&lt;strong&gt;SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases and Cancer-Associated Mutations&lt;/strong&gt;

Mustafa, Mujahed I.; Makhawi, Abdelrafie M.

doi:10.20944/preprints202004.0080.v1

Cited by 3 publications

(2 citation statements)

References 62 publications

(90 reference statements)

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“…Метод позволяет проводить мультиплексный анализ. Чувствительность и скорость у метода довольно высокие, однако для работы Cas12a требуется PAM [25,37].…”

Section: платформы диагностики на основе систем Crispr/cas V типаunclassified

CRISPR/Cas-based diagnostic platforms

Volkov

Dolgova

Dedkov

2022

Russian Journal of Infection and Immunity

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Over the last few years, CRISPR/Cas systems have been extensively studied and used for a wide range of applied purposes. The variety of their applications is accounted for by the ability of Cas-type nucleases to targetly cleave specified nucleic acid sequences. In this case, the researcher might set the necessary sequence of the guiding elements in the CRISPR/Cas system, played by so-called single guide RNAs allowing it to act on select targets. This potential underlies one of the reasons for exerting interest in CRISPR/Cas systems. One of the first areas for applying these systems was its use for genomic editing. Later, the list of potential opportunities has been expanded: e.g., they can be used in gene therapy and epigenetic research. It is possible to create sgRNA libraries which might be used to create a pool of viral vectors applied for bacterial cell transformation with subsequent cas-protein transduction that cause target gene knockout. This approach allows finding genes responsible for resistance or sensibility to diverse substances. Using such systems in molecular diagnostics of infectious diseases is considered as one of the most promising directions allowing to detect even extremely low concentrations of pathogenic organisms in samples due to their specific nucleotide sequences. Simultaneously, such assays turn out to be accurate, rapid and easy to utilize. In addition, some platforms may work without using expensive equipment, because methods for fast and simple sample preparation have already been developed, whereas modern preamplification approaches allow to avoid applying thermocycling devices. Interestingly, a great amount of diverse types of natural CRISPR/Cas systems have been already discovered. Such abundance promotes development of multiple artificial systems, each of which exerting own unique characteristics. Therefore, a variety of diagnostic platforms with different properties are created on their basis that allows researchers and physicians to choose an optimal approach for performing specific tasks. For this reason, insights into structure and operation of CRISPR/Cas systems are necessary for selecting a suitable platform. The current classification of systems is based on such principles serving as the basis, in turn, for convenient evaluation of the very variety of molecular diagnostics platforms and presentation of the typical technical characteristics and nuances for each method. Thus, this review, which is mainly devoted to the platforms for molecular diagnostics of infectious diseases, also touches upon the issues of functioning, devices, and classification of CRISPR/Cas systems.

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Section: платформы диагностики на основе систем Crispr/cas V типаunclassified

CRISPR/Cas-based diagnostic platforms

Volkov

Dolgova

Dedkov

2022

Russian Journal of Infection and Immunity

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“…It is based on CRISPR (clustered regularly interspaced short palindromic repeats) technology. SHERLOCK is composed of nucleic acid pre-amplification with CRISPR-Cas enzymology to identify desired DNA or RNA sequences [23] accurately. It affords multiplexed, portable, and highly sensitive detection of RNA from COVID-19 patients samples [24].…”

Section: Molecular Diagnostic Testsmentioning

confidence: 99%

Newly developed diagnostic methods for SARS-CoV-2 detection

Saatçi

2020

Turkish Journal of Biochemistry

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The emergence of SARS-CoV-2, responsible for COVID-19 disease, has caused a substantial worldwide pandemic and has become a significant public health problem. World Health Organization (WHO) has declared COVID-19 as a devastating health emergency for all countries. Public health officials continue to monitor the situation closely to control this new virus-related outbreak. In order to continue to manage this pandemic, a fast and sensitive diagnosis of COVID-19 is attempted. Emerging tests have become an essential part of the management of the COVID-19 crisis. This review article aims to provide a detailed explanation of ongoing and new diagnostic technologies for SARS-CoV-2 and a summary of method principles. Examples of new diagnostic methods for providing efficient and rapid diagnostic tests for managing the SARS-CoV-2 outbreak are also mentioned.

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RNA-guidedAsCas12a- andSpCas9-catalyzed knockout and homology directed repair of theomega-1locus of the human blood fluke,Schistosoma mansoni

Ittiprasert

Chatupheeraphat

Mann

et al. 2021

Preprint

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We compared the efficiency of gene knockout (KO) and precision of insertion (knock-in, KI) of the RNA-guided AsCas12a nuclease of Acidaminococcus sp. with that of SpCas9 from Streptococcus pyogenes, aiming to enhance the functional genomics toolkit for Schistosoma mansoni. Programmed DNA cleavages catalyzed by Cas12a and Cas9 result in staggered and blunt ended strand breaks, respectively. TTTV, the optimal protospacer adjacent motif for AsCas12a would occur frequently within the AT-rich genome of this platyhelminth. We deployed optimized conditions for the ratio of guide RNAs to the nuclease, donor templates, and electroporation parameters, to target a key enzyme termed omega-1 that is secreted by the schistosome egg. AsCas12a was more efficient than SpCas9 for gene knockout of omega-1 as determined by tracking of indels by decomposition (P < 0.001). Resulting from CRISPREsso2 analysis, most mutations were deletions; SpCas9 induced short deletions of 3 nt in length whereas AsCas12a induced deletions of 2 to 26 nt. Knockout efficiency of both nucleases markedly increased in the presence of short, single stranded oligodeoxynucleotide (ssODN) donor templates. With AsCas12a, ssODNs representative of both the non-CRISPR target (NT) and target (T) strands of the targeted gene were tested, resulting in KO efficiencies of 15.67, 28.71 and 21.43% in the SpCas9 plus donor ssODN, AsCas12a plus NT-ssODN, and AsCas12a plus T-ssODN groups, respectively. Trans cleavage activity against the ssODNs by activated AsCas12a was not apparent in vitro. Programmed SpCas9 editing led to more precise transgene insertion than AsCas12a, with KI efficiencies of 17.07% for the KI_SpCas9 group, 14.58% for KI_AsCas12a-NT-ssODN and 12.37% for KI_AsCas12a-T-ssODN. Although AsCas12a induced fewer mutations per genome than SpCas9, the phenotypic impact on transcription and expression of omega-1 was similar for both nucleases. These findings revealed that AsCas12a and SpCas9 both provide tractable routes for RNA-guided programmed mutation of the genome of the schistosome egg.

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<strong>SHERLOCK and DETECTR: CRISPR-Cas Systems as Potential Rapid Diagnostic Tools for Emerging Infectious Diseases and Cancer-Associated Mutations</strong>

Cited by 3 publications

References 62 publications

CRISPR/Cas-based diagnostic platforms

CRISPR/Cas-based diagnostic platforms

Newly developed diagnostic methods for SARS-CoV-2 detection

RNA-guidedAsCas12a- andSpCas9-catalyzed knockout and homology directed repair of theomega-1locus of the human blood fluke,Schistosoma mansoni

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