CRISPR-/Cas12a-Mediated Liposome-Amplified Strategy for the Surface-Enhanced Raman Scattering and Naked-Eye Detection of Nucleic Acid and Application to Food Authenticity Screening.
TL;DR: Wang et al. as mentioned in this paper proposed a sensitive SERS-based on-site nucleic acid detection strategy mediated by CRISPR/Cas12a with trans-cleavage activity on ssDNA linkers utilized to capture liposomes.
Abstract: Surface-enhanced Raman scattering (SERS) has been recognized as a powerful tool for biosensors due to the ultrahigh sensitivity and unique fingerprint information. However, there are some limitations in trace target nucleic acid detection for the restricted signal-transducing and amplification strategies. Inspired by CRISPR/Cas12a with specific target DNA-activated collateral single-strand DNA (ssDNA) cleavage activity and liposome with signal molecule-loading properties, we first proposed a sensitive SERS-based on-site nucleic acid detection strategy mediated by CRISPR/Cas12a with trans-cleavage activity on ssDNA linkers utilized to capture liposomes. Liposomes loading two kinds of signal molecules, 4-nitrothiophenol (4-NTP) and cysteine, could achieve the dual-mode detection of target DNA with SERS and naked eye, respectively. The promptly amplified signals were initiated by the triggered breakdown of signal molecule-loaded liposomes. Emancipated 4-NTP, a biological-silent Raman reporter, would achieve highly selective and sensitive SERS measurement. Released cysteine induced the aggregation of plasmonic gold nanoparticles, leading to an obvious red to blue colorimetric shift to realize portable naked-eye detection. With this strategy, target nucleic acid concentration was dexterously converted into SERS and visualization signals and could be detected as low as 100 aM and 10 pM, respectively. The approach was also successfully applied to determine meat adulteration, achieving the detection of a low adulteration ratio in the complicated food matrix. We anticipate that this strategy will not only be regarded as a universal platform for the on-site detection of food authenticity but also broaden SERS application for the accurate determination of diverse biomarkers.
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54 citations
TL;DR: In this paper , a recombinase polymerase amplification (RPA)-integrated microfluidic paper-based analytical device (μPAD) was designed for supersensitive SERS detection.
45 citations
TL;DR: In this article , an ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection.
Abstract: An ultrasensitive surface-enhanced Raman scattering (SERS) biosensor driven by CRISPR/Cas12a was proposed for on-site nucleic acid detection. We tactfully modified single-strand DNA (ssDNA) with a target-responsive Prussian blue (PB) nanolabel to form a probe and fastened it in the microplate. Attributed to the specific base pairing and highly efficient trans-cleavage ability of the CRISPR/Cas12a effector, precise target DNA recognition and signal amplification can be achieved, respectively. In the presence of target DNA, trans-cleavage towards the probe was activated, leading to the release of a certain number of PB nanoparticles (NPs). Then, these free PB NPs would be removed. Under alkali treatment, the breakdown of the remaining PB NPs in the microplate was triggered, producing massive ferricyanide anions (Fe(CN)64-), which could exhibit a unique characteristic Raman peak that was located in the "biological Raman-silent region". By mixing the alkali-treated solution with the SERS substrate, Au@Ag core-shell NP, the concentration of the target DNA was finally exhibited as SERS signals with undisturbed background, which can be detected by a portable Raman spectrometer. Importantly, this strategy could display an ultralow detection limit of 224 aM for target DNA. Furthermore, by targeting cow milk as the adulterated ingredient in goat milk, the proposed biosensor was successfully applied to milk authenticity detection.
25 citations
TL;DR: In this article , a review on CRISPR-Cas-based detection and its current status and huge potential specifically for food safety inspection is presented. But, the current food safety detection methods are still unsatisfactory in some ways such as being timeconsuming, displaying unmet sensitivity and specificity standards, and there is a comparative paucity of multiplexed testing and POCT.
Abstract: Food safety is one of the biggest public issues occurring around the world. Microbiological, chemical, and physical hazards can lead to food safety issues, which may occur at all stages of the supply chain. In order to tackle food safety issues and safeguard consumer health, rapid, accurate, specific, and field-deployable detection methods meeting diverse requirements are one of the imperative measures for food safety assurance. CRISPR-Cas system, a newly emerging technology, has been successfully repurposed in biosensing and has demonstrated huge potential to establish conceptually novel detection methods with high sensitivity and specificity. This review focuses on CRISPR-Cas-based detection and its current status and huge potential specifically for food safety inspection. We firstly illustrate the pending problems in food safety and summarize the popular detection methods. We then describe the potential applications of CRISPR-Cas-based detection in food safety inspection. Finally, the challenges and futuristic opportunities are proposed and discussed. Generally speaking, the current food safety detection methods are still unsatisfactory in some ways such as being time-consuming, displaying unmet sensitivity and specificity standards, and there is a comparative paucity of multiplexed testing and POCT. Recent studies have shown that CRISPR-Cas-based biosensing is an innovative and fast-expanding technology, which could make up for the shortcomings of the existing methods or even replace them. To sum up, the implementation of CRISPR-Cas and the integration of CRISPR-Cas with other techniques is promising and desirable, which is expected to provide "customized" and "smart" detection methods for food safety inspection in the coming future.
