LAMP-Coupled CRISPR–Cas12a Module for Rapid and Sensitive Detection of Plant DNA Viruses

Rapid, point-of-care (POC) diagnostics are essential to mitigate the impacts of current (and future) epidemics; however, current methods for detecting severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) require complicated laboratory tests that are generally conducted off-site and require substantial time. CRISPR-Cas systems have been harnessed to develop sensitive and specific platforms for nucleic acid detection. These detection platforms take advantage of CRISPR enzymes’ RNA-guided specificity for RNA and DNA targets and collateral trans activities on single-stranded RNA and DNA reporters. Microbial genomes possess an extensive range of CRISPR enzymes with different specificities and levels of collateral activity; identifying new enzymes may improve CRISPR-based diagnostics. Here, we identified a new Cas13 variant, which we named as miniature Cas13 (mCas13), and characterized its catalytic activity. We then employed this system to design, build, and test a SARS-CoV-2 detection module coupling reverse transcription loop-mediated isothermal amplification (RT-LAMP) with the mCas13 system to detect SARS-CoV-2 in synthetic and clinical samples. Our system exhibits sensitivity and specificity comparable to other CRISPR systems. This work expands the repertoire and application of Cas13 enzymes in diagnostics and for potential in vivo applications, including RNA knockdown and editing. Importantly, our system can be potentially adapted and used in large-scale testing for diverse pathogens, including RNA and DNA viruses, and bacteria.

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“… 45 , 46 Therefore, isothermal amplification methods are coupled with CRISPR systems to enhance specificity and LoD. 16 , 21 , 47 , 48 …”

Section: Resultsmentioning

confidence: 99%

A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics

et al. 2021

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“…Recently, CRISPR/Cas12a was applied for the detection of RNA [ 43 , 44 ] and DNA [ 45 ] plant viruses, suggesting its potential application in the detection of plant viral diseases. Here we adopt the CRISPR/Cas12a technology to specifically identify the emerging ToBRFV and to distinguish it from a closely related tobamovirus, ToMV.…”

Section: Introductionmentioning

confidence: 99%

Differential Detection of the Tobamoviruses Tomato Mosaic Virus (ToMV) and Tomato Brown Rugose Fruit Virus (ToBRFV) Using CRISPR-Cas12a

Alon

Hak

Bornstein

et al. 2021

Plants

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CRISPR/Cas12a-based detection is a novel approach for the efficient, sequence-specific identification of viruses. Here we adopt the use of CRISPR/Cas12a to identify the tomato brown rugose fruit virus (ToBRFV), a new and emerging tobamovirus which is causing substantial damage to the global tomato industry. Specific CRISPR RNAs (crRNAs) were designed to detect either ToBRFV or the closely related tomato mosaic virus (ToMV). This technology enabled the differential detection of ToBRFV and ToMV. Sensitivity assays revealed that viruses can be detected from 15–30 ng of RT-PCR product, and that specific detection could be achieved from a mix of ToMV and ToBRFV. In addition, we show that this method can enable the identification of ToBRFV in samples collected from commercial greenhouses. These results demonstrate a new method for species-specific detection of tobamoviruses. A future combination of this approach with isothermal amplification could provide a platform for efficient and user-friendly ways to distinguish between closely related strains and resistance-breaking pathogens.

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“…A number of CRISPR/Cas12a diagnosis assays have been developed for the rapid detection of plant pathogens, such as potato virus X; potato virus Y; tobacco mosaic virus [ 25 ]; tomato mosaic virus; tomato brown rugose fruit virus [ 26 ]; tomato yellow leaf curl virus; tomato leaf curl New Delhi virus [ 27 ]; and beet necrotic yellow vein virus [ 28 ]. Recently, in citrus, RPA-CRISPR/Cas12a detecting method for Candidatus Liberibacter asiaticus, which is the causal agent of citrus greening—Huanglongbing, was developed [ 29 ], however, CRISPR/Cas12a diagnosis assays for fungal plant pathogens have not yet been developed, even though such pathogens are easily widely spread and require early diagnosis for efficient control.…”

Section: Introductionmentioning

confidence: 99%

Sensitive and Rapid Detection of Citrus Scab Using an RPA-CRISPR/Cas12a System Combined with a Lateral Flow Assay

Shin

Kwon

Lee

et al. 2021

Plants

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Citrus is the most extensively produced fruit tree crop in the world and is grown in over 130 countries. Fungal diseases in citrus can cause significant losses in yield and quality. An accurate diagnosis is critical for determining the best management practices and preventing future losses. In this study, a Recombinase polymerase amplification (RPA)-clustered regularly interspaced short palindromic repeats (CRISPR)/associated (Cas) system was established with the integration of a lateral flow assay (LFA) readout system for diagnosis of citrus scab. This detection can be completed within 1 h, is highly sensitive and prevents cross-reactions with other common fungal citrus diseases. Furthermore, the detection system is compatible with crude DNA extracted from infected plant tissue. This RPA-CRISPR/Cas12a-LFA system provides a sensitive, rapid, and cost-effective method with promising and significant practical value for point-of-care diagnosis of citrus scab. To our knowledge, this is the first report to establish an RPA- and CRISPR-based method with LFA for fungal diseases in plants.

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LAMP-Coupled CRISPR–Cas12a Module for Rapid and Sensitive Detection of Plant DNA Viruses

Cited by 72 publications

References 23 publications

A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics

A Novel Miniature CRISPR-Cas13 System for SARS-CoV-2 Diagnostics

Differential Detection of the Tobamoviruses Tomato Mosaic Virus (ToMV) and Tomato Brown Rugose Fruit Virus (ToBRFV) Using CRISPR-Cas12a

Sensitive and Rapid Detection of Citrus Scab Using an RPA-CRISPR/Cas12a System Combined with a Lateral Flow Assay

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