Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection

Zeng, Muchu; Ke, Yuqing; Zhuang, Zhiyi; Qin, Chao; Li, Lai Yan; Sheng, Gaoyuan; Li, Zhuoru; He, Meng; Li, Yiyang

doi:10.1021/acs.analchem.2c01588

Cited by 38 publications

(31 citation statements)

References 30 publications

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“…RAVI-CRISPR achieves accurate colorimetric naked-eye detection using a ROX-labeled reporter [ 61 , 76 ]. The CRISPR/Cas12a/multiplex-crRNA system was designed for direct detection of ASFV DNA without nucleic-acid preamplification [ 79 ]. Its detection limit (~1 pM) is 6–64 times stronger than that of the conventional single-crRNA CRISPR/Cas12a system [ 79 ].…”

Section: Current Applications Of Crispr/cas-based Nucleic Acid Detect...mentioning

confidence: 99%

“…The CRISPR/Cas12a/multiplex-crRNA system was designed for direct detection of ASFV DNA without nucleic-acid preamplification [ 79 ]. Its detection limit (~1 pM) is 6–64 times stronger than that of the conventional single-crRNA CRISPR/Cas12a system [ 79 ]. It also reduces the possibility of detecting losses due to naturally occurring mutations in viral genes [ 79 ].…”

Section: Current Applications Of Crispr/cas-based Nucleic Acid Detect...mentioning

confidence: 99%

“…Its detection limit (~1 pM) is 6–64 times stronger than that of the conventional single-crRNA CRISPR/Cas12a system [ 79 ]. It also reduces the possibility of detecting losses due to naturally occurring mutations in viral genes [ 79 ]. In the future, the CRISPR/Cas12a/multiplex-crRNA method could be further combined with naked-eye colorimetric detection using a ROX-labeled reporter [ 61 , 76 ], making the diagnostic platform more deployable in the field.…”

Section: Current Applications Of Crispr/cas-based Nucleic Acid Detect...mentioning

confidence: 99%

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Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

Zhang¹

2022

Genes

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The rapid rate of virus transmission and pathogen mutation and evolution highlight the necessity for innovative approaches to the diagnosis and prevention of infectious diseases. Traditional technologies for pathogen detection, mostly PCR-based, involve costly/advanced equipment and skilled personnel and are therefore not feasible in resource-limited areas. Over the years, many promising methods based on clustered regularly interspaced short palindromic repeats and the associated protein systems (CRISPR/Cas), i.e., orthologues of Cas9, Cas12, Cas13 and Cas14, have been reported for nucleic acid detection. CRISPR/Cas effectors can provide one-tube reaction systems, amplification-free strategies, simultaneous multiplex pathogen detection, visual colorimetric detection, and quantitative identification as alternatives to quantitative PCR (qPCR). This review summarizes the current development of CRISPR/Cas-mediated molecular diagnostics, as well as their design software and readout methods, highlighting technical improvements for integrating CRISPR/Cas technologies into on-site applications. It further highlights recent applications of CRISPR/Cas-based nucleic acid detection in livestock industry, including emerging infectious diseases, authenticity and composition of meat/milk products, as well as sex determination of early embryos.

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Section: Current Applications Of Crispr/cas-based Nucleic Acid Detect...mentioning

confidence: 99%

Section: Current Applications Of Crispr/cas-based Nucleic Acid Detect...mentioning

confidence: 99%

Section: Current Applications Of Crispr/cas-based Nucleic Acid Detect...mentioning

confidence: 99%

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Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

Zhang¹

2022

Genes

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“…Cas12 and Cas13 effectors both possess multiple turnover trans-cleavage activity that can intrinsically realize signal amplification, enabling preamplification-free detection of nucleic acids [55][56][57]. Targeting multiplex sites can increase activation of Cas12 or Cas13 effectors, benefitting sensitivity improvement [58,59]. Nuclease-dead engineering of Cas effectors coverts them from an RNA-guided nucleases to simple binding proteins.…”

Section: Class 2 Crispr Toolboxmentioning

confidence: 99%

“…Therefore, CRISPR/Cas-based nucleic acid diagnostics have frequently reported for SARS-CoV-2, and other reported target viruses include ASFV [59,76], Zika virus [6,73,77], Dengue virus [6], Ebola virus [74] and hepatitis B virus [78].…”

