Cas12a/blocker DNA-based multiplex nucleic acid detection system for diagnosis of high-risk human papillomavirus infection

Han, Jiqing; Shin, Jiye; Lee, Eun Sung; Seok, Byung; Kim, Seokjoon; Jang, Youngjun; Kim, Seokhwan; Park, Sun-Hee

doi:10.1016/j.bios.2023.115323

Cited by 17 publications

(5 citation statements)

References 47 publications

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“…The accurate and timely detection of disease biomarkers is essential to ensure the effective treatment and epidemiological surveillance. − Nucleic acid testing (NAT) capable of detecting trace amount of disease-related DNA or RNA via nucleic acid amplification has become the gold standard for various infectious and somatic diseases. ,− However, standard NATs relying on polymerase chain reaction (PCR) require costly equipment and trained personnel and thus can only be used in centralized facilities . The ongoing need to enable accurate testing at home and at the point-of-care (POC) demands the field-deployable NATs with high assay performance but low infrastructural requirements. − Toward this goal, clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated (Cas) systems are currently revolutionizing NATs by offering exceptional sensitivity, specificity, and accessibility. − A parrel effort was also made to engineer deoxyribozymes (DNAzymes) into CRISPR-like tools for gene editing and disease diagnostics because DNAzymes also possess RNA/DNA-cleaving activities but with no need for protein components. − However, existing DNAzymes do not possess sequence selectivity to nucleic acid targets. To address this challenge, Mokany et al introduced a multicomponent DNAzyme (MNAzyme) design that allowed the detection of any nucleic acid target by splitting the catalytic core of a DNAzyme into two parts, each of which was extended with a complementary domain to a given target nucleic acid .…”

Section: Introductionmentioning

confidence: 99%

Engineering Gene-Specific DNAzymes for Accessible and Multiplexed Nucleic Acid Testing

Gao,

Yi,

Tan

et al. 2024

JACS Au

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The accurate and timely detection of disease biomarkers at the point-of-care is essential to ensuring effective treatment and epidemiological surveillance. Here, we report the selection and engineering of RNA-cleaving DNAzymes that respond to specific genetic markers and amplify detection signals. Because the target-specific activation of genespecific DNAzymes (gDz) is like the trans-cleavage activity of clustered regularly interspaced short palindromic repeats (CRISPR) CRISPRassociated (Cas) machinery, we further developed a CRISPR-like assay using RNA-cleaving DNAzyme coupled with isothermal sequence and signal amplification (CLARISSA) for nucleic acid detection in clinical samples. Building on the high sequence specificity and orthogonality of gDzs, CLARISSA is highly versatile and expandable for multiplex testing. Upon integration with an isothermal recombinase polymerase amplification, CLARISSA enabled the detection of human papillomavirus (HPV) 16 in 189 cervical samples collected from cervical cancer screening participants (n = 189) with 100% sensitivity and 97.4% specificity, respectively. A multiplexed CLARISSA further allowed the simultaneous analyses of HPV16 and HPV18 in 46 cervical samples, which returned clinical sensitivity of 96.3% for HPV16 and 83.3% for HPV18, respectively. No false positives were found throughout our tests. Besides the fluorescence readout using fluorogenic reporter probes, CLARISSA is also demonstrated to be fully compatible with a visual lateral flow readout. Because of the high sensitivity, accessibility, and multiplexity, we believe CLARISSA is an ideal CRISPR-Dx alternative for clinical diagnosis in fieldbased and point-of-care applications.

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Section: Introductionmentioning

confidence: 99%

Engineering Gene-Specific DNAzymes for Accessible and Multiplexed Nucleic Acid Testing

Gao,

Yi,

Tan

et al. 2024

JACS Au

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“…Although the CRISPR/Cas12a method has several turnovers, it can only detect DNA at a limit of 0.1 nM, which is not enough to detect the extremely low levels of the target . In order to enhance the ability to identify small amounts of genetic material, some techniques for amplifying nucleic acids, including as polymerase chain reaction, rolling circle amplification, , and enzyme assisted chain replacement, were incorporated into the CRISPR/Cas12a system. Nevertheless, the stages after target identification using Cas12a/crRNA, which facilitate successive signal cycles, are susceptible to nucleic acid sequence discrepancies, resulting in inaccurate positive outcomes.…”

