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/lp/springer-journals/aptamer-based-crispr-cas-powered-diagnostics-of-diverse-biomarkers-and-NJwBW7pUOE
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- Springer Journals
- Copyright
- Copyright © The Author(s) 2023
- ISSN
- 2468-0834
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- 2468-0842
- DOI
- 10.1186/s13765-023-00771-9
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Abstract
CRISPR-Cas systems have been widely used in genome editing and transcriptional regulation. Recently, CRISPR-Cas effectors are adopted for biosensor construction due to its adjustable properties, such as simplicity of design, easy operation, collateral cleavage activity, and high biocompatibility. Aptamers’ excellent sensitivity, specificity, in vitro synthesis, base-pairing, labeling, modification, and programmability has made them an attractive molecular recogni- tion element for inclusion in CRISPR-Cas systems. Here, we review current advances in aptamer-based CRISPR-Cas sensors. We briefly discuss aptamers and the knowledge of Cas effector proteins, crRNA, reporter probes, analytes, and applications of target-specific aptamers. Next, we provide fabrication strategies, molecular binding, and detec- tion using fluorescence, electrochemical, colorimetric, nanomaterials, Rayleigh, and Raman scattering. The application of CRISPR-Cas systems in aptamer-based sensing of a wide range of biomarkers (disease and pathogens) and toxic contaminants is growing. This review provides an update and offers novel insights into developing CRISPR-Cas-based sensors using ssDNA aptamers with high efficiency and specificity for point-of-care setting diagnostics. Keywords Biomarker, Pathogen, Disease diagnostics, CRISPR-Cas, Cas12a, Cas13a, Cas14a, Aptamer, Fluorescence, Colorimetric detection which protects the host from invading genetic materi- Introduction als, like bacteriophages or plasmids [2]. In principle, the In recent years, novel diagnostic tools empowered by the CRISPR-associated proteins (Cas protein) use specific integration of CRISPR-Cas proteins (clustered regularly sequences that make up the guide RNA (gRNA) to cleave interspaced short palindromic repeats-CRISPR asso- recognition sites of the foreign DNA under the control ciated) have fueled several applications for food sens- of gRNA. This effectively silences the exogenously intro - ing and biosafety analysis [1]. CRISPR-Cas is part of the duced genetic elements and protects the host organism. adaptive immune system of the bacteria and archaea, Further, advancements in the revolutionary CRISPR- Cas-based gene editing system won it a Nobel Prize in *Correspondence: Chemistry in 2020. This biotechnological tool has been Ulhas Sopanrao Kadam ukadam@gnu.ac.kr widely adopted in genomic editing for insertion, knock- Jong Chan Hong out, fusion, gene regulation, epigenetic modification, tar - jchong@gnu.ac.kr geted mutagenesis, localization, and crop improvement. Division of Life Science and Division of Applied Life Science (BK21 Four), Plant Molecular Biology and Biotechnology Research Center, Gyeongsang Several CRISPR-Cas systems have been shown to have National University, Jinju, Gyeongnam-do 52828, Republic of Korea specific (cis-cleavage) or nonspecific (trans-cleavage or Graduate School of Education, Yonsei University, Seoul 03722, Republic collateral-damage) degrading activity on dsDNA, ssDNA, of Korea Division of Plant Sciences, University of Missouri, Columbia, MO 65211, or ssRNA. The discovery of the unusual spread of repeti - USA tive DNA elements in bacteria led to concurrent series of © The Author(s) 2023. Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article’s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article’s Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http:// creat iveco mmons. org/ licen ses/ by/4. 0/. Kadam et al. Applied Biological Chemistry (2023) 66:13 Page 2 of 15 revelations regarding the multifunctional role of CRISPR- (DNA or RNA). This indiscriminate nucleic acid degra - Cas proteins [1]. Later findings on CRISPR-Cas9 fueled dation potential is coupled with fluorescence labeling of the race to understand and develop CRISPR-Cas technol- DNA probes as reporter molecules and for signal amplifi - ogy in gene editing under the guidance of gRNA. Sub- cation (Table 1). sequently, outstanding application of the CRISPR-Cas9 The collateral cleavage of nucleic acids has opened a system for genome editing was evidenced [3], which fur- new chapter in sensing of diverse targets such as genetic ther catapulted elaborate studies and novel applications elements, disease markers, pathogenic agents, and other in microbiology, plant biology, and biomedical sciences, biomolecules using nucleic acids as molecular recog- specifically genomic editing and molecular diagnostics nition elements. For example, CRISPR-Cas12a-based [4]. The RNA-guided and specific-targeted CRISPR effec - DETECTR, HOLMES, and CRISPR-Cas13a-based tors like Cas9, Cas12, Cas13, and Cas14 (Fig. 1) were suc- SHERLOCK assays (Fig. 2) are designed for this purpose cessively discovered [2, 5]. [5, 6]. The CRISPR-Cas tools are easy to design and con - In cis-cleavage, Cas proteins (CRISPR-Cas9) first rec - struct, moreover, it possesses high specificity and sensi - ognizes the protospacer-adjacent motif (PAM) in spe- tivity. Therefore, these assays could be incorporated into cific dsDNA and then uses guide CRISPR-RNA (crRNA) a portable format