CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a

Moon, Jeong; Zhang, Jiongyu; Guan, Xin; Yang, Rui; Guo, Chong; Schalper, Kurt T.; Avery, Lori; Banach, David; LaSala, Rocco; Warrier, Ranjit; Liu, Changchun

doi:10.1038/s41467-025-64205-4

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Published: 15 October 2025

CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a

Nature Communications volume 16, Article number: 9142 (2025) Cite this article

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Abstract

The CRISPR/Cas13a enzyme serves as a powerful tool for RNA detection due to its RNA-targeting capabilities. However, simple and highly sensitive detection using Cas13a faces challenges, such as the need for pre-amplification and elevated reaction temperatures. In this study, we investigate the allosteric regulation mechanism of Cas13a activation by target RNAs with various structures containing the CRISPR anti-tag sequence. We discover that the target RNA secondary structure and anti-tag sequences inhibit the trans-cleavage reaction of Cas13a. By designing and introducing a specific CRISPR anti-tag hairpin, we develop CRISPR Anti-tag Mediated Room-temperature RNA Detection (CARRD) using a single CRISPR/Cas13a enzyme. This method enables one-step cascade signal amplification for RNA detection without the need for pre-amplification. We apply the CARRD method to detect human immunodeficiency virus (HIV) and hepatitis C virus (HCV), achieving a detection sensitivity of 10 aM. Furthermore, we validate its clinical feasibility by detecting HIV clinical plasma samples, demonstrating a simple, affordable, and efficient approach for viral RNA detection. Due to its simplicity, sensitivity, and flexible reaction temperature, the CARRD method is expected to have broad applicability, paving the way for the development of field-deployable diagnostic tools.

Introduction

In recent years, the CRISPR/Cas (clustered regularly interspaced short palindromic repeats/CRISPR-associated) system has emerged as a powerful tool for nucleic acid detection due to its high specificity and programmability^1,2. Among the various CRISPR/Cas systems, Cas13a has garnered significant attention for its RNA-targeting capabilities^3,4,5. Cas13a, an RNA-guided ribonuclease, forms a ribonucleoprotein complex (RNP) with CRISPR RNA (crRNA)^6,7. Upon binding to complementary target RNA, Cas13a activates its HEPN (higher eukaryotes and prokaryotes nucleotide-binding domain) motifs, leading to indiscriminate cleavage of surrounding single-stranded RNAs (ssRNAs)⁸. This collateral cleavage activity, coupled with a fluorescent-labeled RNA reporter, enables sensitive and specific molecular diagnosis^6,9.

Cas13a has been reported to detect RNA with attomolar sensitivity when used in conjunction with nucleic acid preamplification strategies, including reverse-transcription recombinase polymerase amplification (RT-RPA) and reverse-transcription loop-mediated isothermal amplification (RT-LAMP)^10,11. However, incorporating preamplification steps into CRISPR-based detection requires multiple manual interventions and can increase detection costs¹². To address these challenges, researchers are exploring amplification-free CRISPR-based molecular diagnostic technologies. These approaches include digital microfluidic technology and the application of multiple guide RNAs^13,14,15,16. Another strategy involves using Cas tandem systems (e.g., Cas13–Cas12, Cas13–Cas14, and Cas13–Csm6), in which one Cas protein is responsible for recognizing the target, while a second Cas protein amplifies the signal through a cascade mechanism^17,18,19. Compared to single Cas13a reaction systems, which rely on a single target to activate a single Cas13a RNP, Cas tandem systems can achieve higher sensitivity. However, using multiple Cas enzymes introduces challenges related to optimization, scalability, and buffer compatibility, as well as increased costs and processing time.

Recent functional studies of Cas13a have revealed critical features governing its target recognition and specificity. The crRNA spacer, which guides target RNA binding, plays a pivotal role in initiating the allosteric response²⁰. Within the spacer, the seed region (nucleotides 9–14) requires perfect base pairing for stable target binding, while the adjacent switch region (nucleotides 5–8) induces activation of the catalytic cleft upon target RNA binding²¹. Moreover, extending complementarity between the 3’-flank of the crRNA spacer (referred to as the tag) and the target RNA (anti-tag) can inhibit type VI-A Cas13’s nuclease activity^8,22. Studies have shown that an 8-nt complementary anti-tag sequence inhibits both cis- and trans-RNase activities of Cas13a^8,22. This extended pairing enhances specificity by distinguishing self from non-self targets, a feature critical for Cas13-based diagnostics²⁰. Insights into seed, switch, and tag: anti-tag pairing mechanisms provide a deeper understanding of Cas13a’s regulatory architecture, offering valuable perspectives for precise RNA targeting and control.

