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      Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

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          Abstract

          With the trend of moving genomic test from (medical) laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established genomic diagnostics with the ease, cost effectiveness and speed of isothermal amplification methods. A promising new genomic sensing method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing.

          This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives.

          Graphical abstract

          Highlights

          • Rapid and reliable detection of nucleic acid is critical in many different fields.

          • CRISPR/Cas effector protein complexes could be used for specific DNA detection without the need of complicated machinery and trained personnel in so-called point-of-care testing.

          • A short and comprehensive introduction to CRISPR sensing is given.

          • We review recent developments of integrating CRISPR sensing in Point-of-care devices.

          • The challenges of (point-of-care) CRISPR sensing are discussed.

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          Most cited references70

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          CRISPR-Cas12–based detection of SARS-CoV-2

          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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            Intervening sequences of regularly spaced prokaryotic repeats derive from foreign genetic elements.

            Prokaryotes contain short DN repeats known as CRISPR, recognizable by the regular spacing existing between the recurring units. They represent the most widely distributed family of repeats among prokaryotic genomes suggesting a biological function. The origin of the intervening sequences, at present unknown, could provide clues about their biological activities. Here we show that CRISPR spacers derive from preexisting sequences, either chromosomal or within transmissible genetic elements such as bacteriophages and conjugative plasmids. Remarkably, these extrachromosomal elements fail to infect the specific spacer-carrier strain, implying a relationship between CRISPR and immunity against targeted DNA. Bacteriophages and conjugative plasmids are involved in prokaryotic population control, evolution, and pathogenicity. All these biological traits could be influenced by the presence of specific spacers. CRISPR loci can be visualized as mosaics of a repeated unit, separated by sequences at some time present elsewhere in the cell.
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              Clustered regularly interspaced short palindrome repeats (CRISPRs) have spacers of extrachromosomal origin.

              Numerous prokaryote genomes contain structures known as clustered regularly interspaced short palindromic repeats (CRISPRs), composed of 25-50 bp repeats separated by unique sequence spacers of similar length. CRISPR structures are found in the vicinity of four genes named cas1 to cas4. In silico analysis revealed another cluster of three genes associated with CRISPR structures in many bacterial species, named here as cas1B, cas5 and cas6, and also revealed a certain number of spacers that have homology with extant genes, most frequently derived from phages, but also derived from other extrachromosomal elements. Sequence analysis of CRISPR structures from 24 strains of Streptococcus thermophilus and Streptococcus vestibularis confirmed the homology of spacers with extrachromosomal elements. Phage sensitivity of S. thermophilus strains appears to be correlated with the number of spacers in the CRISPR locus the strain carries. The authors suggest that the spacer elements are the traces of past invasions by extrachromosomal elements, and hypothesize that they provide the cell immunity against phage infection, and more generally foreign DNA expression, by coding an anti-sense RNA. The presence of gene fragments in CRISPR structures and the nuclease motifs in cas genes of both cluster types suggests that CRISPR formation involves a DNA degradation step.
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                Author and article information

                Contributors
                Journal
                Biosens Bioelectron
                Biosens Bioelectron
                Biosensors & Bioelectronics
                Published by Elsevier B.V.
                0956-5663
                1873-4235
                26 July 2020
                26 July 2020
                : 112445
                Affiliations
                [a ]BIOS Lab on a Chip group, Technical Medical Centre, MESA+ Institute for Nanotechnology, University of Twente, P.O. box 217, 7500, AE, Enschede, the Netherlands
                [b ]Department of Pathology, Amsterdam University Medical Centers, Vrije Universiteit Amsterdam, Cancer Center Amsterdam, Amsterdam, the Netherlands
                [c ]Department of Clinical Genetics, section oncogenetics, Cancer Center Amsterdam, Amsterdam University Medical Centers, De Boelelaan 1118, 1081, HV, Amsterdam, the Netherlands
                Author notes
                []Corresponding author. j.e.vandongen@ 123456utwente.nl
                Article
                S0956-5663(20)30439-5 112445
                10.1016/j.bios.2020.112445
                7382963
                32758911
                500f1e7f-4655-4ec8-b9fe-40c15db68e12
                © 2020 Published by Elsevier B.V.

                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.

                History
                : 19 May 2020
                : 2 July 2020
                : 10 July 2020
                Categories
                Article

                Biomedical engineering
                point-of-care,crispr/cas,dna sensing,genomic diagnostic tools,crispr sensing,diagnosis

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