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      Potential of CRISPR/Cas system as emerging tools in the detection of viral hepatitis infection

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          Abstract

          Viral hepatitis, the most common cause of inflammatory liver disease, affects hundreds of millions of people worldwide. It is most commonly associated with one of the five nominal hepatitis viruses (hepatitis A–E viruses). HBV and HCV can cause acute infections and lifelong, persistent chronic infections, while HAV and HEV cause self-limiting acute infections. HAV and HEV are predominantly transmitted through the fecal-oral route, while diseases transmitted by the other forms are blood-borne diseases. Despite the success in the treatment of viral hepatitis and the development of HAV and HBV vaccines, there is still no accurate diagnosis at the genetic level for these diseases. Timely diagnosis of viral hepatitis is a prerequisite for efficient therapeutic intervention. Due to the specificity and sensitivity of clustered regularly interspaced short palindromic repeat (CRISPR)/CRISPR-associated sequences (Cas) technology, it has the potential to meet critical needs in the field of diagnosis of viral diseases and can be used in versatile point-of-care (POC) diagnostic applications to detect viruses with both DNA and RNA genomes. In this review, we discuss recent advances in CRISPR–Cas diagnostics tools and assess their potential and prospects in rapid and effective strategies for the diagnosis and control of viral hepatitis infection.

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          CRISPR-Cas12a target binding unleashes indiscriminate single-stranded DNase activity

          CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems. Like CRISPR-Cas9, Cas12a has been harnessed for genome editing based on its ability to generate targeted, double-stranded DNA (dsDNA) breaks. Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules. We find that target-activated, non-specific ssDNase cleavage is also a property of other type V CRISPR-Cas12 enzymes. By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA Endonuclease Targeted CRISPR Trans Reporter (DETECTR), which achieves attomolar sensitivity for DNA detection. DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics.
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            Cpf1 is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system.

            The microbial adaptive immune system CRISPR mediates defense against foreign genetic elements through two classes of RNA-guided nuclease effectors. Class 1 effectors utilize multi-protein complexes, whereas class 2 effectors rely on single-component effector proteins such as the well-characterized Cas9. Here, we report characterization of Cpf1, a putative class 2 CRISPR effector. We demonstrate that Cpf1 mediates robust DNA interference with features distinct from Cas9. Cpf1 is a single RNA-guided endonuclease lacking tracrRNA, and it utilizes a T-rich protospacer-adjacent motif. Moreover, Cpf1 cleaves DNA via a staggered DNA double-stranded break. Out of 16 Cpf1-family proteins, we identified two candidate enzymes from Acidaminococcus and Lachnospiraceae, with efficient genome-editing activity in human cells. Identifying this mechanism of interference broadens our understanding of CRISPR-Cas systems and advances their genome editing applications.
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              CRISPR provides acquired resistance against viruses in prokaryotes.

              Clustered regularly interspaced short palindromic repeats (CRISPR) are a distinctive feature of the genomes of most Bacteria and Archaea and are thought to be involved in resistance to bacteriophages. We found that, after viral challenge, bacteria integrated new spacers derived from phage genomic sequences. Removal or addition of particular spacers modified the phage-resistance phenotype of the cell. Thus, CRISPR, together with associated cas genes, provided resistance against phages, and resistance specificity is determined by spacer-phage sequence similarity.
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                Author and article information

                Contributors
                abbashajizade@gmail.com
                ahmadian@nigeb.ac.ir
                Journal
                Virol J
                Virol J
                Virology Journal
                BioMed Central (London )
                1743-422X
                8 May 2023
                8 May 2023
                2023
                : 20
                : 91
                Affiliations
                [1 ]GRID grid.419420.a, ISNI 0000 0000 8676 7464, Department of Industrial and Environmental and Biotechnology, , National Institute of Genetic Engineering and Biotechnology (NIGEB), ; Tehran, 1497716316 Iran
                [2 ]GRID grid.419420.a, ISNI 0000 0000 8676 7464, Department of Animal Biotechnology, , National Institute of Genetic Engineering and Biotechnology (NIGEB), ; Tehran, 1497716316 Iran
                [3 ]GRID grid.411521.2, ISNI 0000 0000 9975 294X, Applied Microbiology Research Center, Systems Biology and Poisonings Institute, , Baqiyatallah University of Medical Sciences, ; Tehran, 1435916471 Iran
                Article
                2048
                10.1186/s12985-023-02048-5
                10165583
                37158910
                46d46347-91b3-4e7c-b996-01b9e4967e4b
                © The Author(s) 2023

                Open AccessThis article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 4 February 2023
                : 23 April 2023
                Categories
                Review
                Custom metadata
                © BioMed Central Ltd., part of Springer Nature 2023

                Microbiology & Virology
                viral hepatitis,crispr/cas system,virus diagnosis tools
                Microbiology & Virology
                viral hepatitis, crispr/cas system, virus diagnosis tools

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