Here, we investigate the allosteric effects of target RNA secondary structure and the influence of anti-tag sequences on RNA detection using Leptotrichia wadei (Lwa)Cas13a. We discover that the presence of anti-tag sequences and target RNA secondary structures inhibits the trans-cleavage activity of Cas13a. Building on this finding, we design a specific CRISPR anti-tag hairpin containing secondary structure and anti-tag sequences. Using this design, we develop a simple and sensitive CRISPR anti-tag mediated room temperature RNA detection (CARRD) assay with a single LwaCas13a/crRNA complex. In the presence of target RNA, the CRISPR anti-tag hairpin mediates CRISPR cascade signal amplification, enabling highly sensitive, one-pot, amplification-free RNA detection at room temperature. Using this assay, we successfully detect target viral RNAs, including HIV and HCV. Furthermore, we clinically validate the CARRD assay by detecting HIV viral RNA extracted from clinical plasma samples, demonstrating its potential for simple and accurate viral RNA detection in resource-limited settings.

Results

Working principle of the CARRD assay

In a conventional CRISPR/Cas13a activation reaction, the Cas13a endonuclease and crRNA form a functional RNP complex that binds RNA targets with sequences complementary to the crRNA spacer region. Upon binding to a target RNA, the Cas13a RNP undergoes a conformational change that activates its collateral cleavage activity²³. The activated Cas13a RNP indiscriminately cleaves ssRNA fluorescent reporters, amplifying the detection signal and enabling RNA detection (Fig. 1a, i). However, in the presence of target RNA containing an anti-tag sequence, this conformational change is inhibited, preventing the activation of the trans-cleavage reaction of CRISPR/Cas13a (Fig. 1a, ii).

**Fig. 1: Schematic of CARRD: a CRISPR anti-tag mediated room temperature RNA detection assay.**

Inspired by the inhibitory role of the anti-tag sequence in Cas13a activation, we designed a specific hairpin-structured target RNA, termed the “CRISPR anti-tag hairpin,” and developed a one-pot, one-enzyme, amplification-free CARRD assay for RNA detection at room temperature. The CRISPR anti-tag hairpin consists of an anti-tag sequence and a double-stranded chimeric region of DNA and target RNA with asymmetric lengths, where the target RNA region is fully complementary to the crRNA (Fig. 1b). Due to its hairpin structure and anti-tag sequence, the CRISPR anti-tag hairpin cannot initially activate the trans-cleavage reaction of Cas13a in the absence of target RNA, even if it contains the target RNA sequence. In contrast, in the presence of target RNA, Cas13a is activated, initiating a trans-cleavage reaction that results in cleavage of the anti-tag sequence (loop region) of the CRISPR anti-tag hairpin. This cleavage exposes the target RNA sequence within the hairpin, allowing it to be recognized by the Cas13a/crRNA complex and further activating Cas13a’s trans-cleavage activity. This results in a cascade signal amplification, enabling sensitive RNA detection (Fig. 1c). Thus, by leveraging the CRISPR anti-tag hairpin, we established a one-pot, amplification-free, sensitive, and specific CRISPR cascade signal amplification method using a single Cas13a enzyme.

Development and optimization of the CARRD assay

Although CRISPR/Cas13a reactions are typically performed at 37 °C in most Cas13a-based diagnostic studies, we conducted in vitro trans-cleavage assays at various reaction temperatures (25, 30, 33, and 37 °C). Surprisingly, we found that the CRISPR/Cas13a reaction targeting ssRNA (Fig. 2a, target RNA) performed more effectively at 25 °C than at 37°C (Fig. 2b, c). This result aligns with a recent study showing that the CRISPR/Cas13a system operates efficiently across a broad temperature range (7–37 °C) while maintaining rapid trans-cleavage kinetics once activated²⁴. This discrepancy from earlier reports suggesting 37 °C as the optimal temperature may result from differences in RNA secondary structure, which influence the efficiency of ternary complex formation. Efficient formation of the Cas13a ternary complex at lower temperatures has been identified as a key factor enabling robust cleavage activity even at room temperature²⁴. This finding significantly simplifies the equipment requirements (e.g., eliminating the need for a heater) and is ideal for simple, minimally instrumented point-of-care diagnostic applications. Thus, we conducted subsequent Cas13a experiments at 25 °C.

