Activation mechanism and integrated one-pot assay of chiral-like cRNA-enhanced CRISPR/Cas12a systems

Domain: neuroscience
Published: 2026-04-08
Editorial outcome: major_revision

Abstract
The 5′-repeat fragments released during pre-crRNA maturation are critical yet understudied components of the CRISPR/Cas12a system. Here, we demonstrate that engineered 5′-repeat fragments can potently activate Cas12a cleavage, with efficiency strongly dependent on the length of the 3′-spacer. Strikingly, complete truncation of the 3′-spacer generates a "chiral-like crRNA" conformation that induces a delayed-switch mode of Cas12a activation, fundamentally distinct from conventional mature crRNA. Leveraging this characteristic, we develop the delayed cleavage feature-mediated one-pot sensing strategy that resolves the long-standing challenge of incompatibility between Cas12a-based cleavage reaction and nucleic acid amplification, achieving a 1,000-fold improvement in sensitivity over that of the conventional mature crRNA-mediated one-pot method. Furthermore, we integrate a cleavage-based one-pot assay with a portable temperature-controlled fluorescence imaging device to create an on-site diagnostic platform for high-throughput screening. Our study further advances the understanding of the crRNA-guided mechanism and facilitates the expansion of its applications in genome editing and molecular diagnostics.

Introduction
The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas12a system mediates precise nucleic acid cleavage, evolving from its native role as a prokaryotic phage defense system to become a versatile tool for genome editing and molecular diagnostics1-3. As a type V CRISPR effector, Cas12a is guided by a single CRISPR RNA (crRNA) and exhibits dual RNase and DNase activities. Its precursor crRNA (pre-crRNA) consists of repeat and spacer sequences4. During its activity, Cas12a first utilizes its RNase activity to cleave the repeat sequences of pre-crRNA, releasing 14–17 nt fragments, whereas the remaining fragments fold into a stem-loop structure5. The mature crRNA produced from this cleavage subsequently guides the DNase activity of Cas12a for targeted DNA cleavage6,7. The crRNA is a critical regulator of the Cas12a system, with its structure and sequence significantly influencing the system's recognition efficiency and cleavage performance8. Owing to its programmability, crRNA can be engineered to enhance the functionality of the Cas12a system, a topic of considerable and growing research interest. Modifications include 5′ or 3′ terminal extensions with single-stranded DNA (ssDNA), single-stranded RNA, or thiophosphate ssDNA to enhance trans-cleavage activity9; 3′ terminal 2′-O-methyl modifications to improve target affinity10; and ortho-nitrobenzyl phosphate esters caging to achieve light-controlled activation11. These strategies have broadened the Cas12a system's applications in non-amplified nucleic acid detection, single-nucleotide polymorphism discrimination, and even non-nucleic acid target sensing12.

Current research has primarily focused on investigating the guiding mechanism and engineering modifications of mature crRNA13-15. A key unanswered question in CRISPR/Cas12a biology is whether the 5′-repeat fragments released during pre-crRNA maturation have functional significance. Evolutionary economics principles in biological systems suggest that products of organismal evolution usually acquire specific functions16. We therefore hypothesize that the released 5′-repeat fragments may uniquely activate latent functions of Cas12a, a possibility not yet fully explored. It is important and interesting to systematically examine the role of these fragments in Cas12a activation and explore their molecular mechanisms.

Here, we engineer the native pre-crRNA by extending the 5′-repeat fragment from 15 nt to 22 nt to meet the sequence length requirement for Cas12a activation. Introducing DNA target complementary only to the released 5′-repeat fragment reveals that these engineered fragments effectively activate Cas12a trans-cleavage activity (Fig. 1a I). Notably, the cleavage activity is modulated by the length of the 3′-spacer in the engineered pre-crRNA (Fig. 1a II). Strikingly, when the 3′-spacer is shortened to 0 nt, the engineered pre-crRNA exhibits guiding activity comparable to that of conventional mature crRNA, with both adopting a structurally chiral-like configuration. Based on this observation, we define the concept of “chiral-like crRNA” (C-crRNA) and systematically investigate its role in guiding Cas12a activity. Further studies demonstrate that chiral-like crRNA can serve as a “delay switch” for Cas12a activity activation. The chiral-like crRNA-guided Cas12a system exhibits distinctive delayed cleavage dynamics and remarkable sensitivity to the length of ssDNA targets. Exploiting these characteristics, we develop a “delayed cleavage” feature-mediated one-pot sensing strategy that resolves the long-standing challenge of incompatibility between Cas12a-based cleavage reactions and nucleic acid amplification within a single reaction vessel (Fig. 1b, c). Additionally, we develop a portable temperature-controlled fluorescence imaging device integrated with this one-pot sensing strategy, constructing a portable diagnostic (DFTFD) platform for on-site detection. This study not only deepens our understanding of the crRNA-guided mechanism of Cas12a but also propels its broader application in fundamental research and accelerates the development of next-generation point-of-care molecular diagnostics.

Results
Engineered pre-crRNA-mediated Cas12a cleavage
Cas12a utilizes its intrinsic RNase activity to process the pre-crRNA, releasing a portion of the repeat sequence and thereby promoting its maturation (Supplementary Fig. 1a). The mechanism by which conventional mature crRNA activates Cas12a has been well characterized17,18. However, it remains unclear whether the 5′-repeat fragments released during pre-crRNA maturation contribute to Cas12a activity activation. To investigate this, we introduced a DNA target complementary to the released 5′-repeat fragments, but observed no activation of Cas12a cleavage activity (Fig. 2a–c). Previous studies have indicated that a minimum of 17 complementary nucleotides is required to activate Cas12a-mediated cleavage4. We hypothesized that the inability of the released repeat fragments (15 nt) to activate Cas12a may result either from insufficient length rather than a lack of intrinsic functional activity. To evaluate this, we engineered pre-crRNAs by extending the released 5′-repeat fragments from 15 to 22 nt and reintroduced the DNA target (Supplementary Fig. 1b). Gel electrophoresis revealed that cis-cleavage activity remained undetectable. Interestingly, however, the addition of an ssDNA fluorescein–quencher (ssDNA-FQ) probe resulted in measurable trans-cleavage activity, as shown by fluorescence signals (Fig. 2c). These findings indicate that native 5′-repeat fragments fail to activate Cas12a due to insufficient length rather than inherent inactivity. When engineering is extended, the released fragments contribute to trans-cleavage activity, revealing previously unrecognized functional potential.