as point-of-care (POC) diagnostics to create a double-stranded break. Whereas, the trans- tools. cleavage activity (collateral damage) occurs when a ter- Before 2019, CRISPR-Cas sensors could only recog- nary complex of Cas, crRNA, and target nucleic acid nize nucleic acid markers. An impediment was develop- (ssDNA or ssRNA) is formed, which then activates indis- ing a system to identify and bind specifically to various criminate nonspecific cleavage of nearby nucleic acids non-nucleic acid targets not directly recognized by Cas Fig. 1 Overview of CRISPR-Cas enzyme activities and their catalytic mechanisms. A Cas9 can cleave the target and non-target strands of DNA; a short trinucleotide PAM is also essential for the initial DNA binding; B Cas12a can cleave dsDNA under the guidance of gRNA. The Cas12a enzyme recognizes the PAM of the original T-rich spacer and then recognizes the target sequence to generate PAM distal dsDNA breaks with staggered 5′ and 3′ ends, and Cas12 has the side chains trans-cleavage activity. At the time that the sgRNA-guided DNA is combined in Cas12, Cas12 will release a powerful, indiscriminate single-stranded DNA (ssDNA) cleavage activity; C Cas13 can activate its single-stranded RNA (ssRNA) cleavage activity by binding to crRNA, and it has a additional cleavage activity triggered by the target RNA; D Cas14 protein is a RNA-guided nuclease and can recognize the target ssDNA without restriction sequences and cleave it, and also can non-specifically cleave the surrounding ssDNA nucleases molecule (Modified after: Li et al., Diagnostics 2022, 12(10); Copyright: CC BY License) K adam et al. Applied Biological Chemistry (2023) 66:13 Page 3 of 15 Fig. 2 CRISPR-based diagnostics. A, B Schematic of DETECTR and SHERLOCK assays; C Sequence-specific target binding. Catalytically inactive Cas proteins bind to the target gene that is complementary to gRNA. D Sequence-specific target cleavage. Cas proteins cleave the target gene, followed by the sequence-specific binding. E Target-specific trans-cleavage. Some Cas proteins such as Cas12a or Cas13a non-specifically cleave the ssDNA or ssRNA nearby upon binding to the target gene. F Three widely-used signal detection techniques: the fluorescence, colorimetric or electrochemical signal can be monitored to detect the existence of the analytes (Figure modified after Kim et al., Biomolecules 2021, 11(8); Copyright: CC BY License) proteins [7, 8]. To overcome this hurdle, a study demon- Optimizing the CRISPR-Cas effectors for aptamer- strated short ssDNA sequences (such as, a fragment of based biosensing has opened new doors in molecular DNA aptamer); can serve as an “activator DNA (acDNA)” diagnostics. The inclusion of aptamers for high-affin - to initiate CRISPR-Cas12a trans-cleavage activity [9]. ity detection of more comprehensive targets enables Use of acDNA molecules catapulted CRISPR-Cas appli- direct measurement of a signal as a result of a binding cations for non-nucleic acid molecules by integrating event of an aptamer to the target molecules and relay- aptamers as molecular recognition elements (Table 2). ing it in CRISPR-Cas supported signal enhancement SELEX is commonly used for aptamer discovery and by collateral cleavage of ssDNA probes. CRISPR-Cas- produces highly specific aptamers against target mol - based diagnostics, aptamers facilitate sensing of non- ecules, where the aptamers are short fragments of nucleic nucleic acid targets. In general, an ssDNA plays the acids (ssDNA or RNA) sequences that attach to their role of activators as crRNA could recognize aptamer; targets with a high binding affinity [10–12]. Aptamer depending on target recognition or binding detec- possesses several merits over other molecular recog- tion and quantification of oligonucleotides is possible; nition elements, for example, ease of in vitro synthesis, and collateral damage provides a direct readout from amplification, sequencing, fluorescent labeling, chemical reporter probes. Here, we provide holistic coverage of modifications, and modular design. The aptamers have advancements in aptamer-based CRISPR-Cas sensors. applications in a wide range of fields. Over several hun - This review presents the basics of the CRISPR-Cas12 dred precise and characterized aptameric sequences are system and aptamer, including the necessary compo- available for the detection of small molecules, proteins, nents of CRISPR-Cas for diagnostics (Fig. 2). Then, we live cells, pathogens, metal ions, pesticides, and antibiot- focus on signal generation strategies using fluorescence ics ([13–16]). Many aptameric sensors are available for modifications, colorimetric assays, electrochemical, screening in biomedical and life sciences and have been nanomaterials (gold nanoparticles, nanosheets, mag- helpful for analytical chemistry, environmental, and food netic particles, etc.), Rayleigh-and Raman scattering for analysis [17] diagnostics. Kadam et al. Applied Biological Chemistry (2023) 66:13 Page 4 of 15 Fundamental concepts of CRISPR‑Cas‑based CRISPR-Cas diagnostics assay, a Cas protein, crRNA, an biosensing activator DNA, a labeled reporter, and the target specific The polymorphic genes and Cas proteins, which form ssDNA aptamers are required. the basis of CRISPR-Cas technology, are characterized The examples of Class 2 Cas proteins include Cas3, by the presence of palindromic sequences, protospacer Cas9, Cas10, Cas12a (Fig. 3), Cas13a (Fig. 4), and Cas14a motifs, and an upstream leader sequence