**Fig. 2: Cas13a activity across temperatures and anti-tag substrate designs.**

Recent in vivo studies have demonstrated that extended complementarity between the target RNA and the 3’-flank of the crRNA (tag:anti-tag pairing) can significantly reduce RNA cleavage by the type VI-A Cas13a system^8,22,25. Here, we investigated the cleavage activity of LwaCas13a, focusing on various RNA structures and the effects of anti-tag sequences (Fig. 2a). First, we compared the trans-cleavage activity of LwaCas13a on target RNA with and without an anti-tag sequence (5’–rGrUrUrUrUrArGrU–3’). The results showed reduced trans-cleavage activity on the target RNA containing the anti-tag (anti-tag target in Fig. 2a) compared to the target RNA without the anti-tag sequence, consistent with previous findings using LseCas13a, LbuCas13a, and LshCas13a^8,22 (Fig. 2d). Moreover, when the target RNA was paired with a complementary DNA sequence to form a double-stranded structure (dsTarget in Fig. 2a), the trans-cleavage activity further decreased compared to the single-stranded RNA target (Target RNA in Fig. 2a, d). This signal reduction effect was even more pronounced when the RNA containing the anti-tag sequence formed a double-stranded structure (dsAnti-tag Target in Fig. 2a, d).

Next, we built a hairpin structure by incorporating the anti-tag sequence into the loop region to compare its trans-cleavage activity. In this configuration, the anti-tag sequence was positioned differently at the 3’ and 5’ ends of the target RNA. We designed two constructs: 5’-DNA tag HP (5’-DNA-8nt anti-tag segment-target RNA) and 5’-RNA tag HP (5’-target RNA-8nt anti-tag segment-DNA) (Fig. 2a). The 5’-DNA tag HP showed a similar result to the double-stranded target RNA:DNA (dsTarget), indicating that the loop structure does not significantly affect its performance (Fig. 2d). In contrast, the 5’-RNA tag HP showed a markedly reduced signal, even lower than that of the double-stranded anti-tag target RNA:DNA (dsAnti-tag Target). This result suggests that the loop structure at the 3’ end of the RNA has a pronounced inhibitory effect on Cas13a’s trans-cleavage activity (Fig. 2d). These findings are consistent with a previous study reporting that the 3’ region of the protospacer exerts an allosteric inhibitory effect²⁶.

To further evaluate the anti-tag sequence’s effect, we designed a hairpin structure where the loop region contained an 8nt rU sequence (used as a cleavage substrate for Cas13a) instead of the anti-tag sequence (named 5’-RNA rU8 HP, Fig. 2a). The results showed that trans-cleavage activity noticeably decreased in the hairpin structure containing the anti-tag sequence compared to the hairpin structure containing the 8nt rU sequence (Fig. 2d). These findings further confirm that the trans-cleavage activity of Cas13a can be effectively inhibited by the cooperative effects of the hairpin structure and the anti-tag sequence. Based on these results, we obtained a comprehensive understanding of the factors affecting Cas13a activation, such as reaction temperature, target RNA secondary structure, and anti-tag sequences. Leveraging these insights, we used a specific CRISPR anti-tag hairpin as the mediator of the cascade signal amplification reaction and developed a simple, sensitive, amplification-free CARRD assay that operates at room temperature.

Cascade signal amplification mechanism induced by the CRISPR anti-tag hairpin

We hypothesized that the RNA sequence within the hairpin containing the anti-tag would not activate the Cas13a reaction (e.g., 5’-RNA tag HP, Fig. 2d). However, after the anti-tag is cleaved, the RNA sequence would activate Cas13a (e.g., dsTarget or dsAnti-tag Target in Fig. 2d), leading to further signal amplification and serving as a cascade signal amplifier. Since the anti-tag sequence contains rU residues, we anticipated these residues would be susceptible to cleavage by the trans-cleavage activity of Cas13a activated by the target RNA²⁷.

To test this hypothesis, we designed a sequential reaction to verify whether the anti-tag cleavage occurs and whether the trans-cleavage reaction of Cas13a can subsequently be activated by the RNA sequence within the CRISPR anti-tag hairpin. In step 1, we conducted the Cas13a/M crRNA reaction in the presence and absence of target M. Instead of using a fluorescent-quencher (FQ) reporter for fluorescence measurement, we added CRISPR anti-tag hairpins containing the anti-tag and HIV RNA:DNA sequences and incubated the reaction at room temperature (25 °C) for 20 min. In step 2, we added the reactant from step 1 as a target to a solution containing Cas13a/HIV crRNA and FQ reporters, and subsequently measured the fluorescence signal (Fig. 3a).

**Fig. 3: Two-step CRISPR/Cas13a activation using anti-tag hairpins with variable DNA blockers.**

First, we compared the trans-cleavage reactions for three target HIV RNA lengths (20, 22, and 24 nt) to investigate the effect of RNA length on incorporation into the hairpin (HP). At a target RNA concentration of 10 pM, the 22- and 24-nt target RNAs exhibited similar signals, whereas the 20-nt target RNA produced a weak fluorescence signal (Supplementary Fig. 1). Based on this result, we selected a 22-nt HIV RNA sequence and designed CRISPR anti-tag HPs with various complementary DNA blocker lengths (14-, 16-, 18-, and 20-bp) to compare the sequential reactions using these four HP types (14D-, 16D-, 18D-, and 20D-HP).