Section: Viral Infection Diagnosismentioning

confidence: 99%

CRISPR-based nucleic acid diagnostics for pathogens

Yang

Zhang

Teng

et al. 2022

Preprint

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Pathogenic infections remain the primary threat for human health, especially the global COVID-19 pandemic. It is important to develop rapid, sensitive and multiplexed tools for detecting pathogens and their mutations, particularly the tailor-made strategies for point-of-care diagnosis allowing for use in resource-constrained settings. The rapidly evolving CRISPR/Cas systems have provided a powerful toolbox for pathogenic diagnostics via nucleic acid tests. In this review, we first describe the resultant promising class 2 (single, multidomain effector) and recently explored class 1 (multisubunit effector complexes) CRISPR tools. We present the diverse engineering nucleic acid diagnostics based on CRISPR/Cas systems for pathogenic viruses, bacteria and fungi, and highlight the application for detecting viral variants and drug-resistant bacteria enabled by CRISPR-based mutation profiling. Finally, we discuss the challenges such as the development of preamplification-free diagnostic assays and present the emerging CRISPR systems and CRISPR cascade that potentially enable multiplexed and preamplification-free pathogenic diagnostics.

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A Simplified Amplification‐Free Strategy with Lyophilized CRISPR‐CcrRNA System for Drug‐Resistant Salmonella Detection

Bao

Sun

et al. 2023

Small

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With the widespread abuse of antibiotics, the problem of bacterial resistance is becoming increasingly serious, resulting in more than 700 000 deaths annually, worldwide. [1] Salmonella, one of the most dangerous and infectious foodborne pathogens, ranks second among pathogens that endanger public food safety. [2] Salmonella accounts for 41% of global diarrhea-related deaths. [1,3] The emergence of drug-resistant Salmonella has increased the danger to public health. Early diagnosis and prompt detection of drug resistance require a rapid, sensitive, and cost-effective detection program from the perspective of prevention, screening for drug-resistant bacteria, and management of food-related accidents. [4,5] Traditional culture-based antibiotic sensitivity testing (AST) methods are complicated and often require 2 to 7 days. [6] In contrast, nucleic acid detection of drug-resistance genes has significant advantages in terms of sensitivity and specificity. [7] The gold standard for nucleic acid detection, quantitative polymerase chain reaction (qPCR), can detect 10 CFU mL −1 of resistant bacteria within a few hours. [8,9] However, it still relies heavily on specialized equipment and expertise and is difficult to use outside a laboratory setting. [4] Lateral flow assay (LFA) is the most promising point-of-care testing (POCT) platform owing to its minimal device dependence, speed, sensitivity, and user-friendliness. [10,11] A visual readout LFA based on antigen capture with a faster and more accurate reading within 15 min was developed. [12] Unfortunately, this immunology-based pathogenic bacterial detection via LFA generally has low sensitivity and cannot be used for AST. [10,13] Combining the CRISPR/Cas system with LFA allows for sensitive and specific detection of targets while maintaining its fast and user-friendly features. For instance. Gootenberg et al [14] proposed a method called SHERLOCK for SARS-CoV-2 detection, [15] Joung et al. [13] demonstrated the direct detection of SARS-CoV-2 samples and further developed the SHINE. [16] All of the LFA-CRISPR methods mentioned above enable Drug resistance in pathogenic bacteria has become a major threat to global health. The misuse of antibiotics has increased the number of resistant bacteria in the absence of rapid, accurate, and cost-effective diagnostic tools. Here, an amplification-free CRISPR-Cas12a time-resolved fluorescence immunochromatographic assay (AFC-TRFIA) is used to detect drugresistant Salmonella. Multi-locus targeting in combination crRNA (CcrRNA) is 27-fold more sensitive than a standalone crRNA system. The lyophilized CRISPR system further simplifies the operation and enables one-pot detection. Induction of nucleic acid fixation via differentially charged interactions reduced the time and cost required for flowmetric chromatography with enhanced stability. The induction of nucleic acid fixation via differentially charged interactions reduces the time and cost required for flowmetric chromatography with enhanced stability. The platform developed for th...

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Harnessing Multiplex crRNA in the CRISPR/Cas12a System Enables an Amplification-Free DNA Diagnostic Platform for ASFV Detection

Cited by 38 publications

References 30 publications

Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

Development of CRISPR-Mediated Nucleic Acid Detection Technologies and Their Applications in the Livestock Industry

CRISPR-based nucleic acid diagnostics for pathogens

A Simplified Amplification‐Free Strategy with Lyophilized CRISPR‐CcrRNA System for Drug‐Resistant Salmonella Detection

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