Exploring the Trans‐Cleavage Activity of CRISPR‐Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor

Dai, Yifan; Somoza, Rodrigo A.; Wang, Liu; Welter, Jean F.; Li, Yan; Caplan, Arnold I.; Liu, Chung Chiun

doi:10.1002/ange.201910772

Cited by 113 publications

(116 citation statements)

References 55 publications

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“…Several sensor types have been created by researchers so far, in which no target-amplification is needed to measure at a very low LOD. These sensors are a graphene-based field effect transistor (gFET) ( Hajian et al, 2019 ), nanopore sensors ( Weckman et al, 2019 ; Yang et al, 2018 ), electrochemical sensors ( Bruch et al, 2019 ; Dai et al, 2019 ; Xu et al, 2020 ; D. Zhang et al, 2020 ) and a conductivity sensor combined with a DNA gel ( English et al, 2019 ).…”

Section: Crispr/cas Sensing In Poc Sensorsmentioning

confidence: 99%

“…7 An overview of the mechanisms in the different electronic sensors described in the literature. A) gFET ( Hajian et al, 2019 ), B) nanopore sensor, adjusted from ( Yang et al, 2018 ), C) different types of electrochemical sensors with MB as an electrochemical tag, based on targeted (II) and collateral cleavage (I & III) ( Dai et al, 2019 ; Xu et al, 2020 ; D. Zhang et al, 2020 ) D) collateral cleavage based sensor with a glucose oxidase electrochemical tag. Adapted from ( Bruch et al, 2019 ), according to Creative Commons Attribution License (CC BY 4.0) E) collateral cleavage of DNA-gel based sensor with combined colorimetric and amperometric readout ( English et al, 2019 ).…”

Section: Crispr/cas Sensing In Poc Sensorsmentioning

confidence: 99%

See 1 more Smart Citation

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

Dongen

Berendsen

Steenbergen

et al. 2020

Biosensors and Bioelectronics

231

164

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With the trend of moving molecular tests from clinical laboratories to on-site testing, there is a need for nucleic acid based diagnostic tools combining the sensitivity, specificity and flexibility of established diagnostics with the ease, cost effectiveness and speed of isothermal amplification and detection methods. A promising new nucleic acid detection method is Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)-associated nuclease (Cas)-based sensing. In this method Cas effector proteins are used as highly specific sequence recognition elements that can be combined with many different read-out methods for on-site point-of-care testing. This review covers the technical aspects of integrating CRISPR/Cas technology in miniaturized sensors for analysis on-site. We start with a short introduction to CRISPR/Cas systems and the different effector proteins and continue with reviewing the recent developments of integrating CRISPR sensing in miniaturized sensors for point-of-care applications. Finally, we discuss the challenges of point-of-care CRISPR sensing and describe future research perspectives.

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Section: Crispr/cas Sensing In Poc Sensorsmentioning

confidence: 99%

Section: Crispr/cas Sensing In Poc Sensorsmentioning

confidence: 99%

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

Dongen

Berendsen

Steenbergen

et al. 2020

Biosensors and Bioelectronics

231

164

View full text Add to dashboard Cite

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“…Overall, LbCas12a showed the highest fluorescence signal, which is consistent with previous studies. 13,18 Through observation of the fluorophore-quencher-based reporter assay and time-dependent gel electrophoresis, we hypothesized that the various extensions of ssDNA on the crRNA induce conformational changes on LbCas12a that result in enhanced endonuclease activity.…”