In the sequential reactions using these four HP types, no fluorescence signal was observed in the absence of HPs, regardless of the presence of target M (Fig. 3c and Supplementary Fig. 2). This indicates that the reaction between Cas13a/M crRNA and target M in step 1 does not affect fluorescence signal generation in step 2. Similarly, when the DNA blocker complementary to the HIV sequence was relatively long (20 bp; 20D HP), the fluorescence signal increase was negligible regardless of the presence of target M (Fig. 3c and Supplementary Fig. 2). This finding suggests that when the DNA blocker is long, the target RNA within the hairpin is not effectively recognized by Cas13a/HIV crRNA, regardless of whether the loop region (anti-tag sequence) is cleaved.

However, when hairpins containing 14, 16, or 18 bp DNA blockers were used, a notable increase in fluorescence signal was detected in the presence of target M. Unlike the 20D HP, when the DNA blocker complementary to the target RNA was shorter, the HIV target RNA within HP could activate Cas13a/HIV crRNA in step 2 after the loop (anti-tag sequence) was cleaved by the activated Cas13a/M crRNA in step 1. In the absence of target M, a weak fluorescence signal was generated, which indicates the background signals resulting from the interactions between HIV target RNA within the HP and Cas13a/HIV crRNA; However, these results demonstrated that signals undetectable in the absence of the hairpin were amplified to observable levels when the hairpin is used (Fig. 3c and Supplementary Fig. 2). In other words, initially undetectable signals from low concentrations of target M were amplified to observable levels using the HP. The optimal DNA blocker length was 14 bp, which effectively blocked the target RNA sequence within the HP before loop cleavage. After loop cleavage, however, Cas13a can effectively recognize the target RNA within the HP and further induce trans-cleavage (Fig. 3c).

To further confirm the hairpin cleavage, we conducted a CRISPR sequential reaction and analyzed the reaction products using PAGE gel analysis. In the presence of target M, two bands corresponding to the 14D HP (hairpin containing a 14-bp DNA blocker) were observed (Fig. 3d), indicating that the HP was cleaved by the trans-cleavage reaction of Cas13a/M crRNA (Fig. 3d, step 1, lane 4). Additionally, FQ reporter cleavage was predominantly observed in the step 1 reactant containing target M and 14D HP; however, the cleavage reaction diminished in the absence of target M (Fig. 3d, step 2, lanes 1 and 2). This result confirms that the HIV sequence in 14D HP is detected by Cas13a/HIV crRNA in step 2 only after 14D HP cleavage. When 14D HP was absent during step 1, FQ reporter cleavage by Cas13a/M crRNA activated by target M was barely detectable due to the tenfold dilution as the reaction proceeded from step 1 to step 2 (Fig. 3d, step 2, lanes 3 and 4). Based on the results of these CRISPR sequential reactions, we concluded that following the cleavage of the anti-tag sequence in the loop region of the hairpin mediator, the target sequence in the stem region is recognized by the Cas13a/crRNA complex. This recognition triggers a trans-cleavage reaction and enables CRISPR cascade signal amplification.

Sensitivity and specificity of the CARRD assay

Next, we investigated whether the CRISPR anti-tag HP, which includes the target RNA sequence, could improve target RNA detection efficiency under a one-step reaction condition using a single Cas13a/crRNA complex. To test this, we prepared solutions containing Cas13a/HIV crRNA, the CRISPR anti-tag hairpin, and the FQ reporter, and measured the fluorescence signal in the presence or absence of the HIV target RNA. We used the same CRISPR anti-tag HPs (14-, 16-, 18-, and 20D HP) containing the HIV target RNA sequence as those used in Fig. 3 and compared their results. Additionally, we evaluated the performance of the CARRD assay against the conventional Cas13a/crRNA reaction without the CRISPR anti-tag HP.

