A critical review of microfluidic systems for CRISPR assays

Avaro, Alexandre S.; Santiago, Juan G.

doi:10.1039/d2lc00852a

Cited by 21 publications

(7 citation statements)

References 128 publications

(244 reference statements)

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“…In this modality, fluorogenic enzymes are directly activated by one-to-one binding to target biomolecules, allowing indirect detection of target molecules from the fluorescent signal generated by the enzymatic reaction. Recently, nucleic acid cleavage enzymes such as CRISPR–Cas proteins, which are activated upon binding to specific nucleic acid sequences, have been widely adopted in biomedical applications 18 , 19 . Digital bioanalysis using these enzymes has attracted great attention in the diagnosis of infectious diseases, including COVID-19, as an innovative genetic testing method that bypasses the amplification process of genetic material 11 , 12 , 20 – 23 .…”

Section: Resultsmentioning

confidence: 99%

Exploring fluoropolymers for fabrication of femtoliter chamber arrays used in digital bioanalysis

Ando,

Murai,

Mori

et al. 2024

Sci Rep

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The global supply of fluoropolymers and fluorinated solvents is decreasing due to environmental concerns regarding polyfluoroalkyl substances. CYTOP has been used for decades primarily as a component of a femtoliter chamber array for digital bioanalysis; however, its supply has recently become scarce, increasing the urgency of fabricating a femtoliter chamber array using alternative materials. In this study, we investigated the feasibility of fabricating a femtoliter chamber array using four types of fluoropolymers in stable supply as candidate substitutes and verified their applicability for digital bioanalysis. Among these candidates, Fluorine Sealant emerged as a viable option for fabricating femtoliter chamber arrays using a conventional photolithography process. To validate its efficacy, we performed various digital bioanalysis using FP-A-based chamber arrays with model enzymes such as CRISPR–Cas, horseradish peroxidase, and β-galactosidase. The results demonstrated the similar performance to that of CYTOP, highlighting the broader utility of FP-A in digital bioanalysis. Our findings underscore the potential of FP-A to enhance the versatility of digital bioanalysis and foster the ongoing advancement of innovative diagnostic technologies.

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Section: Resultsmentioning

confidence: 99%

Exploring fluoropolymers for fabrication of femtoliter chamber arrays used in digital bioanalysis

Ando,

Murai,

Mori

et al. 2024

Sci Rep

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“…As the synthesis of RNA oligos is expensive, these properties will also contribute to reducing the overall cost of the assay. We envision that the integration of microfluidics technology will further advance UNIVERSE toward a valid point‐of‐care testing tool [5b,d,24] …”

Section: Discussionmentioning

confidence: 99%

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR‐Cas12a

Zhang,

Li,

Guo

et al. 2024

Angewandte Chemie

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The CRISPR‐Cas12a system has emerged as a powerful tool for nucleic acid‐based molecular diagnostics. However, it has long been believed to be effective only on DNA targets. Here, we investigate the intrinsic RNA‐enabled trans‐cleavage activity of AsCas12a and LbCas12a and discover that they can be directly activated by fully complementary RNA targets, although LbCas12a exhibits weaker trans‐cleavage activity than AsCas12a on single‐stranded DNA and RNA substrates. Remarkably, we find that the RNA‐activated Cas12a exhibits higher specificity compared to DNA activation. Based on these findings, we develop the “Universal Nuclease for Identification of Virus Empowered by RNA‐sensing” (UNIVERSE) assay for nucleic acid testing. We incorporate a T7 transcription step into this assay, thereby eliminating the requirement for a protospacer adjacent motif (PAM) sequence in the target. Additionally, we successfully detect multiple PAM‐less targets in HIV clinical samples that are undetectable by the conventional Cas12a assay, demonstrating random target selection with the UNIVERSE assay. We further validate the clinical utility of the UNIVERSE assay by testing both HIV RNA and HPV 16 DNA in clinical samples. We envision that the intrinsic RNA targeting capability may bring a paradigm shift in Cas12a‐based nucleic acid detection and further enhance the understanding of CRISPR‐Cas biochemistry.

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“…Recently, CRISPR-Cas has attracted attention as a new genetic test for the detection of viral genomes [ 9 , 10 ]. Cas12a and Cas13a, which are widely used in genetic testing, form a binary complex with CRISPR-RNA (crRNA), which consequently form a specific ternary complex with the target DNA or RNA, having complementary sequences to crRNA.…”

Section: Satori: a Genetic Test Based On Digital Bioanalysis Using Cr...mentioning

confidence: 99%

SATORI: Amplification-free digital RNA detection method for the diagnosis of viral infections

2023

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With the recent global outbreak of COVID-19, there is an urgent need to establish a versatile diagnostic method for viral infections. Gene amplification test or antigen test are widely used to diagnose viral infections; however, these methods generally have technical drawbacks either in terms of sensitivity, accuracy, or throughput. To address this issue, we recently developed an amplification-free digital RNA detection method (SATORI), which can identify and detect viral genes at the single-molecule level in approximately 9 min, satisfying almost all detection performance requirements for the diagnosis of viral infections. In addition, we also developed practical platforms for SATORI, such as an automated platform (opn-SATORI) and a low-cost compact fluorescence imaging system (COWFISH), with the aim of application in clinical settings. Our latest technologies can be inherently applied to diagnose a variety of RNA viral infections, such as COVID-19 and Influenza A/B, and therefore, we expect that SATORI will be established as a versatile platform for point-of-care testing of a wide range of infectious diseases, thus contributing to the prevention of future epidemics. This article is an extended version of the Japanese article published in the SEIBUTSU BUTSURI Vol. 63, p. 115–118 (2023).

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A critical review of microfluidic systems for CRISPR assays

Abstract: We review recent advances in CRISPR-based nucleic acid detection using microfluidic devices and discuss the capabilities, limitations, and potential of this emerging technology.

Cited by 21 publications

References 128 publications

Exploring fluoropolymers for fabrication of femtoliter chamber arrays used in digital bioanalysis

Exploring fluoropolymers for fabrication of femtoliter chamber arrays used in digital bioanalysis

Intrinsic RNA Targeting Triggers Indiscriminate DNase Activity of CRISPR‐Cas12a

SATORI: Amplification-free digital RNA detection method for the diagnosis of viral infections

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