Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13

Marqués, María-Carmen; Sanchez-Vicente, Javier; Ruiz, Raúl; Montagud-Martínez, Roser; Márquez-Costa, Rosa; Gómez, Gustavo; Carbonell, Alberto; Daròs, José‐Antonio; Rodrigo, Guillermo

doi:10.1021/acssynbio.2c00090

Cited by 41 publications

(31 citation statements)

References 45 publications

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“…These results demonstrate the successful activation of RspCas13d with target RNA at a picomolar level, forming the basis for the DATAS‐Cas13d system. Notably, compared with Cas13a‐based RNA sensor with a femtomolar sensitivity level because of its substantial collateral ssRNA cleavage activity, [4b,12b] the RspCas13d is less efficient in collateral trans ‐cleavage activity, [12c] thus indicating the need to integrate an additional signal amplification especially for the detection of trace amounts of targets.…”

Section: Resultsmentioning

confidence: 99%

Harnessing the CRISPR‐Cas13d System for Protein Detection by Dual‐Aptamer‐Based Transcription Amplification

Feng

Liu

et al. 2023

Chemistry A European J

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CRISPR-based biosensing technology has been emerging as a revolutionary diagnostic tool for many diseaserelated biomarkers. In particular, RspCas13d, a newly identified RNA-guided Cas13d ribonuclease derived from Ruminococcus sp., has shown great promise for accurate and sensitive detection of RNA due to its RNA sequence-specific recognition and robust collateral trans-cleavage activity. However, its diagnostic utility is limited to detecting nucleic-acidrelated biomarkers. To address this limitation, herein we present a proof-of-concept demonstration of a target-responsive CRISPR-Cas13d sensing system for protein biomarkers. This system was rationally designed by integrating a dual-aptamer-based transcription amplification strategy with CRISPR-Cas13d (DATAS-Cas13d), in which the protein binding initiates in-vitro RNA transcription followed by the activation of RspCas13d. Using a short fluorescent ssRNA as the signal reporter and cardiac troponin I (cTnI) as the model analyte, the DATAS-Cas13d system showed a wide linear range, low detection limit, and high specificity for the detection of cTnI in buffer and human serum. Thanks to the facile integration of various bioreceptors into the DATAS-Cas13d system, the method could be adapted to detecting a broad range of clinically relevant protein biomarkers, and thus broaden the medical applications of Cas13d-based diagnostics.

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Section: Resultsmentioning

confidence: 99%

Harnessing the CRISPR‐Cas13d System for Protein Detection by Dual‐Aptamer‐Based Transcription Amplification

Feng

Liu

et al. 2023

Chemistry A European J

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“…Schematics of a gene detection approach using CRISPR/Cas13 (SHERLOCK) that combines DNA/RNA extraction and isothermal amplification. Different modes of signal transductions, such as, fluorescent real-time, , multiplexed readouts (e.g., detection of two genes employing two-color fluorescence with Cas13a and Cas13b), and lateral flow detection can be used in this biosensing approch. , …”

Section: Crispr Based Assay For Plant Diagnosticsmentioning

confidence: 99%

“…In addition, a lateral flow readout for visual purposes was generated by dipping the lateral flow strip in a CRISPR/Cas12a combination. In the end, a field-applicable CRISPR/Cas12a system was established by extracting plant total nucleic acid in 5 min with alkaline lysis solution, RT-RPA amplification in 30 min, and fluorescence and biotin labeled ssDNA probe for lateral flow readout . They also utilized CRISPR-Cas13a/d based method for amplification free detection of the viruses with fluorescent signal which can be detected by fluorescent reader.…”

Section: Crispr Based Assay For Plant Diagnosticsmentioning

confidence: 99%

“…For instance, rice blast pathogens and bt-rice genes were efficiently detected using a single method and highly active crRNAs. DNA and RNA can also be targeted on the same platform by switching the isothermal amplification method from RPA, LAMP to RT-RPA, RT-LAMP. , Moreover, DNA and RNA targets can be identified in a single reaction by combining Cas12a and Cas13a with distinct transducing signals . Additionally, distinct orthologues of Cas proteins can perform multiplexing.…”

Section: Universal Applicationmentioning

confidence: 99%

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CRISPR/Cas-Based Diagnostics in Agricultural Applications

Tanny

Sallam

Soda

et al. 2023

J. Agric. Food Chem.

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Pests and disease-causing pathogens frequently impede agricultural production. An early and efficient diagnostic tool is crucial for effective disease management. Clustered regularly interspaced short palindromic repeats (CRISPR) and the CRISPRassociated protein (Cas) have recently been harnessed to develop diagnostic tools. The CRISPR/Cas system, composed of the Cas endonuclease and guide RNA, enables precise identification and cleavage of the target nucleic acids. The inherent sensitivity, high specificity, and rapid assay time of the CRISPR/Cas system make it an effective alternative for diagnosing plant pathogens and identifying genetically modified crops. Furthermore, its potential for multiplexing and suitability for point-of-care testing at the field level provide advantages over traditional diagnostic systems such as RT-PCR, LAMP, and NGS. In this review, we discuss the recent developments in CRISPR/Cas based diagnostics and their implications in various agricultural applications. We have also emphasized the major challenges with possible solutions and provided insights into future perspectives and potential applications of the CRISPR/Cas system in agriculture.

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“…8 Because these methods use an amplification step, they achieve high sensitivity (∼2−20 aM (∼1−10 copies/μL)) equivalent to RT-qPCR; however, it requires more than 30 min for detection. 10 Therefore, even with the use of Cas12a/13a, high sensitivity and rapid detection of viral RNA are not possible.…”

Section: ■ Introductionmentioning

confidence: 99%

Purification/Amplification-Free RNA Detection Platform for Rapid and Multiplex Diagnosis of Plant Viral Infections

et al. 2023

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Genetic tests are highly sensitive, and quantitative methods for diagnosing human viral infections, including COVID-19, are also being used to diagnose plant diseases in various agricultural settings. Conventional genetic tests for plant viruses are mostly based on methods that require purification and amplification of viral genomes from plant samples, which generally take several hours in total, making it difficult to use them in rapid detection at point-of-care testing (POCT). In this study, we developed Direct-SATORI, a rapid and robust genetic test that eliminates the purification and amplification processes of viral genomes by extending the recently developed amplification-free digital RNA detection platform called SATORI, allowing the detection of various plant viral genes in a total of less than 15 min with a limit of detection (LoD) of 98 ∼ copies/μL using tomato viruses as an example. In addition, the platform can simultaneously detect eight plant viruses directly from ∼1 mg of tomato leaves with a sensitivity of 96% and a specificity of 99%. Direct-SATORI can be applied to various infections related to RNA viruses, and its practical use is highly anticipated as a versatile platform for plant disease diagnostics in the future.

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Diagnostics of Infections Produced by the Plant Viruses TMV, TEV, and PVX with CRISPR-Cas12 and CRISPR-Cas13

Cited by 41 publications

References 45 publications

Harnessing the CRISPR‐Cas13d System for Protein Detection by Dual‐Aptamer‐Based Transcription Amplification

Harnessing the CRISPR‐Cas13d System for Protein Detection by Dual‐Aptamer‐Based Transcription Amplification

CRISPR/Cas-Based Diagnostics in Agricultural Applications

Purification/Amplification-Free RNA Detection Platform for Rapid and Multiplex Diagnosis of Plant Viral Infections

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