The results showed that fluorescence signal intensity was significantly higher when CRISPR anti-tag HPs were applied compared to reactions without the HP (Supplementary Fig. 3a). Among the HPs tested, 14D HP exhibited the strongest fluorescence intensity enhancement. Further optimization of the 14D HP concentration revealed that 5 nM of 14D HP produced the greatest fluorescence difference between the presence and absence of the target RNA (Supplementary Fig. 3b). Under optimized conditions, we monitored real-time fluorescence signals for HIV RNA detection across concentrations ranging from 10 aM to 1 pM. As shown in Fig. 4a and Supplementary Fig. 4, the fluorescence signal intensity was greatly enhanced with the addition of HIV 14D HP (CARRD assay), significantly improving detection sensitivity compared to the conventional Cas13a/HIV crRNA reaction without 14D HP. The limit of detection (LOD), calculated as the mean blank + 3σ of the blank, was determined to be 10 aM for the CARRD assay—10,000 times more sensitive than the conventional Cas13a/crRNA reaction (LOD: 100 fM). Additionally, time-dependent fluorescence signal changes for detecting 100 fM of HIV RNA demonstrated that the fluorescence intensity at 1 h with the CARRD assay was 22.9-fold higher than that of the conventional reaction (Fig. 4c).

**Fig. 4: Sensitive and specific detection of HIV and HCV RNA using the CARRD assay.**

To evaluate the assay’s versatility, we applied the CARRD assay to detect hepatitis C virus (HCV) RNA (Fig. 4b). For HCV detection, the CRISPR anti-tag HP was designed with a double-stranded chimeric region composed of the HCV target RNA sequence, complementary DNA, and an anti-tag sequence in the loop region. We optimized the complementary DNA lengths to 14, 15, and 16 nt and compared the results. Among the HPs tested, the 14D HP showed the strongest fluorescence intensity (Supplementary Fig. 5a). Further optimization confirmed that the detection efficiency was optimal at a 14D HP concentration of 1 nM (Supplementary Fig. 5b). Using these optimized conditions, we measured real-time fluorescence signals for HCV RNA detection across a concentration range of 10 aM to 1 pM (Fig. 5b and Supplementary Fig. 6). Similar to the HIV detection results, the CARRD assay with the HCV 14D HP achieved a detection limit of 10 aM for HCV RNA.

**Fig. 5: Comparison of crRNA variants in CARRD and conventional assays.**

To evaluate the specificity of the CARRD assay, we first tested HIV detection using Cas13a/HIV crRNA and HIV 14D CRISPR anti-tag HP. A strong fluorescence increase was observed with the HIV RNA target, while the HCV RNA did not enhance the fluorescence signal (Fig. 4d, black column). Similarly, for HCV detection using Cas13a/HCV crRNA and HCV 14D CRISPR anti-tag HP, a strong fluorescence signal was observed with the HCV target RNA, while the HIV RNA produced no significant fluorescence signal (Fig. 4d, orange column). These results demonstrate the high specificity of the CARRD assay.

Evaluation of crRNA efficiency in HIV detection using the CARRD assay

It is well-known that the binding site and sequence of crRNA can significantly affect detection efficiency^13,28. Therefore, to optimize the CARRD assay for HIV detection, we designed and tested different crRNAs. We designed three crRNAs along the HIV-1 genome (Fig. 5a), and we compared fluorescence signals using the conventional Cas13a/crRNA reaction and the CARRD assay. Figure 5b, c shows the differences in fluorescence signals in the presence and absence of target HIV RNA for the conventional Cas13a/crRNA reaction (without CRISPR anti-tag HP) and the CARRD assay (with CRISPR anti-tag HP) using the three crRNAs (crRNA #1, #2, and #3).

The increase in fluorescence intensity varied depending on the type of crRNA, despite using the same target RNA concentration. When the target RNA was present, crRNA #1 exhibited the highest trans-cleavage activity among the three crRNAs, regardless of the presence or absence of the HP. In contrast, with the conventional Cas13a/crRNA reaction using crRNA #3, no significant fluorescence signal increase was observed, even in the presence of the target, likely due to low or no trans-cleavage activity^13,29. Interestingly, when the CRISPR anti-tag hairpin was applied, clear fluorescence differences were observed between the target and control. An additional increase in fluorescence signal was observed for all three crRNAs in the CARRD assay compared to the conventional Cas13a reaction (Fig. 5b, c). The signal intensity followed the order crRNA #1 > #2 > #3, consistent with the results of the conventional Cas13a/crRNA reaction. Based on these findings, we selected crRNA #1 for subsequent testing of HIV clinical plasma samples.