in the pro- (Fig. 5). Among these, Cas12a is most commonly used moter regions. With unique activity, Cas proteins, and in biosensing, it is a single guide RNA-mediated DNA the mechanism of CRISPR-Cas, it is classified as Class 1 nuclease with two unique domains: a Nuc and a RuvC [2, and Class 2. Class 1 is a multi-factor effector system that 18] RuvC domain is involved in target recognition and necessitates several Cas protein subunits and is less ame- facilitates the cleavage activity by Nuc lobes. Cas12a can nable; however, the Class 2 effectors have a simple com - be activated either by dsDNA or ssDNA and can degrade ponent and depend on a single Cas protein which forms both the specific target sequence and the nonspecifically the basis of diagnostics applications. For the design of the (collateral damage) any sequence. A protospacer motif Fig. 3 Applications of CRISPR/Cas12. A. RAA-based E-CRISPR, uses MB to modify the ssDNA reporter gene and assemble it on the working electrode, the sample is first amplified by RAA, when the target sequence exists, non-specifically cleaves the MB-modified reporter gene on the electrode surface, finally analyzed by SWV to measure the microelectrochemical signal before and after the introduction of the target nucleic acid sequence; B. EIS-CRISPR, fixes ssDNA on a gold electrode to limit the electronic communication between the electrode and the solution; when the target DNA exists, the Cas12/gRNA system binds to the target DNA and trans-cleaves the ssDNA on the gold electrode and accelerates the electron transfer between the electrode and the solution, detecting subtle changes in the electrode surface current at last (Modified after Li et al., Diagnostics 2022, 12(10); Copyright: CC BY License) K adam et al. Applied Biological Chemistry (2023) 66:13 Page 5 of 15 region (PAM) is essential for binding to dsDNA tar- enhancement and diversification of diagnostics for field- gets, while a PAM sequence is not required for ssDNA. level testing. The past couple of years has seen a rise in Among several Cas proteins, Cas12a is commonly used applications of CRISPR-Cas proteins biosensing due in aptamer-based sensing. Another Cas protein, Cas13a, to rapid and specific detection potential [8]. Moreover, recognizes RNA as a target and requires a single RNA; it the combination of CRISPR-Cas with aptamers pro- also possesses two separate domains for target recogni- vides solutions because they are quick, simple, accurate, tion and RNA degradation. Cas13a enzyme digests flank - modular, dynamic, and cheap. Additionally, these sensors ing RNA sequence next to crRNA on complimentary site can be used in compact assembly with portable biosens- and also cleaves ssRNA in a nonspecific manner; com - ing [22, 23]. Several signal generation and transduction monly employed for viral analysis. Another Cas protein, are demonstrated by coupling with aptameric sensors, Cas14a, is highly compact and much smaller than Cas9; including use of novel nanomaterials for electrochemi- can target and degrade ssDNAs nonspecifically without cal, fluorescent, colorimetric, and SERS sensors (Fig. 7). need of a target sequence. Moreover, Cas14a has shown In following section, we discuss some of the most com- a high affinity towards ssDNAs than Cas12a and could monly used signal detection approaches. degrade long ssDNA probes. Cas14a is a newly found enzyme used to analyze various targets [19, 20]. Fluorescence‑based sensing The major limiting factor in the CRISPR-Cas system The development of fluorophore-modified aptameric is the design of effective crRNA, which facilitates target sensors brings agility and ease of conducting assay due to recognition, binding, and cleavage efficiency [21]. The increased sensitivity and availability of wide range of sig- crRNA nucleotide composition, sequence, and length nal detection devices. Fluorescence analysis is one of the need careful evaluation for a successful outcome of the key technique in molecular diagnostics. Several strategies diagnostic assay. The crRNA contains two functional are found to construct aptamer-based CRISPR-Cas fluo - domains of a guide region and an activator sequence. rescent sensors, for instance, direct detection, sandwich Cas12a, the guide region sequence which forms the scaf- design, and allosteric hairpin (AH) mediated detection. fold is 5’-UAA UUU CUA CUA AGU GUA GAU-3’ (Fig. 6). The direct sensing strategy depends on Cas enzymes’ The guide sequence, which forms the basis of crRNA potential to damage collaterally via binding with ssDNA and makes a binding scaffold, helps the Cas enzyme and activator regions to crRNA; there is no need for pre- varies according to the Cas protein. In Cas13a, this seg- amplification steps. Two ways direct binding and detec - ment carries a sequence of 5’-ACC CCA AAA AUG AAG tion can be performed using aptamers: direct-activation GGGA CU AAA A-3’. An ssDNA activator sequence is strategy and locked-activated strategy. This detection used for Cas proteins, usually designed with complemen- strategy uses a short activator ssDNA (acDNA) sequence tary a fragment of target nucleic acids such as aptamer to facilitate CRISPR-Cas binding. The reporter sequences sequences. The molecular identification and efficient are dual labeled with a fluorophore and a quencher at binding to the activator is a prerequisite to proceed col- both ends, and start with the quenched fluorescence. lateral cleavage of fluorophore-modified reporter DNAs. One