We next replaced the conserved repeat fragment of pre-crRNA with a 22-nt random fragment, whereas retaining the intact 3′-spacer (AB-pre-crRNA) (Supplementary Fig. 1b). In this AB-pre-crRNA-guided Cas12a system, the trans-cleavage activity was still activated upon introduction of a complementary target (Supplementary Fig. 1c, d). This observation indicates that the engineered 5′ fragment of pre-crRNA can activate Cas12a's trans-cleavage activity in a manner independent of repeat sequence specificity. Such a sequence-independent activation mechanism significantly expands the potential applications of the Cas12a system. To investigate whether the released 5′ fragment and 3′-spacer could act synergistically, we established three groups: (1) AB-pre-crRNA + target B; (2) AB-pre-crRNA + targets A and B; and (3) BB-pre-crRNA + target B. Equal amounts of ssDNA-FQ probes and target were added to all groups. Fluorescence analysis revealed no significant differences among groups at low target concentrations (10 pM) (Supplementary Fig. 1e-g), suggesting that the 3′-spacer primarily governs endonuclease activation.

To elucidate the role of the released 5′ fragment in enzymatic activation, the 3′-spacer of AB-pre-crRNA was systematically shortened and then tested for its guided Cas12a activity (Fig. 2d). Gel electrophoresis revealed no detectable cis-cleavage activity in the system when the length of the 3′-spacer exceeded 1 nt, with weak activity emerging only after the spacer was reduced to 1 nt. Notably, when the 3′-spacer was eliminated entirely (0 nt), cis-cleavage was strongly activated (Fig. 2e). The addition of the ssDNA-FQ probe further revealed that, in contrast to cis-cleavage, trans-cleavage activity was consistently present across all constructs and gradually increased with the shortening of the 3′-spacer (Fig. 2f). To further explore substrate specificity, the ssDNA was used as the target. As the 3′-spacer was shortened, Cas12a exhibited progressively enhanced cleavage of ssDNA targets, consistent with increased fluorescence signals (Fig. 2g, h). The variation in cleavage efficiency between ssDNA and dsDNA may relate to differences in protospacer adjacent motif (PAM) sequence dependence. These results demonstrate that the 5′ fragments released during engineered pre-crRNA maturation can indeed activate Cas12a cleavage activity, with activity modulated by 3′-spacer length. This finding informs a strategy for regulating Cas12a cleavage activity and engineering crRNAs.

Notably, complete removal of the 3′ spacer produced a crRNA with functional activity comparable to that of a conventional mature crRNA. Strikingly, in this functional molecule, the guide sequence was located at the 5′ end rather than at its canonical 3′ end. These two configurations adopt chiral-like structural states; we therefore refer to this variant as a chiral-like crRNA (Supplementary Fig. 2).

The guiding mode of chiral-like crRNA represents an engineered feature. For different Cas12a orthologs (including LbCas12a, AsCas12a, and FnCas12a), we precisely designed chiral-like crRNA sequences, and the experimental results demonstrated that this guiding mode exhibits good compatibility among these Cas12a orthologs (Fig. 2i–l, Supplementary Fig. 3). Unlike conventional mature crRNA, the Cas12a system guided by chiral-like crRNAs exhibits strict dependence on perfect complementarity between the guide sequence and the dsDNA target (Fig. 2m, n). For ssDNA targets, cleavage efficiency decreases progressively with shortening of the guide sequence (Fig. 2p). Similarly, the trans-cleavage activity of Cas12a declines as the guide length is reduced (Fig. 2o, q). This guiding mode expands the functional landscape of the Cas12a system and may inform molecular diagnostic applications.

Collectively, these findings establish that engineered 5′ fragments actively regulate Cas12a activity, particularly its trans-cleavage function. The Cas12a system guided by these 5′ fragments exhibits unique enzymatic characteristics, which are governed by the 3′-spacer length of the pre-crRNA. These results deepen our mechanistic understanding of crRNA-guided Cas12a, provide a framework for its rational engineering, and will inform strategies for regulating its activity in diagnostics and therapeutics.

The mechanism of Cas12a cleavage mediated by chiral-like crRNA
In the preceding investigation, we found that the chiral-like crRNA exhibits guiding activity similar to that of the conventional mature crRNA, but its underlying mechanism remained unclear. Previous studies have demonstrated that the RNase activity of the Cas12a enzyme exhibits strong sequence and structural specificity5. To investigate the functional coupling between RNase and DNase activities in the chiral-like crRNA-guided Cas12a system, we introduced mutations at the 5′ and 3′of the chiral-like crRNA stem-loop fragment (Fig. 3a). Gel electrophoresis revealed that the mutated crRNA remained intact after incubation with Cas12a, indicating that RNase activity was not activated. When dsDNA targets were subsequently added, no degradation occurred, suggesting that DNase activity was also not triggered. In contrast, wild-type chiral-like crRNA was cleaved into two fragments by Cas12a, and the addition of dsDNA targets led to further degradation (Fig. 3b, c). These results demonstrate that RNase activation is a prerequisite for DNase activation in the chiral-like crRNA-guided Cas12a system.