mentioning

confidence: 99%

Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

Nguyễn

Smith

Jain

2020

Preprint

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The CRISPR/Cas12a RNA-guided complexes have a tremendous potential for nucleic acid detection due to its ability to indiscriminately cleave ssDNA once bound to a target DNA. However, the current CRISPR/Cas12a systems are limited to detecting DNA in a picomolar detection limit without an amplification step. Here, we developed a platform with engineered crRNAs and optimized conditions that enabled us to detect DNA, DNA/RNA heteroduplex and methylated DNA with higher sensitivity, achieving a limit of detection of in femtomolar range without any target pre-amplification step. By extending the 3'-or 5'-ends of the crRNA with different lengths of ssDNA, ssRNA, and phosphorothioate ssDNA, we discovered a new selfcatalytic behavior and an augmented rate of LbCas12a-mediated collateral cleavage activity as high as 3.5-fold compared to the wild-type crRNA. We applied this sensitive system to detect as low as 25 fM dsDNA from the PCA3 gene, an overexpressed biomarker in prostate cancer patients, in simulated urine over 6 hours. The same platform was used to detect as low as ~700 fM cDNA from HIV, 290 fM RNA from HCV, and 370 fM cDNA from SARS-CoV-2, all within 30 minutes without a need for target amplification. With isothermal amplification of SARS-CoV-2 RNA using RT-LAMP, the modified crRNAs were incorporated in a paper-based lateral flow assay that could detect the target with up to 23-fold higher sensitivity within 40-60 minutes. MainClass 2 CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPRassociated proteins) systems, such as Cas12a (Cpf1, subtype V-A) and Cas13a (C2c2, subtype VI), are capable of nonspecific cleavage of ssDNA (single-stranded DNA) and RNA, respectively, in addition to successful gene editing. 1-3 This attribute, known as trans-cleavage, is only activated once bound to an activator (ssDNA or dsDNA) that has complementary base-pairing to the guide crRNA. When combined with a FRET-based reporter, a fluorophore connected to a quencher via a short oligonucleotide sequence, the presence of the target activator can be confirmed. This phenomenon has been efficiently harnessed by SHERLOCK (Specific High-sensitivity Enzymatic Reporter unLOCKing) and DETECTR (DNA Endonuclease Targeted CRISPR Trans Reporter) to reliably detect nucleic acids. 1,[4][5][6][7][8] Research studies have reported that an extended secondary DNA on the guide crRNA for Cas12a or a hairpin RNA structure added to the sgRNA for Cas9 increases the efficiency and specificity of gene editing. 9,10 In addition, chemically modified Cas12a guided-RNA has also been shown to facilitate improved gene correction in mammalian cells through both viral-and non-viral methods compared to the wild-type guide RNA. 11 Though these modifications were employed to utilize the cis-cleavage aspect of the CRISPR/Cas systems, the effects of such alterations on the transcleavage remain unknown.was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

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“…One of the many examples of successful identification of CRISPR proteins with previously unknown properties is Cas13a (previously referred to as C2c2), which has transformed into a powerful molecular tool 16 . Application of CRISPR may go far beyond detection of nucleic acids, as several CRISPR technologies have been devised to identify proteins 66 and could be utilized for detecting small molecules 111 (e.g. toxic agents, opioids, drugs etc.).…”

Section: Resultsmentioning

confidence: 99%

“…Dai and colleagues created E-CRISPR system is equipped with electrochemical signal detection module utilizing Cas12a trans-cleavage activity and modified ssDNA reporter with methylene blue electrochemical tag attached to the sensor surface 66 . In the presence of target sequence, Cas12a proteins cleave ssDNA-MB reporter decreasing the level of electrochemical signal being transduced.…”

Section: Methodsmentioning

confidence: 99%

CRISPR-Cas Systems for Diagnosing Infectious Diseases

Kostyusheva¹,

Brezgin²,

Babin³

et al. 2020

Preprint

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Infectious diseases are a global health problem affecting billions of people. Developing rapid and sensitive diagnostic tools is key for successful patient management and curbing disease spread. Currently available diagnostics are very specific and sensitive but time-consuming and require expensive laboratory settings and well-trained personnel; thus, they are not available in resource-limited areas, for the purposes of large-scale screenings and in case of outbreaks and epidemics. Developing new, rapid, and affordable point-of-care diagnostic assays is urgently needed. This review focuses on CRISPR-based technologies and their perspectives to become platforms for point-of-care nucleic acid detection methods and as deployable diagnostic platforms that could help to identify and curb outbreaks and emerging epidemics. We describe the mechanisms and function of different classes and types of CRISPR-Cas systems, including pros and cons for developing molecular diagnostic tests and applications of each type to detect a wide range of infectious agents. Many Cas proteins (Cas9, Cas12, Cas13, Cas14) have been leveraged to create highly accurate and sensitive diagnostic tools combined with technologies of signal amplification and fluorescent, potentiometric, colorimetric, or lateral flow assay detection. In particular, the most advanced platforms -- SHERLOCK/v2, DETECTR, or CRISPR-Chip -- enable detection of attomolar amounts of pathogenic nucleic acids with specificity comparable to that of PCR but with minimal technical settings. Further developing CRISPR-based diagnostic tools promises to dramatically transform molecular diagnostics, making them easily affordable and accessible virtually anywhere in the world. The burden of socially significant diseases, frequent outbreaks, recent epidemics (MERS, SARS and the ongoing coronoviral nCov-2019 infection) urgently need the developing of express-diagnostic tools. Recently devised CRISPR-technologies represent the unprecedented opportunity to reshape epidemiological surveillance and molecular diagnostics.

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Exploring the Trans‐Cleavage Activity of CRISPR‐Cas12a (cpf1) for the Development of a Universal Electrochemical Biosensor

Cited by 113 publications

References 55 publications

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

Point-of-care CRISPR/Cas nucleic acid detection: Recent advances, challenges and opportunities

Enhancement of trans-cleavage activity of Cas12a with engineered crRNA enables amplified nucleic acid detection

CRISPR-Cas Systems for Diagnosing Infectious Diseases

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