of the most commonly used F:Q pairs is Fluorescein- The reporter modifications vary from fluorophore- Black Hole Quencher 1 (FAM-BHQ1). Upon binding of quencher (F:Q) pair to nanoparticles to antibodies or activator DNA to the ribonucleoprotein complex formed affinity tags at 5’ or 3’-ends (or both terminals). Addition - by Cas12a-crRNA, the activation of Cas enzyme takes ally, the molar ratio of the Cas protein to crRNA has to place, and the collateral cleavage of the F:Q reporter by be carefully adjusted for efficient signal amplification [2, Cas12a begins, which in turn produces intense fluores - 5, 8]. cence. The fluorescence signal is measured and quantified The aptamer-based sensing of the target has reached (or could be used for presence and absence in visual anal- its saturation; hence, there is a necessity for signal ysis). As aptamers are specific to the target molecules, in (See figure on next page.) Fig. 4 Applications of CRISPR/Cas13 and CRISPR/Cas14 technology. A LLPS-CRISPR, combined with the collateral cleavage activity of Cas12a/ Cas13a, cleaves long-chain into short-chain nucleotides when the target sequence is present; then the solution will become clear afterwards; B Light-up aptamer-based-Cas13a introduces a new light-up RNA aptamer broccoli/DFHBI-1T complex; when the target sequence is present, Cas13a digests the aptamer broccoli, and the high-fluorescence bound-state DFHBI-1T becomes the low-fluorescence free state; C APC-Cas’s aptamer domain will specifically recognize and bind to the target pathogen, so that AP expands from a hairpin-like inactive structure and transforms into an active structure; the primer domain can be combined with the primer, and then, with the participation of DNA polymerase, AP is used as the template chain to generate dsDNA, which replaces the target pathogen and realizes the first amplification; then the T7 promoter domain is amplified by T7 RNA polymerase to achieve the second step of amplification; subsequently, the Cas13a/crRNA complex recognizes the ssRNA produced by the second step and non-specifically cleaves a large number of surrounding RNA gene reporter probes, achieving the third step of amplification, finally generating a fluorescent signal (Figure modified after Li et al., Diagnostics 2022, 12(10); Copyright: CC BY License) Kadam et al. Applied Biological Chemistry (2023) 66:13 Page 6 of 15 Fig. 4 (See legend on previous page.) the presence of the target, they would form a high affinity manner, where signal yield is directly proportional to binding complex with the target, and the acDNA would free-aptamer concentration; the approach has been be released, resulting in concentration-dependent cleav- devised for ATP detection [24]. Some factors affect - age by Cas12a. The assay could be used in the opposite ing CRISPR-Cas detection were identified, including K adam et al. Applied Biological Chemistry (2023) 66:13 Page 7 of 15 Fig. 5 . Magnetic-bead-assisted dual-signal-amplification aptasensor for sensitive ZEN detection based on the Nt.AlwI enzyme and the Cas12a enzyme. Step 1: The aptamer probe recognizes the ZEN toxin and causes Z1 to dissociate into solution by competitive binding. Step 2: After Z1 and Z2 were hybridized, the cutting activity of the Nt.AlwI enzyme was activated, the Z2 chain was cut to release Z3, Z1 was self-shed after the cutting was finished and it hybridized with Z2 again, and a large amount of Z3 was released by the enzyme-cutting signal amplification to achieve the first signal amplification. Step 3: The combination of Z3 and the Cas12a-crRNA complex activates trans-cleavage activity, non-specifically cleaving any ssDNA so that the added fluorescent signal molecule was cleaved and the quenched fluorescence was restored (Figure from Yao et al., Foods 2022, 11(3); Copyright: CC BY License) 2+ the presence of the target, the aptamer preferentially the concentration of M g ions and the ratio of acDNA. 2+ binds to the target molecule, and that would release While developing CRISPR-Cas for sensing M g ions acDNA. By direct strategy, the acDNA binds to CRISPR- using aptamers, the effect of ionic strength was noticed, Cas and activates the nuclease activity. The approach which was found to play a role in the conformation of the could differentiate live vs. killed dead bacterial cells using RuvC domain [25]. aptamer-Cas14-a1 [19, 20]. Nonspecific or background signals present unnecessary For successful detection based on acDNA, opti hurdles in fluorescence analysis using aptamer-based mal probe design is essential. Using partial base pair- CRISPR-Cas detection. To overcome this challenge, ing in ATP aptamers (Fig. 6B, C) to lock acDNA a locked-activated approach was designed in which a with a sandwich probe of a1-acDNA-a2 (aptamer1- complementary strand of aptamer acts as an acDNA. In acDNA-aptamer2) [19]. Similarly, an excellent onsite this design, the structure-switching approach of ssDNA aptasensor toolkit was developed that displayed high sen- aptamer is exploited, where a complementary acDNA sitivity of 38 nM to melamine, compared to single acDNA probe is allowed to hybridize with the aptamer [26]. In Kadam et al. Applied Biological Chemistry (2023) 66:13 Page 8 of 15 Fig. 6 The small molecule diagnostics. A Generalized schematic of the molecular radar strategy for small molecules diagnostics (Figure from Niu et al., Biosensors and Bioelectronics 183 (2021) 113196; Copyright by Elsevier, used with permission). B Proposed CRISPR-Cas12a