To further investigate the guiding mechanism, we labeled the guide fragment of the chiral-like crRNA with carboxyfluorescein (FAM) and incubated it with Cas12a. High-temperature denaturation followed by gel electrophoresis showed that the guide fragment was gradually cleaved over time and became fully cleaved after 30–40 min (Fig. 3d). To maintain the integrity of the Cas12a enzyme-crRNA complex, we terminated the reaction with ethylenediaminetetraacetic acid (EDTA) and performed gel electrophoresis analysis. A portion of FAM-labeled guide RNA remained bound to Cas12a even after prolonged incubation (Fig. 3e). Given that Cas12a can completely cleave crRNA within 40 min, this suggests that the released guide fragments may rebind to the Cas12a. In contrast, when FAM was labeled on the 5′-guide fragment of the AB-per-crRNA, only a low amount of the Cas12a-FAM complex was detected after prolonged incubation. This suggests that the 3′-spacer may hinder the recombination of the cleavage products (Fig. 3f). Supplementing the pre-crRNA system with free stem-loop fragments moderately increased the fraction of FAM-labeled Cas12a (Fig. 3g), confirming that a vacant binding site at the 3′ end promotes recombination of the guide fragment. Consistently, the addition of free stem-loop fragments enhanced Cas12a trans-cleavage activity, as shown by ssDNA-FQ probe assays (Fig. 3h). These findings collectively support the hypothesis that recombination of released guide fragments contributes to Cas12a activation.

We further investigated the positional dependence of PAM recognition in the Cas12a system guided by chiral-like crRNA. To this end, we designed two double-stranded DNA targets: one with the PAM sequence positioned at the distal end of the stem-loop structure (PAM-L), and the other with the PAM located proximally (PAM-R). The results showed that, unlike the conventional mature crRNA-guided system, the chiral-like crRNA-mediated Cas12a system exhibits a unique site-selective preference, effectively recognizing only targets in which the PAM resides at the distal end of the stem-loop (Supplementary Fig. 4).

Together, these results reveal that the mechanism of chiral-like crRNA-guided Cas12a begins with RNase-mediated cleavage of the 5′-guide fragment, which is subsequently released and reassembled with Cas12a. The reassembled complex then recognizes the DNA target and activates cleavage. This model explains why Cas12a loses cis-cleavage activity when the engineered pre-crRNA retains more than 1 nt at the 3′ end, or when the 5′ end of the chiral-like crRNA guide fragment is truncated (Fig. 2e, n). In these cases, the recombined guide fragment lacks a complete seed region, impairing accurate PAM recognition. To further confirm this, we progressively shortened the guide fragment adjacent to the stem-loop region in chiral-like crRNA and tested Cas12a activity with dsDNA target (Fig. 3i). Gel electrophoresis revealed a gradual decline in cleavage activity rather than an abrupt loss (Fig. 3j). These observations support a model in which recyclable guide fragments mediate target recognition, with structural integrity being essential for maintaining Cas12a cleavage activity.

Chiral-like crRNA-guided Cas12a activity characteristics
The cleavage kinetics of the chiral-like crRNA-guided Cas12a system were first compared with the conventional mature crRNA-guided system. Unlike mature crRNA, chiral-like crRNA requires an initial cleavage and recombination step after enzyme binding (Fig. 4a). To ensure complete crRNA reassembly, we then incubated the chiral-like crRNA with Cas12a for sufficient time before comparing nuclease activity with the canonical crRNA–guided system. Once fully reassembled, the chiral-like crRNA–guided system exhibited only slightly lower activity than the standard crRNA system, likely due to minor loss of guide fragments during reassembly (Supplementary Fig. 5a, b). Under conditions in which the chiral-like crRNA is not yet fully reassembled, activity comparison with the canonical crRNA system produces the delayed-cleavage pattern shown in Fig. 4b-d. Upon addition of dsDNA targets, the conventional mature crRNA-guided Cas12a system achieved complete cleavage within 10 min, while the chiral-like crRNA-guided Cas12a system required approximately 30 min to reach a comparable level (Fig. 4b, c). The trans-cleavage activity has also been evaluated using an ssDNA-FQ reporter probe. The mature crRNA-guided Cas12a system exhibited robust cleavage activity immediately at the onset of the reaction, reaching peak fluorescence within 20 min. In contrast, the chiral crRNA-like guided Cas12a system displayed delayed cleavage activity, initiating fluorescence enhancement only after 12 min and achieving maximal signal intensity comparable to the mature crRNA system after 40 min (Fig. 4d; Supplementary Fig. 5c, d).

We employed molecular dynamics simulations to compare the binding patterns of chiral-like crRNA and conventional mature crRNA with Cas12a. The root mean square deviation (RMSD) of the crRNA–Cas12a complex initially increased rapidly during the simulation before reaching equilibrium. The post-equilibrium RMSD values differed by only 0.1 nm between the conventional mature crRNA-Cas12a complex and the chiral-like crRNA-Cas12a complex, indicating that both systems can form similarly stable structural configurations (Supplementary Fig. 6a). The solvent accessible surface area (SASA) represents the contact surface area between the simulated target and solvent molecules. A lower SASA value indicates a more compact and relatively more stable structure. Both the conventional mature crRNA-Cas12a complex and the chiral-like crRNA-Cas12a complex exhibited gradually decreasing SASA values (Supplementary Fig. 6b). This observation further confirms that both systems can form similarly stable structural configurations. Previous studies have highlighted the importance of the stem-loop in binary complex formation6. As shown in Fig. 4e, f, after system stabilization, the stem-loop fragments of both the chiral-like crRNA and conventional mature crRNA bind to the oligonucleotide-binding domain (OBD) of Cas12a. Detailed structural alignments revealed highly conserved interaction sites between the nucleobases in the stem-loop fragments and amino acid residues in Cas12a for both crRNA variants (Supplementary Fig. 6c-e). We next calculated the root mean square fluctuation (RMSF) of amino acid residues in Cas12a. The analysis revealed highly consistent residue fluctuations in the OBD region between both systems (Fig. 4g). These results indicate that the positioning of the guide fragment does not alter the binding site of the crRNA stem-loop fragment to Cas12a.

Furthermore, we calculated the binding free energy and hydrogen bond formation in both systems (Fig. 4h, Supplementary Table 1). Compared to the conventional mature crRNA system, the chiral-like crRNA molecule demonstrated comparable binding affinity with Cas12a. This result demonstrates that the positioning of the guide fragment does not affect crRNA-Cas12a binding. The molecular dynamics simulations demonstrate that the chiral-like crRNA can bind to Cas12a's target domain with stability and a high affinity equivalent to conventional mature crRNA, while the differential cleavage kinetics between the two systems originate from Cas12a's distinct processing of the chiral-like guide fragment.