Section: Introductionmentioning

confidence: 99%

CRISPR/Cas12a-Based APE1 Enzyme Cleavage Assay for Drug Resistance Analysis of Staphylococcus aureus-Related Pneumonia

Lai,

Xia,

Lin

et al. 2024

ACS Omega

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Drug resistance analysis of Staphylococcus aureus is responsible for generating significant mortality and morbidity in numerous diseases. However, sensitive and accurate analysis of drug resistance of S. aureus remains a huge challenge. In this study, we present the development of a fluorescence biosensor based on the CRISPR/Cas12a system that enables label-free and ultrasensitive detection of the mecA gene in methicillin-resistant S. aureus (MRSA). The biosensor identified the mecA gene in MRSA using Cas12a/ crRNA. This recognition triggered the trans-cleavage activity of Cas12a and the release of RNA1, which subsequently induced Apurinic/apyrimidinic endonuclease 1 (APE1) enzyme-assisted target recycling and G-quadruplexes/ Thioflavin T-based signal reaction. Based on this, the biosensor effectively detects the mecA gene with a low limit of detection of 212 aM and a high degree of selectivity, even toward single base mutations. Compared with the traditional CRISPR-Cas12a system-based methods, in which the signal amplification process is prone to generate nucleic acid sequence mismatch, which causes errors, the biosensor used APE1 to improve nucleic acid sequence recognition specificity to ensure that the RNA1 sequence released after Cas12a/crRNA cleavage can specifically guide the signal cycle. In addition to enhancing the CRISPR toolkit, the developed biosensor offers a novel method for the precise and sensitive identification of drug-resistant microbes that cause infections.

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“…Clustered regularly interspaced short palindromic repeats/CRISPR-associated (CRISPR/Cas), which is an RNA-guided component from the immune system of bacteria, has been extensively harnessed for developing novel biosensors. − For example, Shin et al developed an enhanced CRISPR/Cas-based fluorescence assay for reliable and sensitive detection of bacteria . In addition, Han et al proposed an isothermal amplification strategy by employing Cas12a-based primer production for sensitive nucleic acid quantification .…”

Section: Introductionmentioning

confidence: 99%

Sensitive and Direct Analysis of Pseudomonas aeruginosa through Self-Primer-Assisted Chain Extension and CRISPR-Cas12a-Based Color Reaction

Hu,

Liang,

et al. 2023

ACS Omega

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Pseudomonas aeruginosa ( P. aeruginosa ) is a common opportunistic Gram-negative pathogen that may cause infections to immunocompromised patients. However, sensitive and reliable analysis of P. aeruginosa remains a huge challenge. In this method, target recognition assists the formation of a self-primer and initiates single-stranded chain production. The produced single-stranded DNA chain is identified by CRISPR-Cas12a, and consequently, the trans -cleavage activity of the Cas12a enzyme is activated to parallelly digest Ag + aptamer sequences that are chelated with silver ions (Ag + ). The released Ag + reacted with 3,3′,5,5′-tetramethylbenzidine (TMB) for coloring. Compared with the traditional color developing strategies, which mainly rely on the DNA hybridization, the color developing strategy in this approach exhibits a higher efficiency due to the robust trans -cleavage activity of the Cas12a enzyme. Consequently, the method shows a low limit of detection of a wide detection of 5 orders of magnitudes and a low limit of detection of 21 cfu/mL, holding a promising prospect in early diagnosis of infections. Herein, we develop a sensitive and reliable method for direct and colorimetric detection of P. aeruginosa by integrating self-primer-assisted chain production and CRISPR-Cas12a-based color reaction and believe that the established approach will facilitate the development of bacteria-analyzing sensors.

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Cas12a/blocker DNA-based multiplex nucleic acid detection system for diagnosis of high-risk human papillomavirus infection

Cited by 17 publications

References 47 publications

Engineering Gene-Specific DNAzymes for Accessible and Multiplexed Nucleic Acid Testing

Engineering Gene-Specific DNAzymes for Accessible and Multiplexed Nucleic Acid Testing

CRISPR/Cas12a-Based APE1 Enzyme Cleavage Assay for Drug Resistance Analysis of Staphylococcus aureus-Related Pneumonia

Sensitive and Direct Analysis of Pseudomonas aeruginosa through Self-Primer-Assisted Chain Extension and CRISPR-Cas12a-Based Color Reaction

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