Clinical validation with HIV clinical plasma samples

We applied the CARRD assay to analyze HIV clinical plasma samples and validate its clinical feasibility. We obtained 30 deidentified clinical plasma specimens and extracted RNA from 140 µL of plasma using commercial nucleic acid extraction kits (Fig. 6a). Before testing these RNA samples with the CARRD assay, we performed real-time reverse transcription-quantitative polymerase chain reaction (RT-qPCR) to confirm the presence of nine HIV-positive samples and 21 HIV-negative samples (Fig. 6b, c). The fluorescence intensity results analyzed using the CARRD assay are shown in Fig. 6d. The fluorescence threshold was determined using background-subtracted fluorescence from negative control samples and set to the median of the negative samples (mean + 3σ), indicated by the blue line (Fig. 6d)⁴. In RT-qPCR, a Cq value of 40 or higher was considered negative (Supplementary Table 1). Comparing the results of RT-qPCR and the CARRD assay revealed that all results matched except for samples 24 and 25 (Fig. 6b, c). Based on the confusion matrix in Fig. 6b, the assay demonstrated a sensitivity of 77.8%, a specificity of 100%, and an overall accuracy of 93.3%. The positive predictive value (PPV) and negative predictive value (NPV) were calculated to be 100 and 91.3%, respectively. These results confirm the assay’s high precision and specificity, demonstrating its robust ability to differentiate true positives from true negatives in clinical plasma samples. The scatter plot in Fig. 6e compares the fluorescence intensity of samples from two groups: positive samples (n = 9) and negative samples (n = 21). Gray dots represent negative samples, showing generally lower fluorescence intensity, while red dots represent positive samples, showing higher fluorescence intensity. The black dot with error bars highlights a significant difference in the mean fluorescence values between the two groups (Fig. 6e). Additionally, we generated a receiver operating characteristic (ROC) curve to evaluate the diagnostic performance of the CARRD assay, using RT-qPCR as the reference standard (Fig. 6f). The area under the curve (AUC) was 0.9709, demonstrating an excellent diagnostic performance.

**Fig. 6: Detection of HIV clinical samples by the CARRD assay.**

Discussion

In this study, we investigated the effects of chimeric DNA/RNA and CRISPR anti-tag sequences on the allosteric regulation of Cas13a for RNA detection. Our results show that the presence of anti-tag sequences and secondary structures in target RNA significantly inhibits the trans-cleavage activity of Cas13a. Building on this discovery, we proposed a cascade amplification method using a CRISPR anti-tag hairpin mediator and developed a simple, sensitive, and affordable CARRD assay for RNA detection at room temperature with a LOD of 10 aM (10,000 times more sensitive than the conventional Cas13a/crRNA detection). This method employs a single CRISPR/Cas13a enzyme, eliminating the need for preamplification and heating equipment. By leveraging the inherent inhibitory effect of the anti-tag sequence, the CRISPR anti-tag hairpin initially prevents Cas13a’s cleavage activity, minimizing background noise and false-positive signals. In the presence of target RNA, the Cas13a enzyme is specifically activated, initiating the non-specific cleavage of ssRNA sequences, releasing the target RNA sequence from the CRISPR anti-tag hairpin. The released target RNA sequence further activates Cas13a’s trans-cleavage reactions, enabling cascade signal amplification. Unlike other approaches that typically require multiple types of enzymes for cascade amplification^{3,9,17,18,30,31,32,33,34}, our study demonstrates that CRISPR cascade amplification can be achieved using a single Cas13a/crRNA complex, achieving an attomolar sensitivity for RNA detection. This significantly simplifies the assay and reduces costs (Supplementary Table 2).

The development of the CARRD assay addresses several key challenges in previous CRISPR/Cas13a-based diagnostic methods. By eliminating the need for pre-nucleic acid amplification and reverse transcription, the CARRD assay streamlines the detection process and lowers costs, making it more suitable for field testing and resource-limited settings. Operating effectively at room temperature, the assay reduces the complexity and time associated with multiple handling steps while eliminating the need for high-temperature incubators. This demonstrates the potential for simple, sensitive, and minimally instrumented detection of viral RNAs, such as HIV and HCV. Notably, the CRISPR anti-tag hairpin structure retains its signal amplification effect across diverse RNA targets, underscoring the versatility of the design. Consequently, the CARRD assay provides a promising solution for improving current molecular diagnostics and expanding the accessibility of RNA virus detection in diverse clinical and environmental contexts. Future research will focus on adapting the CARRD assay to alternative readout formats, such as paper-based detection or integration with portable, low-cost fluorescence readers to enhance its suitability for point-of-care use in low-resource settings.

Despite its advantages, the CARRD assay has room for improvement. The design of the CRISPR anti-tag hairpin significantly influences background noise levels, which may compromise the assay’s accuracy. Variations in hairpin design can lead to inconsistent results, underscoring the need for further optimization to minimize background signals. Addressing these challenges will be crucial for improving the assay’s specificity and reliability in clinical applications. Future work could focus on optimizing crRNA design, refining hairpin structures, or enhancing single-nucleotide discrimination. For instance, employing advanced computational models to predict and design more effective crRNA and hairpin structures could prove valuable. Overall, this study advances our understanding of CRISPR/Cas13a biochemistry and lays the groundwork for more accessible and efficient RNA detection methods, with significant implications for next-generation molecular diagnostics.