We then examined the dependence of cleavage activity on ssDNA target length (Fig. 4i). With a short ssDNA target (22 nt), the cleavage activity of the Cas12a system guided by conventional mature crRNA was fully activated, whereas the chiral-like system exhibited minimal activity (Fig. 4j, k). As the ssDNA target length increased, cleavage activity in the conventional system remained stable, whereas the chiral-like system exhibited substantial activation only when the target exceeded 32 nt, with activity further increasing with target length. These findings indicate that the chiral-like crRNA-guided Cas12a system exhibits a stronger dependence on target length, an attribute that favors high-fidelity detection of long DNA substrates. Moreover, the unique property may be further leveraged to develop next-generation biosensing platforms with improved sensitivity and specificity.

In the chiral-like system, the crRNA is introduced as a single molecule, relying on Cas12a’s intrinsic RNase activity to cleave and release the guide fragment, which subsequently re-associates with the enzyme. This guiding mode differs from the split crRNA-guided system described in recent studies (Fig. 4l)19,20. It simplifies the reaction system while fully utilizing the cleavage potential of Cas12a. Gel electrophoresis revealed that the chiral-like crRNA-guided system exhibits superior cleavage efficiency compared to the split crRNA-guided system. (Fig. 4m).

To investigate the underlying mechanism, we fluorescently labeled the guide fragments of conventional mature crRNA, chiral-like crRNA, and split crRNA with FAM, followed by incubation with Cas12a. Gel electrophoresis analysis revealed that the conventional mature crRNA-guided system exhibited the strongest fluorescence signal, while the chiral-like crRNA system showed comparable signal intensity. In contrast, the split crRNA system exhibited markedly weaker fluorescence (Fig. 4n, o). These results indicate that the guide fragment of chiral-like crRNA retains binding affinity comparable to mature crRNA, whereas the split crRNA guide fragment binds much less efficiently. Previous studies have shown that the stem–loop structure of crRNA plays a critical role in the formation of the Cas12a–crRNA binary complex21. Upon formation of this complex, Cas12a undergoes structural rearrangements that facilitate stable binding of the guide sequence. To test this conclusion, we first incubated the stem-loop sequence with Cas12a enzyme, and then added the FAM-labeled guide sequence. Gel electrophoresis results showed a stronger fluorescence signal compared to the split crRNA system (Fig. 4n, o).

These findings validate a mechanistic model in which, after the chiral-like crRNA binds to Cas12a, the enzyme undergoes a corresponding conformational change, allowing the released guide sequence to rapidly and stably rebind to Cas12a over a shorter spatial distance. In contrast, for split crRNA, the stem–loop and guide sequences are introduced separately, and their binding follows a strict sequential order: the stem–loop must first bind Cas12a before the guide sequence can associate, which reduces overall binding efficiency. This sequential dependency, combined with the full dispersion of the guide sequence in solution, leads to increased spatial separation from the enzyme and consequently reduces overall binding efficiency.

“Delayed cleavage” feature-mediated one-pot Cas12a-based assay
Limited sensitivity remains a major challenge in Cas12a-based molecular diagnostic assays22-24. To enhance detection sensitivity, pre-amplification of the DNA target is usually required25. To minimize aerosol contamination during the amplification, researchers have attempted to integrate the nucleic acid amplification and CRISPR/Cas12a detection into a single reaction 26-28. However, incompatibility between the two systems remains the primary obstacle to achieving a robust one-pot assay (Fig. 5a). Previous studies have proposed several solutions, including spatial separation, photoactivation, and bioengineering approaches29-31. Although these strategies can address the incompatibility issues in one-pot detection systems, they still face challenges such as operational inconvenience, synthetic complexity, and high technical barriers (Supplementary Fig. 7).

Chiral-like crRNA functions as a “delay switch” for Cas12a activity activation, resulting in a characteristic delayed cleavage profile. Leveraging this characteristic, we developed a one-pot sensing strategy in which the chiral-like crRNA-guided Cas12a system and the RPA system were co-encapsulated in a single tube. Following Cas12a enzyme binding, the chiral-like crRNA requires additional cleavage and reorganization steps, during which Cas12a remains silent. This additional silent period provides a critical time window for RPA-mediated target amplification. Once reassembled, Cas12a binds the abundant products and swiftly activates cleavage (Fig. 5b). This delayed cleavage feature-mediated one-pot sensing strategy resolves the long-standing incompatibility between Cas12a cleavage and nucleic acid amplification within a single vessel.

The inert response of chiral-like crRNA-guided Cas12a to short ssDNA fragments minimizes interference from abundant non-target DNA in complex biological samples, thereby greatly enhancing assay robustness. Because this property depends only on Cas12a and the conserved stem-loop fragment, it is independent of target sequence, conferring excellent universality.

We first tested the feasibility of this approach using Salmonella (Gram-negative bacteria) as the target. To achieve optimal performance of the one-pot detection strategy, we systematically optimized the concentration of RPA primers. The results demonstrated that the strategy achieved the highest detection sensitivity when the primer concentration was set at 1 μM, as either excessively high or low primer concentrations significantly affected the amplification efficiency of RPA and consequently the detection sensitivity (Supplementary Fig. 8a). We incorporated an additional pre-incubation step prior to adding the RPA components and other reagents, and optimized its duration. If the pre-incubation step is omitted, Cas12a cleavage activity is activated too late, and the complex reaction environment interferes with guide sequence reassembly. These effects lead to reduced cleavage efficiency and disrupt the balance between amplification and cleavage. Conversely, excessively long pre-incubation compresses the available time window, thereby affecting RPA amplification. Through optimization, we determined that a 15-min pre-incubation provides the best balance between RPA amplification and Cas12a activation. (Supplementary Fig. 8b).