biosensor for ATP detection; C The schematic of target ssDNA as well as crRNA used; the target site is highlighted in red (Figure from Peng et al., Sensors & Actuators: B. Chemical 320 (2020) 1281642; Copyright by Elsevier, used with permission) Fig. 7 The principle of Raman spectrometer-read CRISPR/Cas biosensor for nucleic acids detection of pathogenic bacteria. A The activation of CRISPR/Cas12a for trans-cleavage. The green ribbon represents single-stranded DNA subject to trans-cleavage. B The preparation of gold nanostar@4-mercaptobenzoic acid@goldnanoshell structures (AuNS@4-MBA@Au) and their utility in combination with CRISPR/Cas12a for SERS-based bacterial detection for both in-tube and μPAD detection. DNA1 and DNA2 were colored as blue and red, respectively and linker ssDNA was green. C The schematics of the biosensing processes with the estimated assay time for each step. D The nucleic acid sequences required for the proposed biosensor and the hybridization of linker ssDNA with DNA1 and DNA2. AA ascorbic acid. (Figure from Zhuang et al., Biosensors and Bioelectronics 207 (2022) 114167; Copyright by Elsevier;used with permission) activation approach [27]. Thus, the sandwich probe in ELISA has used aptamer as an alternative to antibod- technique was proven to be better for increased sensi- ies (ELASA) [29]. Aptamers are easier to load onto a tivity. In another study, dsDNA as the acDNA elevated plate and label with a variety of reporters, linkers, and the collateral cleavage ability of Cas12a than ssDNA to a functional groups, making signal transformation more higher level [18]. efficient than an antibody. CRISPR-Cas coupled with Antibody-based enzyme-linked immunosorbent assay ELASA, now called CLASA, provides even more sensi- (ELISA) is a popular analytical approach [28]. Modified tive and practical applications [29, 30]. K adam et al. Applied Biological Chemistry (2023) 66:13 Page 9 of 15 Three types of sandwich design strategies are employed in situ RCA amplification on a gold electrode to detect in ELISA—antibody-target-antibody (anti-T-anti), anti- nucleocapsid protein at picogram per mL concentrations. T-aptamer (anti-T-apt), and apt-T-apt. The indiscrimi - A preassembled EC module was used to increase sig- nate cleavage activity of the Cas enzyme can overcome nal [38, 39], where the HCR (hybridization chain reac- HRP’s detection limit in ELISA. For example, using an tion) product peripheral is exposed to a lot of acDNA to anti-T-anti sandwich biosensor and antibody-dsDNA promote collateral cleavage activity. A modified approach as the acDNA for human IL-6 and VEGF, a highly accu- of immuno-RCA assembly multiplies signals from long rate detection with more than 100 times powerful com- ssDNA for bacterial strain-specific aptamers and targets pared to ELISA was achieved [31]. Similarly, Li et al. [29] repetitive acDNAs. Further, a sandwich-type “apt1-T- adopted the apt1-T-apt2 sandwich strategy to improve apt2” CRISPR sensing on AuNPs@Ti C T -Mxene sur- 3 2 x upon this technology. In some cases, when targets have face and aptamer for VEGF could detect sub-picomolar multiple aptamers, the “apt1-T-apt2” strategy becomes range [31]. To overcome some of the limitations of these obsolete. Therefore, an “anti-T-apt” sandwich was pro - assays, an immobilization-free EC sensor with stacking posed in combination with Cas enzymes [32]. Most sig- interaction between DNA molecules and the reduced nificantly, optical fiber instead of PS was used to form a GO/GCE was established [40] and demonstrated for suc- sandwich of fiber/anti-T-apt/ Cas-crRNA, which was cessful detection of thrombin with as low as single fem- able to detect interferons with over 1000-fold higher sen- tomoles. Large particle size modifications detach the sitivity compared to ELISA [33]. To combine the Cas sen- substrate from the electrode, which hinders electron con- sitivity with PCR technology, in situ PCR amplification duction and performance. Ultra-thin two-dimensional after sandwich formation to increase acDNA and CD109 covalent organic framework nanosheets may have supe- aptamers served as templates [29]. The PCR dsDNA rior application in modifications due to their shorter product and a crRNA activated the downstream Cas12a charge transfer durations and distances, high exposure to system. surfaces, and active binding sites. To make use of aptam- ers and CRISPR-Cas effectors with HDA probe-triggered single-circle amplification, the detection of PD-L1 in Electrochemical‑based sensing exosomes at 38 particles per mL was recorded [41]. Being highly sensitive, easy to handle, cheap, modular assembly, portability, and rapid signal detection, the elec- trochemical sensors have captivated researchers’ atten- Nanotechnology‑based sensing tion and made waves in CRISPR-Cas-based analysis for There are several nanotechnological strategies evolved aptamer [34]. For electrochemical sensing, direct target for biomolecular detection. For example, use of gold recognition, label-free analysis, pre-amplification free, nanoparticles (AuNPs) in the biological analysis is well and availability of novel electrode materials make it a known [42–47]. Zhao et al. designed an AuNPs-based lucrative option for integration in aptamer coupled with nanoprobe for Cas sensing to improve acDNA carrier Cas sensing. For example, Dai et al. [9] created a Cas12a- to gain fluorescence yield [48]. A sandwich structure of based EC sensor with aptamer as the acDNA, captured anti-T-aptamer/AuNP/acDNA