Method

Ethical statement

Deidentified clinical plasma samples were collected and provided by the Clinical Microbiology Laboratory in compliance with ethical regulations and under the approval of the Institutional Review Board of the University of Connecticut Health Center (protocol no.: 21–014–2). In accordance with IRB regulations, the study was approved with a waiver of informed consent, as only deidentified clinical plasma samples without any patient identifiers were used.

Materials

All oligonucleotide sequences used in this study were obtained from Integrated DNA Technologies (IDT, USA) and are listed in Supplementary Table 3 and Supplementary Data 1. RNase-free water (Cat# 11-05-01-14) was purchased from IDT. LwCas13a, active (Cat# CS13A-E321H), was purchased from Signalchem Diagnostics (Canada). The 10× rCutSmart buffer (Cat# B6004S) and 10× NEBuffer r2.1 were purchased from New England Biolabs (NEB, USA). The 10× TBE (tris borate EDTA) buffer (Cat# 1610770), 40% acrylamide/bis solution (Cat# 1610146), ammonium persulfate (Cat# 1610700), TEMED (Cat# 1610800), and Reliance One-Step Multiplex RT-qPCR Supermix (Cat# 12010176) were purchased from Bio-Rad (USA). SYBR gold nucleic acid gel stain (Cat# S11494), Orange DNA Loading Dye (6X) (Cat# R0631), and O’RangeRuler 10 bp DNA Ladder (Cat# SM1313) were purchased from Thermo Fisher Scientific (USA). The QIAamp Viral RNA Mini kit (Cat# 52904) was purchased from Qiagen (USA).

Conventional CRISPR/Cas13 assay

The 200 nM Cas13a/crRNA complex was constructed by mixing 2.6 μL LwCas13a (7.68 μM), 2 μL crRNA (10 μM), and 95.4 μL buffer (20 mM HEPES, 50 mM KCl, 5 mM MgCl₂, and 5% glycerol). A reaction solution containing 1 μL Cas13a/crRNA (200 nM), 1 μL fluorescence-quencher probe (10 μM), 2 μL 10× CutSmart buffer, and 2 μL target nucleic acid was prepared, and RNase-free water was added to a final volume of 20 μL. Double-stranded targets and HPs used in Fig. 2 (1 μM in 1× NEBuffer 2.1) were heated at 90 °C for 5 min, gradually cooled to 25 °C, and incubated at 4 °C before use. Fluorescence signals were measured in real time using a CFX Opus 96 real-time PCR system (Bio-Rad, USA) or an Infinite 200 PRO microplate reader (TECAN, Switzerland).

Sequential CRISPR reaction using different types of CRISPR anti-tag HP

First, the reaction solution was prepared by mixing 1 μL Cas13a/M crRNA (200 nM), 2 μL various types of HP (100 nM), and 2 μL different combinations of target M (100 pM) in 1× rCutSmart buffer and nuclease-free water to a final volume of 20 μL. The reactant solution was incubated at 25 °C for 20 min. After incubation, 2 μL of the reactant was mixed with 1 μL Cas13a/HIV crRNA (200 nM), 1 μL fluorescence-quencher reporter (10 μM), and 1× rCutSmart buffer with nuclease-free water to a final volume of 20 μL. Fluorescence signals were measured in real-time at 25 °C using a CFX Opus 96 real-time PCR system (Bio-Rad, USA).

Gel electrophoresis

Step 1: Cas13a/M crRNA and CRISPR anti-tag hairpin reaction

For step 1, a total of 20 μL of reaction solution was prepared by mixing 1 μL Cas13a/M crRNA (200 nM), 2 μL CRISPR anti-tag HP (1 μM), and 2 μL of different combinations of target M (1 nM) in 1× rCutSmart buffer and nuclease-free water. After incubating the reaction solution at 25 °C for 20 min, each reaction solution was resolved on a 10% polyacrylamide gel using 1× TBE as the running buffer at a constant voltage of 100 V for 100 min. Gels were scanned using a ChemiDoc Imaging System (Bio-Rad).

Step 2: Reactant from step 1 and FQ reporter reaction

For step 2, a total of 20 μL of reaction solution was prepared by mixing 1 μL Cas13a/HIV crRNA (200 nM), 1 μL FQ reporter (10 μM), and 2 μL of the step 1 reactant in 1× rCutSmart buffer with nuclease-free water. The solution was incubated at 25 °C for 40 min. After incubation, the reaction was resolved on a 10% polyacrylamide gel using 1× TBE as the running buffer at a constant voltage of 100 V for 100 min. Gels were scanned using a ChemiDoc Imaging System (Bio-Rad).