In the conventional mature crRNA-guided one-pot CRISPR-Cas12a system, the robust cleavage activity of Cas12a toward dsDNA targets and primers severely compromises RPA amplification efficiency, ultimately limiting detection sensitivity (LOD of 10⁵ CFU/mL) (Supplementary Fig. 8c-e). Under the same conditions, in a one-pot system mediated by chiral-like crRNA, the delayed-cleavage property of the chiral-like RNA creates an activity balance phase, allowing RPA amplification to proceed efficiently. As a result, the system achieves a detection sensitivity of 10² CFU/mL, representing a 1,000-fold improvement over conventional one-pot systems (Fig. 5c; Supplementary Fig. 8f-h). Thus, our method successfully integrates CRISPR-Cas12a cleavage with RPA amplification in a single pot, eliminating extra handling steps while significantly boosting detection sensitivity and simplifying the workflow.

To evaluate the universality of the sensing strategy, we extended the assay to Staphylococcus aureus (Gram-positive bacteria) and Lactobacillus rhamnosus (probiotic). Both strains were reliably detected under identical conditions, with limits of detection (LOD) of 10² CFU/mL (Fig. 5d, e). Specificity was then assessed using multiple non-target strains, including Escherichia coli, Vibrio parahaemolyticus, Listeria monocytogenes, Lactobacillus plantarum, and Saccharomyces cerevisiae. Only the presence of the target strain yielded enhanced fluorescence signals (Fig. 5f–h). This specificity arises from RPA primers designed against conserved target sequences, combined with the intrinsic cleavage fidelity of chiral-like crRNA-guided Cas12a.

Therefore, we have developed a fully compatible one-pot sensing strategy that integrates CRISPR/Cas12a cleavage with RPA amplification. This approach achieves a 1000-fold improvement in sensitivity over conventional mature crRNA-mediated one-pot assays. Unlike previous strategies that depend on Cas enzyme engineering or crRNA chemical modification, our method leverages the intrinsic properties of Cas12a through a chiral-like crRNA design. By exploiting these innate features, the strategy offers significant advantages in cost, simplicity, and versatility across diverse targets, highlighting its strong potential for large-scale application and clinical translation.

Real samples testing
To facilitate on-site detection, we developed a portable temperature-controlled fluorescence imaging device (Fig. 6a). The reaction module incorporates a 96-well PCR plate design, enabling high-throughput screening. A rapid-heating module ensures efficient thermal cycling, whereas the precision control system (±0.1 °C) provides stable reaction conditions. The fluorescence detection module features an optimized optical path, enabling high excitation efficiency. The generated signal intensity is directly observable by the naked eye and supports smartphone imaging recording, enabling rapid experimental data acquisition (Fig. 6b, Supplementary Fig. 9a, Supplementary Movie 1). Through meticulous component arrangement and a 3D-printed custom housing, the device volume was reduced fivefold compared with conventional real-time quantitative polymerase chain reaction (qPCR) instruments.

We integrated the delayed-cleavage feature-mediated one-pot assay with this portable device to construct a portable diagnostic (DFTFD) platform for on-site detection. Salmonella was selected as the detection target due to its wide existence in the food chain and serious harmfulness to human health. Clinical samples (serum and urine) and food products (sea cucumber, shrimp, sea bass, and salmon) were analyzed (Supplementary Fig. 9b). The qPCR test served as the gold standard for comparison. The DFTFD platform achieved complete concordance with qPCR in both positive and negative samples, confirming excellent detection accuracy and reliability (Fig. 6c-e). Compared with qPCR, the DFTFD platform exhibits a lower cost, a shorter turnaround time, and superior portability, all these advantages making it particularly suitable for on-site rapid detection (Fig. 6f).

Methods
Ethical statement
In this study, we exclusively utilized retrospectively collected, de-identified secondary-use patient samples or residual clinical samples. The authors did not have access to any identified patient information, nor did they recruit any subjects specifically for the purposes of this study. Approval for this study was obtained from the Medical Ethics Committee of the General Hospital of the Central Theater Command of the Chinese People's Liberation Army (approval ID: 2023-026-01).

The cleavage characteristics of the Cas12a enzyme on crRNA
All reactions were carried out in 1 × Cas12a reaction buffer (5 mM Tris-HCl (pH 9.0), 20 mM NaCl, 5 mM MgCl2). The Cas12a enzyme and crRNA were incubated at 37°C for 1 h, and then denatured at 85°C for 5 min. The obtained products were analyzed by 10% polyacrylamide gel electrophoresis (PAGE) gel electrophoresis.

Verification of cis-cleavage activation of Cas12a
Initially, the dsDNA template was subjected to hybridization. The hybridization system had a total volume of 20 μL. The final concentrations of the components were as follows: 50 nM Tris-HCl (pH 8), 5 mM KCl, 100 mM NaCl, 1 mM MgCl₂, and 2 μM ssDNA template. The hybridization system was incubated at 95 °C for 5 min for denaturation. Subsequently, it was rapidly transferred to an ice bath for 5 min for annealing. Finally, the hybridization process was carried out at 25 °C for 1 hour.

The Cas12a and crRNA were mixed and incubated at 37°C for pre-assembly for 20 min. Then, the dsDNA target or ssDNA target was added to the above mixture solution for 60 min. After the incubation, denaturation is performed at 85°C for 5 min. The obtained products are analyzed using 10% PAGE gel electrophoresis. The final reaction system is 10 μL, and the final concentrations of each component are 1 × Cas12a reaction buffer, 0.05 mg/mL Cas12a, 50 nM dsDNA target or 100 nM ssDNA target, 250 nM crRNA.

Verification of trans-cleavage activation of Cas12a
The pre-assembly of Cas12a and crRNA was first completed, then the FQ probe and different concentrations of dsDNA targets and ssDNA targets were added to the above mixture. A fluorescence microplate reader was used to record the fluorescence signal in real time, collecting the fluorescence signal every two minutes. The final reaction system is 20 μL, and the final concentrations of each component are 1 × Cas12a reaction buffer, 2 μg/mL Cas12a, 500 nM FQ probe, 100 nM crRNA, and different concentrations of DNA targets.

Analysis of the binding state of the guide fragment to the Cas12a enzyme
The FAM-labeled crRNA was incubated with the Cas12a enzyme for a specific period of time, then it was denatured at 85°C for 5 min or terminated by adding EDTA. The obtained products were analyzed using 10% PAGE gel electrophoresis. The entire process was carried out under light-protective conditions. The final reaction system was 15 μL, and the final concentrations of each component were 1 × Cas12a reaction buffer, 0.1 mg/mL Cas12a, 700 nM crRNA-FAM, 40 mM EDTA (with a pH of 8), and 1.4 μM of free stem-loop fragments were added in some systems.

