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      Towards CRISPR powered electrochemical sensing for smart diagnostics

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          Abstract

          Even though global health has been steadily improved, the global disease burden associated with communicable and non-communicable diseases extensively increased healthcare expenditure. The present COVID-19 pandemic scenario has again ascertained the importance of clinical diagnostics as a basis to make life-saving decisions. In this context, there is a need for developing next-generation integrated smart real-time responsive biosensors with high selectivity and sensitivity. The emergence of clustered regularly interspaced short palindromic repeats (CRISPR)/Cas biosensing systems has shown remarkable potential for developing next-generation biosensors. CRISPR/Cas integrated electrochemical biosensors (E-CRISPR) stands out with excellent properties. In this opinionated review, we illustrate the rapidly evolving applications for E-CRISPR-integrated detection systems towards biosensing and the future scope associated with E-CRISPR based diagnostics.

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

          Enbo Ma, Jennifer Doudna, Joel Palefsky … (2018)
          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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            Nucleic acid detection with CRISPR-Cas13a/C2c2

            Jonathan S. Gootenberg, Omar O Abudayyeh, Jeong Wook Lee … (2017)
            Rapid, inexpensive, and sensitive nucleic acid detection may aid point-of-care pathogen detection, genotyping, and disease monitoring. The RNA-guided, RNA-targeting CRISPR effector Cas13a (previously known as C2c2) exhibits a “collateral effect” of promiscuous RNAse activity upon target recognition. We combine the collateral effect of Cas13a with isothermal amplification to establish a CRISPR-based diagnostic (CRISPR-Dx), providing rapid DNA or RNA detection with attomolar sensitivity and single-base mismatch specificity. We use this Cas13a-based molecular detection platform, termed SHERLOCK ( S pecific H igh Sensitivity E nzymatic R eporter Un LOCK ing), to detect specific strains of Zika and Dengue virus, distinguish pathogenic bacteria, genotype human DNA, and identify cell-free tumor DNA mutations. Furthermore, SHERLOCK reaction reagents can be lyophilized for cold-chain independence and long-term storage, and readily reconstituted on paper for field applications.
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              Multiplexed and portable nucleic acid detection platform with Cas13, Cas12a, and Csm6

              Omar Abudayyeh, Max J. Kellner, Julia Joung … (2018)
              Rapid detection of nucleic acids is integral for clinical diagnostics and biotechnological applications. We recently developed a platform termed SHERLOCK (Specific High Sensitivity Enzymatic Reporter UnLOCKing) that combines isothermal pre-amplification with Cas13 to detect single molecules of RNA or DNA. Through characterization of CRISPR enzymology and application development, we report here four advances integrated into SHERLOCKv2: 1) 4-channel single reaction multiplexing using orthogonal CRISPR enzymes; 2) quantitative measurement of input down to 2 aM; 3) 3.5-fold increase in signal sensitivity by combining Cas13 with Csm6, an auxilary CRISPR-associated enzyme; and 4) lateral flow read-out. SHERLOCKv2 can detect Dengue or Zika virus ssRNA as well as mutations in patient liquid biopsy samples via lateral flow, highlighting its potential as a multiplexable, portable, rapid, and quantitative detection platform of nucleic acids.
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                Author and article information

                Journal
                Journal ID (nlm-ta): Curr Opin Electrochem
                Journal ID (iso-abbrev): Curr Opin Electrochem
                Title: Current Opinion in Electrochemistry
                Publisher: Elsevier B.V.
                ISSN (Print): 2451-9103
                ISSN (Electronic): 2451-9111
                Publication date PMC-release: 12 August 2021
                Publication date (Print): December 2021
                Publication date (Electronic): 12 August 2021
                Volume: 30
                Page: 100829
                Affiliations
                [1 ]Nanomaterial Research Laboratory (NMRL), Nano Division, Yenepoya Research Centre, Yenepoya (Deemed to be University), Deralakatte, Mangalore, 575 018, India
                [2 ]Centre for Nutrition Studies, Yenepoya (Deemed to be University), Deralakatte, Mangalore, 575 018, India
                Author notes
                [∗ ]Corresponding author: Prasad, K. Sudhakara
                [a]

                These authors contributed equally.

                Article
                Publisher Item ID: S2451-9103(21)00143-5 Publisher ID: 100829
                DOI: 10.1016/j.coelec.2021.100829
                PMC ID: 8660062
                PubMed ID: 34909513
                SO-VID: 63203a37-49fa-432a-b36a-51add13bbcfe
                Copyright © © 2021 Elsevier B.V. All rights reserved.
                License:

                Since January 2020 Elsevier has created a COVID-19 resource centre with free information in English and Mandarin on the novel coronavirus COVID-19. The COVID-19 resource centre is hosted on Elsevier Connect, the company's public news and information website. Elsevier hereby grants permission to make all its COVID-19-related research that is available on the COVID-19 resource centre - including this research content - immediately available in PubMed Central and other publicly funded repositories, such as the WHO COVID database with rights for unrestricted research re-use and analyses in any form or by any means with acknowledgement of the original source. These permissions are granted for free by Elsevier for as long as the COVID-19 resource centre remains active.

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                Categories
                Subject: Review Article

                Keywords: crispr/cas,electrochemical,biosensing,point-of-care,e-crispr
                Data availability:
                Keywords: crispr/cas, electrochemical, biosensing, point-of-care, e-crispr

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