical University , Hangzhou, China
                Author notes

                Edited by: Huiming Lu, University of Texas Southwestern Medical Center, United States

                Reviewed by: Chao Wu, University of Texas MD Anderson Cancer Center, United States

                Jinzhen Guo, University of Texas Southwestern Medical Center, United States

                *Correspondence: Saber Imani, saber.imani@ 123456zjsru.edu.cn ; Shusen Zheng, shusenzheng@ 123456zju.edu.cn ; Jianhui Li, surgeonlee@ 123456126.com
                [ † ]

                These authors share first authorship

                Article
                Publisher ID: 1260883
                DOI: 10.3389/fmolb.2023.1260883
                PMC ID: 10552857
                PubMed ID: 37808520
                SO-VID: f9fb7e2a-1c23-4fcf-af2f-69814582c632
                Copyright © Copyright © 2023 Li, Zhong, Li, Qiao, Mao, Fan, Zhong, Imani, Zheng and Li.
                License:

                This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

                History
                Date received : 06 August 2023
                Date accepted : 06 September 2023
                Funding
                The authors declare financial support was received for the research, authorship, and/or publication of this article. This study was financially supported by 2023 National Student Innovation and Entrepreneurship Training Program Project (Item Number: 202311842021X), Major Science and Technology Projects of Hainan Province (ZDKJ2019009), the Public Projects of Zhejiang Province (LGF21H030006), a Research Project of Jinan Microecological Biomedicine Shandong Laboratory (JNL-2022002A and JNL-2022023C), Key Laboratory of Biomarkers and In Vitro Diagnosis Translation of Zhejiang province (KFJJ2023005). The funder did not participate in the designing, performing or reporting in the current study.
                Categories
                Subject: Molecular Biosciences
                Subject: Review
                Custom metadata
                section-at-acceptance Molecular Diagnostics and Therapeutics

                Keywords: crispr-cas,cas9,cas12,cas13,cas14,pathogen nucleic acid,rapid detection
                Data availability:
                Keywords: crispr-cas, cas9, cas12, cas13, cas14, pathogen nucleic acid, rapid detection

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