Bacterial culture and DNA extraction
The strains stored at -80℃ were activated, and then streaked on plates, and single colonies were picked for pre-enrichment. 100 μL of the pre-enriched bacterial suspension was added to 5 mL of specific medium (TSB medium for Salmonella, LB medium for Staphylococcus aureus, and MRS medium for Lactobacillus rhamnosus) and cultured for 12 hours. DNA was extracted from the enriched bacterial suspension using a commercial kit, and the number of colonies was determined by plating.

Molecular Dynamics Simulation
The protein–RNA aptamer complex was modeled using BoltzS-2 (AlphaFold3)34,35 . Molecular dynamics (MD) simulations were performed with GROMACS 2022.436. The AMBER14SB_OL15 force field was used for both the protein and RNA, and the TIP3P water model was employed37,38. The simulation system was solvated in a periodic water box with a salt concentration of 0.15 mol/L MgCl₂. Counterions, including Cl⁻ and Mg²⁺, were added to neutralize the system. Energy minimization was carried out in two steps: the first using the steepest descent algorithm for 10,000 steps, followed by the conjugate gradient method for 5,000 steps. After minimization, the system underwent 500 ps of NVT equilibration and 500 ps of NPT equilibration. Subsequently, a 200 ns production MD simulation was conducted under constant temperature and pressure conditions. Temperature was maintained at 310.15 K using the V-rescale thermostat, and pressure was controlled using the Parrinello–Rahman barostat. The simulation time step was 2 fs. Long-range electrostatics were treated using the Particle Mesh Ewald (PME) method, and both Coulomb and van der Waals interactions were truncated at 12 Å. LINCS was used to constrain all bonds involving hydrogen atoms. Trajectory analysis was performed using built-in GROMACS tools. Structural visualization and interaction analysis were conducted in PyMOL v2.4.0a0 . Binding free energy calculations were carried out using gmx_MMPBSA v1.6.1 39.

Performance of chiral-like crRNA-guided one-pot assay.
First, the primer concentration of RPA and the pre-incubation time for Cas12a and chiral-like crRNA were optimized. The performance of the one-pot assay was then evaluated under these optimal conditions. Specifically, Cas12a and crRNA were pre-incubated for 15 min, followed by the addition of the RPA reagents and other components. The final volume of the one-pot method was 15 μL, and the final concentrations of each component were as follows: 2 μg/mL Cas12a, 0.1 μM crRNA, 2 μM FQ probe, 1 μM RPA primer, 280 mM MgOAC, 10 mM PEG, RPA enzyme, and a certain concentration of nucleic acid target. The conventional mature crRNA-mediated one-pot system was used as the control group, and all components were consistent with those of the one-pot system mediated by chiral-like crRNA. All reagents were placed in the same eight-tube vial and incubated at 37°C. Fluorescence signals were collected using qPCR once every minute.

The integrated temperature-controlled fluorescence equipment
The equipment mainly consists of a temperature control module and a fluorescence imaging module. In the temperature control module, two separate semiconductor heating plates are added to jointly heat the upper and lower sides of the reaction chamber. Multiple high-precision temperature sensors are placed on both sides of the reaction chamber, and together with the temperature control module, they achieve precise temperature control. A heat dissipation module is integrated at the lower end of the reaction chamber to ensure the stable operation of the device. In the fluorescence imaging module, a high-power excitation light source and a narrow-band filter are combined to ensure efficient excitation of the fluorescent groups and effective filtering.

Real samples analysis
The samples were purchased in the market, and these samples were pre-treated according to the national standard method (GB 4789.4-2024). In an aseptic environment, 2.5 g of the sample was taken and placed in 25 mL of buffered peptone water (10 mg/mL peptone, 5 mg/mL sodium chloride, 9 mg/mL disodium hydrogen phosphate, 1.5 mg/mL dipotassium hydrogen phosphate). The sample was then homogenized using a homogenizer for 1-2 min and incubated at 37 ℃ for 8-12 hours for pre-culture. DNA was extracted from the samples using commercial kits, and the obtained DNA targets were added to the aforementioned chiral-like crRNA-mediated one-pot assay. After inoculation, the samples were extracted for DNA using commercial kits. The mixture was incubated in the built temperature-controlled fluorescence imaging integrated device for 1 hour, and then the fluorescence intensity was observed. The same sample was also subjected to qPCR detection. The reaction conditions for the qPCR method are as follows: (1) 95 ℃, 3 min; (2) 94 ℃, 10 s; (3) 55 ℃, 20 s; (4) 72 ℃, 20 s, scanning, returning to step 2, and repeating 40 cycles.