22 citations
TL;DR: In this paper, the authors summarize the advances of nanolabels in immunoassays, covering nanomaterials as nanozymes, direct signal indicators, enzyme carriers, etc.
Abstract: Food safety issues pose a serious threat to human health, and appropriate detection technologies can effectively ensure food safety. Immunoassay is the gold standard for highly sensitive detection of biochemical targets, providing a good platform for food contaminants detection. In immunoassay, the antibody or antigen conjugated labels are the most important units because they play roles to convert the information of target analytes to easily detected signal. Recently, various efforts have been paid into the label design to enhance the detection sensitivity and stability. Nanomaterials own advantages such as facile preparation, excellent stability, low cost and multi-category signal output, showing great potential as novel labels. Herein, we summarize the advances of nanolabels in immunoassays, covering nanomaterials as nanozymes, direct signal indicators, enzyme carriers, etc. Moreover, applications of nanolabels-based immunoassays in the detection of food contaminants (pathogenic microorganisms, allergic proteins, biotoxins, and others) are comprehensively concluded. Finally, challenges and perspectives on the future trends are also proposed.
16 citations
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TL;DR: It is shown that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA cleavage activity by Cas12a that completely degrades ssDNA molecules, which is also a property of other type V CRISPR-Cas12 enzymes.
Abstract: CRISPR-Cas12a (Cpf1) proteins are RNA-guided enzymes that bind and cut DNA as components of bacterial adaptive immune systems Like CRISPR-Cas9, Cas12a has been harnessed for genome editing on the basis of its ability to generate targeted, double-stranded DNA breaks Here we show that RNA-guided DNA binding unleashes indiscriminate single-stranded DNA (ssDNA) cleavage activity by Cas12a that completely degrades ssDNA molecules We find that target-activated, nonspecific single-stranded deoxyribonuclease (ssDNase) cleavage is also a property of other type V CRISPR-Cas12 enzymes By combining Cas12a ssDNase activation with isothermal amplification, we create a method termed DNA endonuclease-targeted CRISPR trans reporter (DETECTR), which achieves attomolar sensitivity for DNA detection DETECTR enables rapid and specific detection of human papillomavirus in patient samples, thereby providing a simple platform for molecular diagnostics
1,989 citations
TL;DR: An outlook of the key challenges in bioanalytical SERS, including reproducibility, sensitivity, and spatial and time resolution is given.
Abstract: Surface-enhanced Raman spectroscopy (SERS) inherits the rich chemical fingerprint information on Raman spectroscopy and gains sensitivity by plasmon-enhanced excitation and scattering. In particular, most Raman peaks have a narrow width suitable for multiplex analysis, and the measurements can be conveniently made under ambient and aqueous conditions. These merits make SERS a very promising technique for studying complex biological systems, and SERS has attracted increasing interest in biorelated analysis. However, there are still great challenges that need to be addressed until it can be widely accepted by the biorelated communities, answer interesting biological questions, and solve fatal clinical problems. SERS applications in bioanalysis involve the complex interactions of plasmonic nanomaterials with biological systems and their environments. The reliability becomes the key issue of bioanalytical SERS in order to extract meaningful information from SERS data. This review provides a comprehensive over...
1,073 citations
TL;DR: In a recent study, it was found that Cas 12a, which belongs to the class 2 type V-A CRISPR-Cas system, performed collateral cleavage on non-targeted ssDNAs upon the formation of the Cas12a/crRNA/target DNA ternary complex.