was created that acti- by crRNA to start collateral cleavage. In which methylene vated the trans-cleavage system. Higher loading on the blue was attached to one end for electrical signal trans- AuNP surface leads to three times more sensitivity than duction, while a thiol moiety helped to link another end free acDNA wither better accuracy. Li et al. assembled on the electrode. Cas12 cleaved off the methylene blue apt-acDNA as in hybrid DNA architecture (HDA) struc- (redox probe) and detached from the electrode surface, ture, with partial ssDNA sequences [29], carrying PAM reducing the signal; using this strategy, TGF-b1 protein sequence specific to promote cis-cleavage by Cas12a with was detected with a sensitivity of 0.2 nM [35]. Addition- the potential of 1000 times sensitivity over traditional ally, an electrochemiluminescence (ECL) sensor using Cas enzyme. Cas and aptamer sensing was designed [36]. Electro- In addition to AuNPs, magnetic nanoparticles, such as chemical sensing usually needs electroactive labels and magnetic beads (MB) (Figs. 7 and 8) are popularly used in a sensitive interface. Abnous et al. created a label-free diagnostics [49, 50]. The use of MB for capture or carrier aptamer-based CRISPR-Cas supported EC sensor by and enrichment in aptamer sensing is highly beneficial. employing acDNA with TdT [37], which allowed the 3−/4− While combining aptamers with CRISPR-Cas, MB can redox probe of [Fe(CN)6] to react with the surface, be used to convert signals, separate, or reject non-target producing a quantifiable signal of cocaine binding to the molecules such as DNA or RNA. MB with a high surface- aptamer. Using a similar approach, Liu et al. [38] designed to-volume ratio can potentially increase acDNA trans- EC impedance spectroscopy with Cas12a substrate and port [49]. Kadam et al. Applied Biological Chemistry (2023) 66:13 Page 10 of 15 Fig. 8 The characterization of AuNS@4-MBA@Au and AuNS@4-MBA@Au@DNA (thiolated ssDNA conjugates). Raman spectra (A) and histogram of SERS signals at wavenumber of 1075 cm-1 (B) for AuNSs, 4-MBA, physically mixed solution of AuNSs together with 4-MBA and AuNS@4-MBA@Au. C UV–Vis absorbance spectrum of each sample. D DLS profile of each sample. E Picture of each sample. 1: AuNPs; 2: AuNSs; 3: AuNS@4-MBA; 4: AuNS@4-MBA@Au; 5: AuNS@4-MBA@Au@DNA. TEM images of AuNPs (F), AuNSs (G), AuNSs@4-MBA (H), AuNS@4-MBA@Au (I) and the crosslinked AuNS@4-MBA@Au@DNA (J) (Figure from Zhuang et al., Biosensors and Bioelectronics 207 (2022) 114167, Copyright by Elsevier; used with permission) Linking of ssDNA aptamer to MBs via streptavidin alpha-fetoprotein, and SARS-CoV-2 viral particles [52, (SA)-biotin binding was found to outbid MB-HDA dis- 53]. sociation [50]. Upon magnetic sorting, the conjugates Connecting a target to higher CRISPR-Cas activators retained free-complementary strands and retained (ssDNA or dsDNA) improves sensitivity, towards this acDNA collateral cleavage activity. Such magnetic sort- rolling circle amplification (RCA) was employed [54], ing made sure acDNA is capable of catalysis without where SA/MB/Apt-A captured protein A-positive bac- off-target or unexpected cleavage by inappropriate DNA teria by magnetic separation, and then target-specific hybridized structure formation with crRNA. The MB methicillin-resistant staphylococcus aureus (MRSA) nanoparticle-assisted method has demonstrated great were identified by enrichment of the penicillin-binding promise for several targets, such as microcystin-LR proteins 2a (PBP2a) with apt-B. In turn, complemen- detection, toxic lead ion detection, and miRNAs analysis tary DNA was released and involved in cyclized padlock [51]. Furthermore, using a modification of DNA hybridi - by hybridizing with its two terminals and triggering the zation to MB and Cas enzymes, several aptamers were following RCA assisted by T4 DNA ligase. Moreover, employed to detect variable targets such as cocaine, the strategy was exploited using Nt.AlwI endonuclease to obtain multiple copies of acDNA, which improved the sensitivity of ZEN toxin [55]. Similarly, Wang et al. used hydrazone ligation in a three-dimensional DNA- zyme walking nanomachine to generate more acDNAs to Table 1 Salient features of various Cas proteins used in diagnostics amplify trans-cleavage activity [56]. It is a versatile tool for understanding molecular behavior and mobility. Its Cas Protein Class Target PAM Collateral Refs. high nanoparticle surface-to-volume ratio enabled signal Activity enhancement and freely available acDNA boosted down- Cas9 Class 2 dsDNA NGG No [85] stream collateral damage after magnetic separation that Cas12a Class 2 Both (ss/ TTTN Yes (ssDNA) [5, 18, 86] could detect lipopolysaccharide with 7.31 fg/mL detec- dsDNA) tion limit [57]. Cas12b Class 2 Both (ss/ TTN Yes (ssDNA) [6] Recently, an MB-multivalent duplexed aptamer mod- dsDNA) ule has been shown to detect PTK7, a cancer biomarker Cas13a Class 2 ssRNA – Yes (ssRNA) [5] using Cas enzyme. Using rolling circle amplification Cas13d Class 2 ssRNA – Yes (ssRNA) [87] (RCA) and preassembled target-specific aptamer on the Cas14a Class 2 ssDNA – Yes (ssDNA) [3] K adam et al. Applied Biological Chemistry (2023) 66:13 Page 11 of 15 Table 2 Key representative examples of CRISPR-Cas proteins and aptamers in diagnostic assays of variety of targets Target Signal CRISPR-Cas Eec ff tor LOD Refs. –8 DNA methylation Fluorescence Cas12b 10 nM [6] Extracellular vesicle Fluorescence Cas12a 100 particles/mL [88] Extracellular vesicles Fluorescence Cas12a 100 particles/µL [89] ATP Fluorescence Cas12a 0.39 μM [67] Na Fluorescence Cas12a 0.21 μM [67] Aflatoxin B1 (AFB1) Biolayer interferometry (BLI) Cas12a 0.8 ng mL − 1 [90] Salmonella typhimurium Electrochemical Cas12a 20 CFU/mL [38] Bacillus Fluorescence/RNA Light-Up Cas13a 10 CFU [91] cereus PDGF-BB Fluorescence Cas12a 0.75 pM [29] Telomere Fluorescence Cas9 – [92] 17β-estradiol Raman sensing/LFA Cas12a 10 pM [93] Thrombin Electrochemical Cas12a 1.26 fM [40] ATP and Na LRET Cas12a ~ 18 nM and ~ 0.37 μM [68] Prostate-specific antigen (PSA) Colorimetric/AuNPs Cas12a 0.030 ng/ mL [69] Cardiac troponin I (cTnI) Fluorescence Cas13d 12.6 pM [87] surface of MB to elongate ssDNA strands; resulted in very such as metal carbide (MXene) nanosheets with high high collateral damage activity. Similarly, to overcome the surface area, act as efficient quenchers [63] and minimize slow release of acDNA, an assay performed using hybrid background signals. Sheng et al. designed a flexible PAM DNA for exponential signal improvement; repeated acD- domain with dsDNA probes as the acDNA achieved NAs enhanced frequency and accessibility to Cas12a/ super-quenching to quantify picograms of lipopolysac- crRNA complex and increased sensitivity [58]. Using this charide and two-digit Gram-negative bacteria. Further, approach, SARS-CoV-2 RNA was detected to be as low 2D nanosheet and Cas14a coupled to aptamer and por- as ~ 42 copies/mL. To simplify the multi-step process as phyrin metal–organic framework nanosheets as the described earlier, a wash-free homogeneous allosteric quencher was able to detect MC-LR at very low levels hairpin probe (using single, dual, and ternary) circle [64–66]. amplification was proposed. Using single-circle ampli - fication [59, 60], an AH probe mediates strand displace- Colorimetry‑based sensing ment amplification with aptamer, nicking enzyme cutting The fluorescence and EC assays are dependent on elec - site, and signal transduction. The aptamer could find the tronic devices and expensive designs. A signal readout target and unzip the AH probe, revealing two regions to that the naked eye can visualize makes appealing alterna- allow the formation of a primer junction. Employing KF tives for resource-limited point-of-care settings [46, 47]. polymerase catalysis, dsDNA was generated and could Several colorimetric assays with DNAzyme-based color- be recognized by Nt. BbvCI to be digested as an acDNA imetry, nanoparticle aggregation, and colorimetric strips fragment, further amplified collateral damage. This per - are being developed [45, 46]. mitted detection of tobramycin with high sensitivity up Integration of optical and visual detection into to picomolar range. Enzyme-free dynamic DNA network CRISPR-Cas12a using an HRP-mimicking DNAzyme catalysis was used in another study to multiply acDNA that formed the sandwich complex of PS/apt1-T-apt2/ copies [61], bypassing the complicated polymerase/enzy- acDNA and activated the cascade reaction of hemin-per- matic reaction. The inclusion of T7 RNA polymerase and oxide, tetramethyl benzidine (TMB) [67] for visualizable CRISPR-Cas13a triggered the reaction, as demonstrated color change produced sensor with 1.5 X 10 times sen- in aptamer application for bacterial detection of 1 CFU, sitivity for ATP detection [24]. Moreover, this approach a level 40 times better than RT-PCR [24, 62]. Similarly, was used in a sandwich design of PS/antibody-T-apt/ dual and ternary circle-based the CRISPR-Cas sensor acDNA to detect several targets such as CEA protein, detected various targets raging from extracellular vesicles bacteria, and norovirus [68]. Additionally, due to the trace level of ATP [24]. peroxidase-mimic activity and distance-dependent opti- The sequential mixing reduced the number of preparatory cal behavior of AuNPs, they have been found in use in steps and increased reproducibility. 2D nanomaterials, the construction of colorimetric sensing. For example, Kadam et al. Applied Biological Chemistry (2023) 66:13 Page 12 of 15 AuNPs coupled with Cas12a collateral digestion and achieved ultra-sensitive detection of ATP [68] and car- RCA amplification were used for colorimetric CRISPR- diac troponin I (cTnI) [76] For ochratoxin A (OTA) Cas sensing, where aptamer/crRNA/Cas12a ternary detection, Mao et al. developed a UCNP-MB probe complexes cleave primer sequences and padlock probes [77] making feasible for OTA bound with aptamer and modified on AuNPs. Wang et al. [69] used distance- unfolded HDA probes to release complementary DNA dependent optical properties of AuNPs and nicking and initiate trans-cleavage action. After magnetic separa- enzyme-free amplification to produce more acDNA and tion, OTA was detected with the sub-ppb level of sensi- detected aflatoxin M1 (AFM1) with ppb level of accuracy tivity in CRISPR supported assay. and sensing of serum PSA [69, 70]. Introducing RNA reporter probes like Broccoli that Lateral flow assays (LFA) or paper-strip designs based could bind DFHBI-1 T dye and switch on its fluorescence on CRISPR-Cas effectors can cleave products and incor - [78] with Cas13a by careful designing the crRNA pro- porate AuNPs for colorimetric readout signals. For vides [79, 80] the light-up RNA aptamer-based CRISPR instance, an MC-LR strip using FAM and biotin dual- sensor. It has the potential to replace expensive chemi- modified ssDNA as the intermediate reporter was devel - cal modification and extensive synthesis steps with