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

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "Li and colleagues investigated the impact of sequence fragments released during the maturation of pre-crRNA on the cleavage activity of the CRISPR/Cas12a system. This is an important yet previously overlooked issue, that can provide new insights into the guiding mechanism of crRNA in the CRISPR/Cas12a system.\nThe study is the first to discover that, once the released sequences exceed a certain length threshold during pre-crRNA maturation, they can spontaneously recombine to activate Cas12a, thereby exhibiting unique cleavage characteristics distinct from those of the conventional crRNA-guided Cas12a system. This finding provides a novel strategy for the precise regulation of Cas12a activity and lays the groundwork for broader applications in future research contexts. Notably, the proposed “chiral-like crRNA” strategy introduces a delayed cleavage activity that elegantly addresses key challenges faced by current one-pot CRISPR-based detection methods. This approach takes advantage of the intrinsic activity of Cas12a, eliminating the need for additional chemical modifications and thereby reducing operational complexity while preserving broad target adaptability. Through methodological innovation and device integration, the developed so-called DFTFD detection platform shows great potential for on-site rapid testing applications.\nOverall, the manuscript is developed timely, and experimental data supplied relatively strong support to the conclusions. We recommend that the authors further improve the following aspects before the manuscript can be accepted for publication in Nature Communications:\nMajor technical criticisms or questions\n1. About the processing of pre-crRNA. This study first investigated the roles of sequence fragments located at both ends of the stem–loop region in natural and engineered pre-crRNAs in the activation of Cas12a. The authors assumed that the 5’ fragment of the stem–loop sequence would be cleaved and released (shows in Fig. 1a). Although the processing of pre-crRNA by Cas12a has been reported in previous studies, it would still be valuable to include additional verification for different crRNA sequences. We suggest that the authors consider adding experiments on Cas12a processing of the various pre-crRNAs mentioned in the manuscript to further strengthen the completeness of the study.\n2. The crRNA typically prefers a TTTN PAM sequence when guiding Cas12a-mediated target cleavage. The chiral-like crRNA proposed in this study represents a novel guiding strategy distinct from natural crRNA. According to the authors’ findings, chiral-like crRNA undergoes additional cleavage and recombination, eventually adopting a structure consistent with conventional crRNA to guide subsequent cleavage. However, the dependence of chiral-like crRNA on the PAM sequence position has not been explored. Performing additional experiments to investigate this dependence would help further validate the robustness of the current conclusions.\n3. This study investigates the compatibility of the chiral-like crRNA guiding mode across multiple Cas12a orthologs. Because the crRNA sequences of different Cas12a proteins vary slightly, providing detailed illustrations of the chiral-like crRNAs for LbCas12a, FnCas12a, and AsCas12a would greatly aid readers in understanding the underlying mechanism.\n4. The manuscript mentions that the loss or mutation of the seed region prevents certain crRNAs from activating Cas enzyme cleavage (line 220). It would be helpful to annotate the seed region in Figure 2h for clarity. Additionally, we are curious whether the presence of mismatches between the pre-crRNA and the target DNA could impact the maturation of pre-crRNA. If so, could the sensitivity of the maturation process to base mismatches potentially be utilized as a method to detect DNA mutations?\n5. About the illustration of the assay principle, it is suggested that Figure 1a be redrawn. The delay of trans-cleavage by engineering pre-crRNA is not well illustrated in Fig. 1a, and its contribution towards one-pot detection is also not well demonstrated.\nMinor technical criticisms or questions\n1. The manuscript includes schematic illustrations to show the structural similarities between conventional mature crRNA and chiral-like crRNA. While these figures are helpful, they do not allow for a clear comparison. Including more comprehensive sequence comparisons would better illustrate the relationship between the two.\n2. Some of the data in this study lack statistical analysis, such as those shown in Fig. 4f–h, making it necessary to supplement these analyses. In addition, some of the existing statistical analyses are not fully compliant with standard reporting practices—for example, in Fig. 5b and 5d, the exact p-values should be reported rather than using general statements such as “P<0.01” or “P<0.001”.\n3. Several key reaction components were optimized when developing the one-pot detection method based on chiral-like crRNA; however, the manuscript does not explain how these components influence the detection performance. Additional clarification on this point is recommended.\n4. The delayed cleavage characteristics exhibited by chiral-like crRNAs offer a novel approach to addressing the challenge associated with one-pot detection. Provide comparison with other reported one-pot CRISPR assays, such as J. Am. Chem. Soc. 2025, 147, 31004-31015; J. Am. Chem. Soc. 2025, 147, 31349-31364; Nature Communications, 2025, 16, 5939; PNAS, 2022, 119, e2202034119.\n5. Fig. 3e and f presents a detailed comparison of the interactions between the guide sequences of conventional mature crRNA and chiral-like crRNA with Cas enzyme amino acids. However, the labels in the current figure are too small to identify.\n6. The manuscript describes a portable fluorescence imaging device coupled with a one-pot detection method to facilitate on-site detection. However, what are the protocols for sample pretreatment, such as nucleic acid extraction? It would be beneficial if the process were simplified to ensure ease of use for on-site detection.\n7. The guiding sequence of the chiral-like crRNA exhibits a stronger affinity with the Cas12a enzyme than that of the split crRNA, resulting in enhanced cleavage activity. It is suggested that the authors further elucidate the underlying mechanism of this phenomenon.\n8. The chiral-like crRNA-guided CRISPR/Cas12a system shows a pronounced dependence on the length of the ssDNA target. It would be valuable for the authors to elaborate on whether and how this property could be harnessed for future applications.\n9. Is there a quantitative criterion for the delayed cleavage activity observed with the chiral-like crRNA? For instance, how long can the cleavage reaction be delayed? The authors should provide further clarification on this point.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "In this manuscript, the authors demonstrate that the 5′-repeat fragment released during pre-crRNA processing functions as a spacer that directs target recognition and cleavage, and they leverage this finding to develop a one-pot CRISPR/Cas assay. While the experimental design and technical integration are robust, the work lacks sufficient novelty, mechanistic depth, and methodological rigor. The activity of chiral-like crRNA (with swapped repeat/ spacer sequences) has been reported previously (10.1021/acs.analchem.4c05399), as has the ability of the resulting split crRNA to guide cleavage (10.1093/nar/gkab1227), thus the innovative contribution is limited. Specific comments are listed below:\n1.The authors attribute the improved sensitivity of their chiral-like crRNA one-pot assay to a \"delayed activation\" caused by the necessary engineering processing step. This interpretation is unconvincing. Figure 3 shows that the split crRNA generated after processing has intrinsically lower activity than canonical crRNA; thus, the apparent sensitivity enhancement likely results from slower consumption of amplicons by the low-activity split crRNA rather than temporal delay. Moreover, the stated lag of 10–15 min lacks kinetic support. The authors should measure the processing kinetics of pre-crRNA by Cas12a directly. And Figures 2d–f show that most chiral-crRNA-FAM is already cleaved by 5 min, indicating processing times well below 15 min. A control experiment in which only crRNA and Cas12 are mixed should be performed to confirm whether RNase activity is triggered during sample preparation.