A CRISPR Test for Rapidly and Sensitively Detecting Circulating EGFR Mutations

Tsou, Jen-Hui; Leng, Qixin; Jiang, Feng

doi:10.3390/diagnostics10020114

Cited by 23 publications

(22 citation statements)

References 22 publications

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“…27,28 One of the main advantages of the IMPACT chip compared to traditional CRISPR assays is its ability to limit the background caused by dye-quencher probes, which is typically seen in CRISPR detection in the liquid state and needs to be designed around to lower the detection limit. 29,30 Our device utilizing solid-phase CRISPR does not need to tether a quencher on the probe. The cleaved CRISPR products are sent to a separate reservoir for detection thus completely avoiding the fluorescence background caused by the tradition "onepot" detection.…”

Section: Discussionmentioning

confidence: 99%

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

Bao

et al. 2020

Preprint

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A fully Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) system is developed for viral DNA detection. This powerful system is patterned with high-aspect ratio micropillars to enhance reporter probe binding. After surface modification and probe immobilization, CRISPR Cas12a/crRNA complex is injected into the fully enclosed system. With the presence of double-stranded DNA target, the CRISPR enzyme is activated and non-specifically cleaves the ssDNA reporters initially immobilized on the micropillars. This collateral cleavage releases fluorescence dyes into the assay, and the intensity is linearly proportional to the target DNA concentration ranging from 0.1 to 10 nM. Importantly, this system does not rely on traditional dye-quencher labeled probe thus eliminating the fluorescence background presented in the assay. Furthermore, our one-step detection protocol is performed at isothermal conditions (37°C) without using complicated and time-consuming off-chip probe hybridization and denaturation. This miniaturized and fully packed IMPACT chip demonstrates rapid, sensitive, and simple nucleic acid detection and is an ideal candidate for the next generation molecular diagnostic platform for point-of-care (POC) applications, responding to emerging and deadly pathogen outbreaks.

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Section: Discussionmentioning

confidence: 99%

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

Bao

et al. 2020

Preprint

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“…Plasmids of KRAS mutation G12C and G12S (#83167 and #83144) in Escherichia coli DH5α were obtained from Addgene (Addgene, Watertown, MA, USA) and grown in LB medium with 50 µg/mL Streptomycin in a shaking incubator. Plasmid DNA was isolated by using our previously developed protocols [ 9 , 10 ]. DNA concentration was measured by using the Quantifiler Human DNA Quantification kit (Applied Biosystems, Foster City, CA, USA).…”

Section: Methodsmentioning

confidence: 99%

“…DNA extraction. We extracted DNA using the DNeasy kit (Qiagen, Valencia, CA, USA) as described in our previous publications [ 9 , 10 , 11 , 12 , 13 , 14 ]. We eluted DNA with 50 μL of elution buffer (10 mmol/L Tris-Cl, pH 8.5) (Sigma-Aldrich Corporation).…”

Section: Methodsmentioning

confidence: 99%

“…For instance, we recently demonstrated that CRISPR–Cas12a could sensitively detect nuclei acids of exogenous viruses, such as human papillomavirus, in plasma without requiring ancillary machineries [ 9 ]. We have also demonstrated that CRISPR–Cas12a can discover endogenous DNA mutations of tumor-related genes in plasma [ 10 ]. In this study, we investigated whether CRISPR–Cas12a could sensitively detect KRAS mutations.…”

Section: Introductionmentioning

confidence: 99%

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Sensitive Detection of KRAS Mutations by Clustered Regularly Interspaced Short Palindromic Repeats

et al. 2021

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Kirsten rat sarcoma viral oncogene (KRAS) is the isoform most frequently mutated in human tumors. Testing for activating KRAS mutations has important implications for diagnosis and the personalized medicine of cancers. The current techniques for detecting KRAS mutations have moderate sensitivity. The emerging clustered regularly interspaced short palindromic repeats (CRISPR) system shows great promise in the detection of nucleic acids and is revolutionizing medical diagnostics. This study aimed to develop CRISPR–Cas12a as a sensitive test to detect KRAS mutations. Serially diluted DNA samples containing KRAS mutations are subjected to CRISPR–Cas12a and polymerase chain reaction (PCR). CRISPR–Cas12a and PCR can specifically detect 0.01% and 0.1% mutant KRAS DNA in the presence of wild-type KRAS DNA, respectively. Twenty pairs of lung tumor and noncancerous lung tissues are tested by CRISPR–Cas12a, PCR, and direct sequencing. CRISPR–Cas12a could identify the G12C mutation in five of 20 tumor tissues, while both PCR and direct sequencing discovered the KRAS mutation in three of the five tumor tissues. Furthermore, the results of CRISPR–Cas12a for testing the mutation could be directly and immediately visualized by a UV light illuminator. Altogether, CRISPR–Cas12a has a higher sensitivity for the detection of KRAS mutations compared with PCR and sequencing analysis, and thus has diagnostic and therapeutic implications. Nevertheless, the technique needs to be validated for its clinical significance in a large and prospective study.

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“…Our previous study has demonstrated that CRISPR-Cas12a can detect nucleic acids of exogenous viruses, such as human papillomavirus, in the raw plasma of cervical cancer patients without requiring RNA extraction [ 12 ]. Our research has also demonstrated that the CRISPR-based test can rapidly and sensitively detect endogenous DNA mutations of EGFR and KRAS in plasma and tissue specimens of lung cancer patients [ 13 , 14 ]. Recently, we developed CRISPR-Cas12a as a rapid test for sensitive detection of SARS-CoV-2 [ 15 ].…”

Section: Introductionmentioning

confidence: 99%

Detection and Differentiation of SARS-CoV-2, Influenza, and Respiratory Syncytial Viruses by CRISPR

et al. 2021

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SARS-CoV-2, influenza, and respiratory syncytial viruses (RSVs) cause acute respiratory infections with similar symptoms. Since the treatments and outcomes of these infections are different, the early detection and accurate differentiation of the viruses are clinically important for the prevention and treatment of the diseases. We previously demonstrated that clustered regularly interspaced short palindromic repeats (CRISPR) could rapidly and precisely detect SARS-CoV-2. The objective of this study was to develop CRISPR as a test for simultaneously detecting and accurately distinguishing the viruses. The CRISPR assay with an RNA guide against each virus was performed in the reference standards of SARS-CoV-2, influenza A and B, and RSV. The CRISPR assay had a limit of detection of 1–100 copies/µL for specifically detecting SARS-CoV-2, influenza A and B, and RSV without cross-reaction with other respiratory viruses. The validation of the test in nasopharyngeal specimens showed that it had a 90–100% sensitivity and 100% specificity for the detection of SARS-CoV-2, influenza A and B, and RSV. The CRISPR assay could potentially be used for sensitive detection and specific differentiation of the respiratory viruses.

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A CRISPR Test for Rapidly and Sensitively Detecting Circulating EGFR Mutations

Cited by 23 publications

References 22 publications

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

Integrated Micropillar Polydimethylsiloxane Accurate CRISPR Detection (IMPACT) System for Rapid Viral DNA Sensing

Sensitive Detection of KRAS Mutations by Clustered Regularly Interspaced Short Palindromic Repeats

Detection and Differentiation of SARS-CoV-2, Influenza, and Respiratory Syncytial Viruses by CRISPR

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