bet - oped [65]. The target caused the cascade reaction and ter quantification potential. Cas13a-catalyzed products Cas12a trans-cleavage, resulting in FAM- and biotin- cannot interact with DFHBI-1 T dyes, resulting in a ssDNA segments. The reporter and cleaved FAM-ssDNA “turn-off ” signal. The light-up RNA sensor could detect were conjugated to anti-FAM-coated AuNPs as they bacteria and was useful for the differentiation of living vs. migrated along the strip. Additionally, the common preg- dead bacterial cells with very low CFUs. nancy strip tests (PST) targeted at the detection of human Gao et al. introduced a G-wire assisted non-cross- chorionic gonadotropin (hCG) [ 22, 71, 72], have found linking HCR reaction to create a label-free resonance different usage. Like, Tang et al. [73] developed a novel Rayleigh scattering (RRS) CRISPR-effector powered NHP probe that could hybridize with cauliflower-like aptameric sensor system that could reveal the molecular large-sized DNA assemblies (CLD). The target-induced size, shape, conformation, and interfacial features [81]. cleavage event prevented the complex CLD-NHP from When the target was present, the aptamer containing the forming, and the cleaved NHP probe migrated on PST PAM segment specifically recognized the target rather with a red T line. This clever design detected adenosine than crRNA/Cas12a system, suppressing trans-cleavage in colorimetric fashion using the naked eyes. activity and triggering the HCR reaction by automati- cally aggregating reporter probes. The hyperbranched Other sensing approaches product produced signal amplification and very high In addition to the above approaches, several CRISPR-Cas RRS intensity. This approach detected LPS with accuracy target sensing mechanisms are integrated with ssDNA and high specificity. Recently, Li et al. created a dual sig - aptamer to generate unique signal transduction modules nal detection aptamer-based CRISPR-enzymatic paper that provide precise and reliable analytical alternatives. strip for colorimetric and Raman scattering-based diag- To name a few methods include luminescence reso- nosis; for which biotin-ssDNA-digoxin as the interme- nance energy transfer (LRET), light-up RNA, resonance diate reporter was used and digoxin antibody-SERS tags Rayleigh scattering (RRS), and surface-enhanced Raman (Au@BDT@Au) were exploited for generation of read- scattering (SERS). out signals [82–84]. Such Raman sensing strips are easy Luminescence resonance energy transfer (LRET) based for batch production, long-term stability, short sample sensing can overcome background interference, offer - demand, and cost-effectiveness; however, the unavailabil - ing a strong enough capability to handle complicated ity of hand-held Raman devices may limit the diffusion of biological samples [74]. Lin et al. used ssDNA-UCNPs this innovative technology. as reporters and gold nanoparticle-modified Ti C T 3 2 x MXene-AuNP nanosheets as quenchers to create an Future directions and conclusions LRET adsorptive quenching sensor [75]. in the absence The high affinity and specific binding properties of of target, the acDNA interact and initiate Cas12a to per- aptamers along with versatile CRISPR-Cas effectors form collateral digestion of ssDNA conjugated to upcon- make it idealistic sensors for detection and quanti- verted nanoparticles (UCNPs), and get adsorbed on fication. CRISPR-Cas enzymes are poised to impact MXene-AuNPs that would retain upconverted of lumi- and advance aptamer-based detection of various nescence (UCL). While Cas12a action is blocked if the biomarkers and small toxic compounds. Although target is present, non-cleaved reporters bind to MXene- both technologies have seen exponential growth in a AuNP, resulting in a quenching effect. Deoxy-nivalenol proof-of-concept, it would be interesting to see how was detected at 0.64 ng/mL by the sensor; the method field-level applications evolve in the coming future. K adam et al. Applied Biological Chemistry (2023) 66:13 Page 13 of 15 Competing interests The sandwich-type CLASA using nanoprobes for sig - The authors declare that they have no known competing financial interests nal enhancement and preassembly for amplification- or personal relationships that could have appeared to influence the work free detection will continue to develop in the coming reported in this paper. years. Colorimetric strips, portable designs, and smart- phone-based optical sensors are desired to simplify Received: 21 January 2023 Accepted: 3 February 2023 detection in POC settings. The aptamer-based CRISPR- Cas platforms have been demonstrated for analysis in biomedical sciences, environmental monitoring, food safety, and clinical diagnosis. Although several biosen- References 1. Kozovska Z, Rajcaniova S, Munteanu P et al (2021) CRISPR: History and sors have adopted CRISPR-Cas effectors and exhibited perspectives to the future. Biomed Pharmacother 141:111917. https:// doi. their performance, critical technical issues still need to org/ 10. 1016/j. biopha. 2021. 111917 be addressed. For example, Cas effector proteins use 2. Zetsche B, Gootenberg JS, Abudayyeh OO et al (2015) Cpf1 Is a single RNA-guided endonuclease of a class 2 CRISPR-Cas system. 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Journal
Applied Biological Chemistry – Springer Journals
Published: Feb 18, 2023
Keywords: Biomarker; Pathogen; Disease diagnostics; CRISPR-Cas; Cas12a; Cas13a; Cas14a; Aptamer; Fluorescence; Colorimetric detection