\n2.Since the same one-pot assay outcome could be achieved by using split crRNA directly, the specific advantage of employing chiral-like crRNA is not demonstrated.\n3.The authors should clarify the advantages of their one-pot assay over previously reported versions and justify the necessity of developing this new approach.\n4.In Supplementary Fig. 6 the pre-incubation time is optimized to 15 min. Please specify explicitly where this step occurs in the workflow—i.e., whether it is a separate incubation before addition of RPA components and other reagents.\n5.Lines 186–197 state that RNase activity is a prerequisite for DNase activity. The authors should mutate the repeat region to verify that Cas12 RNase activity is indeed abolished.\n6.Figures 3l–o show stronger cleavage signals for chiral-crRNA than for split crRNA, which the authors attribute to higher Cas-protein affinity. While unprocessed chiral-crRNA may bind more tightly because of its intact repeat, it must still be processed into split crRNA to become active; at that stage the two species are structurally identical and should exhibit the same activity. The origin of the signal difference therefore requires further investigation."}, "author_response": "We sincerely appreciate the reviewers’ recognition of our work and their insightful suggestions, which have greatly strengthened the quality of this manuscript. We are also grateful for the time and effort they invested in the review process. In response to the comments provided, we have prepared detailed point-by-point replies and further improved the manuscript with additional experimental data.\n\n[... Author responses to all points ...]"}, {"round": 2, "reviewer_comments": {"reviewer_1": "We have carefully reviewed the revised manuscript and the authors' point-by-point responses, and the key concerns have now been satisfactorily addressed. The authors further investigated the processing of different pre-crRNAs by Cas12a and the dependence of the chiral-like crRNA-guided Cas12a system on the positioning of PAM sequences in the target, providing further evidence supporting the mechanistic basis of chiral-like crRNA. In the revised manuscript, the authors further clarified the effects of RPA primer concentration and pre-incubation time on the sensitivity of the one-pot detection assay, and provided a more in-depth discussion on the advantages of the “delayed cleavage” feature-mediated one-pot assay. Additional experiments further strengthened the mechanistic explanation for the high guiding activity of chiral-like crRNA, indicating that conformational changes in Cas12a and the reduced spatial distance contribute to enhanced binding efficiency between the guide sequence and the enzyme. Overall, the revised manuscript shows substantial improvements in logical organization and mechanistic depth, with figures and tables also refined for greater clarity and readability. Overall, I am satisfied that all issues have been comprehensively resolved, and I believe the manuscript is now suitable for publication in Nature Communications.", "reviewer_2": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature Communications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career Researchers who co-review manuscripts.", "reviewer_3": "I have carefully reviewed the revised manuscript and the comments from the other reviewer. Although the authors have adequately addressed some of the concerns, I remain convinced that the work does not meet the standards for publication in Nature Communications for the following key reasons:\nThe central finding of this study—that the proposed chiral-like crRNA can be used for DNA detection—has already been reported (10.1021/acs.analchem.4c05399). While the authors emphasize new mechanistic insights, the processing and maturation of pre-crRNA are well-established concepts in the field. The authors claim that “when the 3′ spacer was reduced to 0 nt, the assembled crRNA exhibited cleavage activity comparable to that of mature crRNA,” but this is an obvious and easily anticipated result, as a 3′ overhang would interfere with split-crRNA assembly and target DNA recognition. Therefore, this observation does not constitute a sufficiently significant new discovery.\nThe authors may reasonably argue that Wen et al.’s work (10.1021/acs.analchem.4c05399) was not applied to develop a one-pot CRISPR assay; however, after careful evaluation of this chiral-like crRNA–based one-pot strategy, I do not believe it is likely to be broadly adopted because of substantial practical limitations. Specifically, the method strictly depends on pre-processing of chiral-like crRNA, including the associated time cost. This requirement is inconvenient for on-site testing, as the additional step increases operational complexity and makes implementation by non-specialists impractical. Moreover, this extra step prolongs the overall assay time. A key advantage of CRISPR diagnostics over other nucleic acid detection technologies, such as PCR, is speed; however, the proposed method undermines this advantage and therefore does not represent a meaningful technical advance. Although the authors may argue that this processing step could be completed in advance, this is difficult to achieve in practice due to the uncontrollable activity of Cas12a. In fact, this strategy resembles recently reported approaches that achieve one-pot CRISPR assays by suppressing Cas12a activity with heparin or by lowering LbCas12a concentrations (Nat Commun 16, 1166 (2025); Sci Adv 11, eadx2067 (2025)), all of which rely on maintaining Cas12a at a low activity level, but the latter approaches are clearly simpler and faster.\nBased on the sensitivity data presented, the reported detection limit of 10² CFU/mL does not appear competitive. Even excluding the additional 15 min required for chiral-like crRNA processing, detection of samples at 10² CFU/mL still requires nearly one hour, which offers no advantage over existing methods and is comparable to PCR in turnaround time.\nOverall, the scientific novelty of this work is limited, and the proposed one-pot CRISPR assay does not demonstrate advantages over existing methods in terms of operational simplicity, sensitivity, or speed.\nAdditional comments:\nThe authors state that “the crRNA adopts a ‘chiral-like’ conformation relative to natural mature crRNA (named chiral-like crRNA). This form does not exist in natural biological systems and represents a rationally engineered guiding mode.” This statement is inaccurate. The authors may state that their designed crRNA adopts a conformation similar to a “chiral-like” structure; however, mature crRNAs are themselves generated through similar sequence architectures, and it is therefore inappropriate to claim that such forms do not exist in nature.\nThe statement that Wen et al.’s work (10.1021/acs.analchem.4c05399) “further corroborates this conclusion” is also inappropriate, as that study was published earlier.\nThe authors present a table comparing different one-pot CRISPR assays and conclude that their method is optimal, but they overlook the possibility that their approach may be less sensitive, more time-consuming, and operationally more complex.\nThe Methods section lacks a clear description of how the one-pot CRISPR assay is actually performed."}, "author_response": "We are grateful for the time you dedicated to reviewing our manuscript. Your suggestions have played a positive role in helping us further improve the quality of the manuscript. With respect to the remaining concerns (particularly regarding the innovation and practical feasibility of the proposed detection method), we have provided a detailed, evidence-based clarification in the response below, including specific comparisons with prior work and additional explanations to avoid any misunderstanding of our manuscript’s primary contributions.\n\n[... Author responses to all points ...]"}]

Decision letter