Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive SARS-CoV-2 detection

Liu, Sitong; Xie, Tie; Huang, Zhaohe; Pei, Xiaojing; Li, Shujing; He, Yifan; Tong, Yigang; Liu, Guoqi

doi:10.1016/j.snb.2022.132746

Cited by 13 publications

(4 citation statements)

References 39 publications

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“…Although the ssDNA reporters are commonly used for research and applications ( Table 2 ), limiting selections of ssDNA reporters from an application and probe diversification standpoint. We recently investigated the properties of various types of fluorescent probes for CRISPR in detail ( Figure 3 ) [ 65 ]. We demonstrated that the trans-cleavage of Cas12a is not limited to ssDNA or dsDNA reporters but can be extended to molecular beacons (MB).…”

Section: Fluorescence Intensity Signalsmentioning

confidence: 99%

Fluorescence Signal-Readout of CRISPR/Cas Biosensors for Nucleic Acid Detection

et al. 2022

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The CRISPR/Cas system is now being used extensively in nucleic acid detection applications, particularly after the trans-cleavage activity of several Cas effectors was found. A CRISPR/Cas system combined with multiple signal-readout techniques has been developed for various molecular diagnostics applications. Fluorescence is now a widely utilized dominant read-out technique in CRISPR biosensors. An in-depth understanding of various fluorescence readout types and variables affecting the fluorescence signals can facilitate better experimental designs to effectively improve the analytical performance. There are the following two commonly used types of CRISPR/Cas detection modes: the first is based on binding activity, such as Cas9 and dCas9; the second is based on cleavage activity, such as Cas12a, Cas12b, Cas13, and Cas14. In this review, fluorescence signal-readout strategies from the last 5 years based on the binding activity and cleavage activity of the CRISPR/Cas system with fundamentals and examples are fully discussed. A detailed comparison of the available fluorescent reporter sequences and design principles is summarized. Current challenges and further applications of CRISPR-based detection methods will be discussed according to the most recent developments.

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Section: Fluorescence Intensity Signalsmentioning

confidence: 99%

Fluorescence Signal-Readout of CRISPR/Cas Biosensors for Nucleic Acid Detection

et al. 2022

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“…28−30 Additionally, standard fluorescent quencher (FQ) probes can cause steric hindrance, which can lower the trans-cleavage activity thus lowering sensitivity. 31 Recently, nanomaterials have demonstrated the ability to offer simpler and more accessible options for fluorescent quenching. 32 Single-layer graphene oxide (GO) has been shown to exhibit extremely specific distance-dependent fluorescent quenching.…”

Section: ■ Introductionmentioning

confidence: 99%

“…This trans -cleavage mechanism has been leveraged for the creation of many relevant CRISPR-Cas12a-based biosensors, , including for the detection of parasitic infections, bacterial infections, drug-resistant bacteria, and more. These systems often utilize fluorescence as a reliable signal transduction method. , While CRISPR-based fluorescent biosensors have successfully been developed for the detection of bacteria, complicated probe designs and expensive fluorescent quenchers have hindered the true portability and affordability for POC detection. − Additionally, standard fluorescent quencher (FQ) probes can cause steric hindrance, which can lower the trans -cleavage activity thus lowering sensitivity . Recently, nanomaterials have demonstrated the ability to offer simpler and more accessible options for fluorescent quenching …”

Section: Introductionmentioning

confidence: 99%

On-Site Fluorescent Detection of Sepsis-Inducing Bacteria using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System

Kasputis,

He,

et al. 2024

Anal. Chem.

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Sepsis is an extremely dangerous medical condition that emanates from the body's response to a pre-existing infection. Early detection of sepsis-inducing bacterial infections can greatly enhance the treatment process and potentially prevent the onset of sepsis. However, current point-of-care (POC) sensors are often complex and costly or lack the ideal sensitivity for effective bacterial detection. Therefore, it is crucial to develop rapid and sensitive biosensors for the on-site detection of sepsisinducing bacteria. Herein, we developed a graphene oxide CRISPR-Cas12a (GO-CRISPR) biosensor for the detection of sepsisinducing bacteria in human serum. In this strategy, single-stranded (ssDNA) FAM probes were quenched with single-layer graphene oxide (GO). Target-activated Cas12a trans-cleavage was utilized for the degradation of the ssDNA probes, detaching the short ssDNA probes from GO and recovering the fluorescent signals. Under optimal conditions, we employed our GO-CRISPR system for the detection of Salmonella Typhimurium (S. Typhimurium) with a detection sensitivity of as low as 3 × 10 3 CFU/mL in human serum, as well as a good detection specificity toward other competing bacteria. In addition, the GO-CRISPR biosensor exhibited excellent sensitivity to the detection of S. Typhimurium in spiked human serum. The GO-CRISPR system offers superior rapidity for the detection of sepsis-inducing bacteria and has the potential to enhance the early detection of bacterial infections in resourcelimited settings, expediting the response for patients at risk of sepsis.

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“…However, its limited catalytic speed (with reported values from 0.07 to 7 turnovers per sec 14 ) is insu cient to realize e cient nucleic acid detection without additional ampli cation strategies. Although various ideas have been introduced to increase the overall endonuclease trans-cleavage levels, such as gRNA modi cations, sequence optimization 15 , trans-cleavage substrate modi cations 16,17 , and chemical enhancers 18 , the overall catalytic e ciency of a single Cas12a RNP remains limited, which is challenging for certain application. More importantly, the conventional one-to-one correspondence between target molecules and activated RNPs limits the overall enzymatic activity level in CRISPR/Cas systems which hinders potential applications in and beyond diagnostics.…”

Section: Introductionmentioning

confidence: 99%

Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis

Li,

Yang,

Deng

et al. 2023

Preprint

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Trans-cleavage rates of Cas endonucleases such as Cas12a remain limited despite optimisation efforts. We found that Cas12a ribonucleoproteins poorly bind to, and are only minimally activated by small circular DNA nanostructures which partially match the gRNA sequence. However, activation is restored when the nanostructure is linearised. Building on this finding, we established a novel Autocatalytic CRISPR/Cas12a Amplification Reaction (AutoCAR) system which allows a single nucleic acid target to activate multiple ribonucleoproteins, and increase the achievable trans-cleavage rates per target by over 3 orders of magnitude. A rate equation-based model was built to interpret the observed nonlinear, near-exponential rate trends. The AutoCAR system was applied for genomic DNA (H. Pylori) and RNA (SARS-CoV-2) diagnostics at 1 aM level sensitivity (1.4 copies/µL and 0.36 copies/µL, respectively), comparable to the polymerase chain reaction without any additional signal amplification at room temperature. The RNA detection with Cas12a RNPs did not require reverse transcription.

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Systematically investigating the fluorescent signal readout of CRISPR-Cas12a for highly sensitive SARS-CoV-2 detection

Cited by 13 publications

References 39 publications

Fluorescence Signal-Readout of CRISPR/Cas Biosensors for Nucleic Acid Detection

Fluorescence Signal-Readout of CRISPR/Cas Biosensors for Nucleic Acid Detection

On-Site Fluorescent Detection of Sepsis-Inducing Bacteria using a Graphene-Oxide CRISPR-Cas12a (GO-CRISPR) System

Topological barrier to Cas12a activation by circular DNA nanostructures facilitates autocatalysis

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