Abstract: Dear Editor, Today, the need for time-effective and cost-effective nucleic acid detection methods is still growing in fields such as human genotyping and pathogen detection. Using synthetic biomolecular components, many methods have been developed for fast nucleic acid detection; however, they may not be able to satisfy specificity, sensitivity, speed, cost and simplicity at the same time. Recently, a very promising CRISPR-based diagnostic (CRISPR-Dx) (namely SHERLOCK) was established, which was based on the collateral effect of an RNA-guided and RNAtargeting CRISPR effector, Cas13a. SHERLOCK is of high sensitivity and specificity, and is very convenient in detection of target RNA. However, to detect DNA sequences, in vitro transcription of DNA to RNA must be conducted prior to the SHERLOCK test, which could be inconvenient. In a recent study, we found that Cas12a, which belongs to the class 2 type V-A CRISPR-Cas system, performed collateral cleavage on non-targeted ssDNAs upon the formation of the Cas12a/crRNA/target DNA ternary complex. Here, with the employment of this feature, we used a quenched fluorescent ssDNA reporter (e.g., HEX-N12-BHQ1 in Supplementary Table S1) as the probe, and developed HOLMES (an one-HOur Low-cost Multipurpose highly Efficient System), which could be used for fast detection of target DNA as well as target RNA. In HOLMES, if a target DNA exists in the reaction system, the Cas12a/crRNA binary complex forms a ternary complex with the target DNA, which will then trans-cleave non-targeted ssDNA reporter in the system, illuminating the HEX fluorescence (or any other fluorescence) (Fig. 1a). We ever purified ten Cas12a proteins (Supplementary Table S3) and found all showed the ssDNA trans-cleavage activity. To find the most suitable Cas12a for HOLMES (i.e., with high signal-to-noise ratios), we tested all ten Cas12a proteins and found Lachnospiraceae bacterium ND2006 Cas12a (LbCas12a), Oribacterium sp. NK2B42 Cas12a (OsCas12a), Lachnospiraceae bacterium NC2008 Cas12a (Lb5Cas12a) and Francisella tularensis Cas12a (FnCas12a) showed good performance, among which LbCas12a was chosen for the following studies (Fig. 1b). To determine the sensitivity of HOLMES, we titrated target DNA, and found the minimum detectable concentration for Cas12a-crRNA was approximately 0.1 nM; however, when combined with PCR, the detectable concentration could be as low as 10 aM (Fig. 1c), which was comparable to the SHERLOCK system and was better than PCR alone or quantitative PCR using the SYBR Green method (Supplementary Figure S1). Therefore, to achieve higher sensitivity, PCR amplification was employed in the HOLMES test thereafter. To test whether HOLMES could discriminate singlebase differences, we made point mutations at different positions in the target DNA sequence, including both the PAM region and the guide sequences (Supplementary Figure S2a). When a full length of crRNA guide sequence (24-nt crRNA, Supplementary Table S2) was used, we found mutations in either the PAM sequences or the region of the 1st–7th bases of the guide sequence resulted in clear decline of the fluorescence signal; however, no significant difference was observed when the mutation was within the region of the 8th–18th bases (Supplementary Figure S2b), which was highly consistent with the previous report that the 5′-end seed region in the crRNA
708 citations
TL;DR: Hydroquinone can be used to produce 50-200 nm gold nanoparticles that are relatively monodispersed in size with nearly spherical shapes that contribute to nanotoxicology and biomedicine.
Abstract: Elucidating the impact of nanoparticle size and shape on biological systems is of fundamental importance to nanotoxicology and biomedicine. Currently, the ability to determine this is limited by the lack of a model nanoparticle system having a narrow size and shape distribution over the relevant size range (2−200 nm). Hydroquinone can be used to produce 50−200 nm gold nanoparticles that are relatively monodispersed in size with nearly spherical shapes.
591 citations
TL;DR: It is found that only the ternary complex of Cas12a/ crRNA/targeted ssDNA (or targeted dsDNA) was able to cleave the 5′-labelled target ss DNA (target-DNMT1-3-R-FAM5′), and the proposed ssDNA cleavage processes were illustrated.