CARRD assay

The CRISPR anti-tag hairpin (1 μM in 1× NEBuffer 2.1) was heated at 90 °C for 5 min, gradually cooled to 25 °C, and incubated at 4 °C before use. The CARRD assay was conducted in a 20 μL reaction volume containing 1 μL Cas13a/crRNA (200 nM), 1.5 μL fluorescence-quencher probe (10 μM), 2 μL CRISPR anti-tag HP, 2 μL 10× CutSmart buffer, 2 μL target nucleic acid at varying concentrations, and 11.5 μL RNase-free water. Fluorescence signals were measured in real-time at 25 °C using an Infinite 200 PRO microplate reader (TECAN, Switzerland).

Viral RNA extraction and detection using the CARRD assay and RT-qPCR

Viral RNA was extracted from 140 μL of input material using the QIAamp Viral RNA Mini Kit (Qiagen) with carrier RNA, following the manufacturer’s instructions. Samples were eluted in 60 μL of elution buffer and stored at -80 °C until use. The extracted viral RNA was tested using both the CARRD assay and RT-qPCR. One-step RT-qPCR was performed using the Reliance One-Step Multiplex RT-qPCR Supermix (Bio-Rad), following the manufacturer’s instructions. In brief, a reverse-transcription reaction was conducted at 50 °C for 10 min, followed by initial denaturation at 95 °C for 10 min. This was followed by 40 cycles of denaturation at 95 °C for 10 s and extension at 60 °C for 30 s. Fluorescence signals were measured at the end of each cycle and analyzed using CFX Maestro Software. Information on primer and probe sets used in this study is provided in Supplementary Table 3 and Supplementary Data 1.

The CARRD assay was conducted in a 20 μL reaction volume containing 1 μL Cas13a/crRNA (200 nM), 1.5 μL fluorescence-quencher probe (10 μM), 2 μL CRISPR anti-tag HP, 2 μL of 10× CutSmart buffer, 4 μL of extracted viral RNA sample, and 9.5 μL of RNase-free water. Each fluorescence signal was measured in real-time at 25 °C using an Infinite 200 PRO microplate reader (TECAN, Switzerland).

Statistics and reproducibility

No statistical method was used to predetermine sample size. No data were excluded from the analysis. The experiments were not randomized. The Investigators were not blinded to allocation during experiments and outcome assessment.

Reporting summary

Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.

Data availability

The main data supporting the findings of this study are available within the article and Supplementary Files. The source data underlying Figs. 2–6 and Supplementary Figs. 1–6 are provided as a Source Data file. Source data are provided with this paper.

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Acknowledgements

This work was partially supported by NIH grant R33AI154642 (to C.L.) and R01AI194917 (to C.L.).

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Authors and Affiliations

Department of Biomedical Engineering, University of Connecticut Health Center, Farmington, CT, 06032, USA
Jeong Moon, Jiongyu Zhang, Xin Guan, Rui Yang, Chong Guo, Kurt T. Schalper & Changchun Liu
Department of Biopharmaceutical Science, Soonchunhyang University, Asan, 31538, Republic of Korea
Jeong Moon
Department of Biomedical Engineering, University of Connecticut, Storrs, CT, 06269, USA
Xin Guan, Rui Yang, Chong Guo & Kurt T. Schalper
Department of Pathology and Laboratory Medicine, University of Connecticut Health Center, Farmington, CT, 06030, USA
Lori Avery & Rocco LaSala
Department of Medicine, Division of Infectious Diseases, University of Connecticut Health Center, Farmington, CT, 06030, USA
David Banach
Centre for Infectious Disease Research in Zambia, Lusaka, 10101, Zambia
Ranjit Warrier

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Contributions

J.M. designed the study, performed the experiment, and drafted the manuscript. J.Z. and X.G. performed the experiment and assisted in editing the manuscript. R.Y., C.G., and K.T.S. assisted in validating and editing the manuscript. L.A. assisted in HIV plasma sample preparation. D.B., R.L., and R.W. contributed to data interpretation. C.L. supervised the concept of the study and reviewed & edited the manuscript.

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Correspondence to Changchun Liu.

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Supplementary Data 1

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Source Data

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Moon, J., Zhang, J., Guan, X. et al. CRISPR anti-tag-mediated room-temperature RNA detection using CRISPR/Cas13a. Nat Commun 16, 9142 (2025). https://doi.org/10.1038/s41467-025-64205-4

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Received: 15 January 2025
Accepted: 08 September 2025
Published: 15 October 2025
DOI: https://doi.org/10.1038/s41467-025-64205-4