Abstract: Dear Editor, The CRISPR-associated protein Cas12a (previously known as Cpf1), which is an endonuclease from the type V-A CRISPR system, has been applied in both in vivo genome editing and in vitro DNA assembly. Cas12a is guided by a single CRISPR RNA (crRNA) with a T-rich protospacer adjacent motif (PAM) sequence to cleave double-stranded DNA (dsDNA) targets, generating sticky ends. Different from Cas9, Cas12a cleaves both the target and non-target strands of a targeted dsDNA by a single active site in the RuvC catalytic pocket (Supplementary information, Figure S12a). Besides, Cas12a also processes precursor crRNAs to generate mature crRNAs. However, the cleavage activity of Cas12a on single-stranded DNA (ssDNA) targets is less understood. To investigate the ssDNA cleavage feature of Cas12a, we employed FnCas12a to cleave short ssDNAs that were labelled with 5(6)-carboxyfluorescein (FAM) on the 3′ terminus and found that the ssDNA cleavage sites were near the 22nd base (i.e., from the 21st to the 23rd), counting from the first 3′-base that was paired with the crRNA guide sequence (Supplementary information, Figure S1a and b and Tables S2 and 3). The cleavage did not require the existence of a PAM sequence in the targeted ssDNA (Supplementary information, Figure S1a and b). In addition, the same cleavage sites were obtained with crRNAs having guide sequences as short as 10 nucleotides (nt) (Supplementary information, Figure S1c and d), which indicates that Cas12a could cleave ssDNA at sites outside of the recognition sequence. We then tested Cas12a cleavage efficiency on ssDNA and dsDNA substrates, and cleavage of ssDNA was slower than that of dsDNA (Supplementary information, Figure S1e and f), whose PAM sequence may account for the higher efficiency. We also performed the Cas12a cleavage experiment with a ssDNA target labelled at its 5′ terminus (target-DNMT1-3-R-FAM5′). Surprisingly, no cleaved bands were observed at the predicted size (20 nt), but short (<6 nt) FAM-labelled products were generated (Fig. 1a). After careful analyses of experimental conditions, we found that only the ternary complex of Cas12a/ crRNA/targeted ssDNA (or targeted dsDNA) was able to cleave the 5′-labelled target ssDNA (target-DNMT1-3), generating short FAM-labelled products (Fig. 1b and Supplementary information, Figure S2). The ternary complex also promiscuously cleaved collateral ssDNAs that had no complementarity to the crRNA guide sequence in the reaction system, generating short products (Fig. 1c and Supplementary information, Figure S3). As it is difficult to distinguish the precise length of the short transcleavage products via polyacrylamide gel electrophoresis, the FAM-labelled short products were purified and analysed by liquid chromatography-mass spectrometry. The results showed that 5′-FAM-labelled substrates were mainly trans-cleaved to 4 nt, while 2-nt products were observed for 3′-FAM-labelled substrates (Supplementary information, Figure S4). We called the promiscuous cleavage of collateral ssDNAs transcleavage to distinguish it from the programmable on-target cleavage of target ssDNA (namely, cis-cleavage), and the proposed ssDNA cleavage processes were illustrated in Fig. 1d. When the ssDNA substrate was labelled at the 5′ terminus, the cis-cleaved 5′-labelled ssDNA products became collateral ssDNAs in the reaction system and were subsequently trans-cleaved into short products, explaining the observed cleavage pattern for 5′-labelled ssDNA substrate. We observed the trans-cleavage products in addition to the cis-products for short 3′-labelled targeted ssDNAs (Fig. 1a). The majority of the ternary complex most likely remained bound to the targeted ssDNAs after cis-cleavage, protecting the labelled 3′-terminus from exposing the trans-activity sites of the Cas12a ternary complex. Next, we tested nine randomly selected Cas12a proteins from different species in addition to the above tested FnCas12a (Supplementary information, Figures S5, 6a and Tables S1, 4 and 5), and all Cas12a proteins exhibited endonuclease activity on plasmid dsDNA (Supplementary information, Figure S6b), cis(Supplementary information, Figure S6c) and trans-cleavage activities on ssDNA (Supplementary information, Figure S6d). This indicates that the cisand trans-cleavage activities on ssDNA might be ubiquitous among Cas12a proteins. When shortened targeted ssDNAs were tested, complexes with 18-nt target ssDNAs that lacked a cleavage site also showed transcleavage activity (Supplementary information, Figure S7a), indicating that cis-cleavage was not a prerequisite for trans-cleavage activity. Trans-cleavage was implemented by the endonuclease activity of the complex, as circular ssDNA (M13mp18) could also be trans-cleaved (Supplementary information, Figure S7b). Moreover, we found that all tested Cas12a complexes except the AsCas12a complex had trans-cleavage activity on collateral dsDNAs (Figure S8), and the activity of the LbCas12a, BoCas12a and Lb4Cas12a complexes was much higher. To identify key residues involved in ssDNA cleavage of both targeted and collateral ssDNAs, we mutated several candidate residues in FnCas12a to alanines, including those related to the RNase activity (H843, K852 and K869) and those responsible for dsDNA cleavage (D917, E1006, D1255 and R1218) (refs. 1,7–9 and Supplementary information, Figure S9). Both cisand transcleavage of ssDNA were unaffected in the RNase activity-related mutants (Supplementary information, Figure S10), but the activities were completely lost or remarkably decreased with mutations in either the RuvC domain (D917A, E1006A and D1255A mutations in FnCas12a) or the Nuc domain (R1218A mutation in FnCas12a) (Fig. 1e, f). Recent studies showed that the RuvC catalytic pocket of both C2c1 and Cas12a was responsible for the cleavage of both strands of targeted dsDNA, leading us to propose that targeted ssDNAs were cis-cleaved by this catalytic pocket (Supplementary information, Figures S11a, b, d, e and S12c). Moreover, according to the structure of the C2c1-crRNAexcess DNA complex (Supplementary information, Figure S11c), trans-cleavage of collateral ssDNAs could also be achieved by the same catalytic pocket (Fig. 1g, and Supplementary information,
522 citations