A Single-Step CRISPR Tool for Clean Detection of Genetic Material

EnGen^® LbaCas12a (M0653s), 10× NEBuffer r2.1, antarctic thermolabile UDG (M0372S), and the LAMP reagents, including Bst 2.0 WarmStart DNA polymerase (M0538S), MgSO₄ (B1003S), dNTPs (N0447S), and 10× LAMP amplification buffer (2 mM MgSO₄, 10 mM (NH₄)₂SO₄, 50 mM KCl, 20 mM Tris-HCl, 0.1% Tween-20), were purchased from New England Biolabs (Ipswich, MA, USA). A 2× Taq PCR reaction mix was acquired from Tiangen Biotech (Beijing, China). Evagreen (20×) was obtained from Biotium (Hayward, CA, USA). A recombinant RNase inhibitor, diethylpyrocarbonate (DEPC)-treated water, and pullulan were acquired from Sangon Biotechnology (Shanghai, China). D-(+)-Trehalose dihydrate, sucrose, and sorbitol were obtained from Sigma (St. Louis, MO, USA). All PCR and LAMP amplifications were performed in a MyGo Pro real-time PCR instrument (IT-IS Life Science Ltd., Middlesbrough, UK). The gel imaging and fluorescence assay were performed on a blue light LED transmission instrument, SMOBIO B-BOX (Model VE0100, Taiwan, China). All primers and ssDNA probes were synthesized by Sangon Biotechnology (Shanghai, China). The gRNA oligonucleotides were synthesized and HPLC-purified by TaKaRa Bio, Inc. (Dalian, China). Data analysis and graphing were performed using OriginPro 9 software (OriginLab Corporation, Northampton, MA, USA).

For the PCR assay, the sequences of specific primers, crRNAs, and the target gene are provided in, respectively. Tables S1, S3 and S4

For the LAMP assay, the sequences of specific primers, crRNAs, and the target gene are provided in, respectively. Tables S2, S3 and S4

The Cas12a assay was carried out in a reaction mixture of 10 μL, incorporating 0.1 μM Cas12a, 1× NEBuffer 2.1 (10 mM MgCl₂, 100 μg/mL BSA, 10 mM Tris-HCl, 50 mM NaCl, pH 7.9), 0.1 μM probe, and 0.1 μM of the appropriate crRNAs. Then, the target DNA (1 μL) was introduced, and the reaction was tested at 37 °C for 30 min.

The thermostability of Cas12a in the presence of various sugars (pullulan, sucrose, trehalose, and sorbitol), including a negative control condition without added sugar, was evaluated by measuring its residual catalytic activity after thermal incubation. Specifically, the Cas12a-crRNA complex was incubated in NEBuffer 2.1 at 37 °C, 45 °C, 54 °C, 60 °C, and 70 °C for 10 min with pullulan, sucrose, trehalose, or sorbitol or without sugar. Then, 100 nM probes and 1 μL of DNA were introduced, and the solutions were incubated at 37 °C for 30 min to activate the trans-cleavage assay. The thermostability was evaluated based on the fluorescence intensity, which reflects the functional integrity of the enzyme after heat treatment. Additionally, 37 °C and 54 °C were also selected as fixed temperatures for testing various concentrations of trehalose (0, 0.3, 0.5, 0.6, 0.7, and 0.9 M) to determine the optimal concentration for maintaining enzyme stability.

To minimize carryover contamination caused by tube opening, we developed a one-pot approach that integrated template amplification with CRISPR-Cas12a-based detection. The principle of this approach is shown in Figure 1. First, we used the dependence of the CRISPR-Cas12a cleavage system on the PAM site to design PCR/LAMP primers, ensuring that the final amplification products included the requisite Cas12a PAM recognition sites (TTT). Prior to the next amplification reaction, the reaction mixture was treated with the Cas12a enzyme at 37 °C for only 10 min. The Cas12a-crRNA1 & 2 complexes specifically recognized contaminating amplicons carrying the introduced PAM sites, thereby triggering cis-cleavage and enabling rapid degradation of carryover contaminants. In contrast, the target DNA remained intact owing to the absence of the corresponding PAM site. Subsequently, the inherent high-temperature PCR/LAMP cycles inactivated the Cas12a enzyme at the bottom of the tube and enriched the amplified product. To achieve closed-tube visual detection, a separate Cas12a-detection complex (comprising Cas12a, crRNA3, trehalose, and a fluorophore-quencher labeled ssDNA probe) was pre-stored on the lid. After amplification, the tube was centrifuged or shaken to thoroughly mix the reaction system. The resulting amplicons then activated the trans-cleavage activity of the Cas12a-crRNA3 complex, leading to nonspecific cleavage of the ssDNA probe. Finally, the fluorescence signal was visualized using a blue light transmission instrument.

As a proof-of-concept approach to testing this assay platform, PCR amplification was performed using both ordinary primers (FP and RP) and primers carrying PAM sites (PAM-FP and PAM-RP). As shown in, amplification curves similar to those obtained with the ordinary primers were observed for the PAM primers, indicating that the PAM primers had no adverse effect on PCR amplification. Furthermore, the amplification products from different primers were verified and analyzed by gel electrophoresis (). Figure S3a Figure S3b

Nevertheless, after recognition and cleavage of contaminating amplicons, activated Cas12a may also exert nonspecific trans-cleavage activity toward ssDNA in the reaction system, including primers, which affected subsequent target enrichment [21]. Therefore, the reaction conditions were further optimized to minimize undesired trans-cleavage while preserving efficient cis-cleavage for contaminant elimination.

To optimize the additional pre-incubation step in our assay, we examined both the pre-incubation time and the crRNA configuration for contaminant removal. This optimization was designed to achieve efficient Cas12a-mediated cis-cleavage of contaminating amplicons, converting the intact contaminant into smaller fragments that would no longer function as templates for subsequent amplification, while minimizing undesired trans-cleavage of primers. As shown in Figure 2a, efficient cleavage of the intact contaminant amplicon was achieved within 10 min, whereas longer incubation did not yield an obvious additional benefit. Notably, previous studies showed that the persistence of Cas12a trans activity resulted in stoichiometric cleavage products accruing with longer working times. However, a short reaction time reduced any unwanted trans activity [22,23]. Several researchers have proposed that multiplex CRISPR/crRNA strategies can confer high levels of sensitivity and specificity [24,25,26]. To further improve contaminant removal, we compared single-crRNA and dual-crRNA systems. The dual-crRNA design, targeting two sites within the same contaminant fragment, showed stronger cleavage efficiency than either single-crRNA condition (Figure 2a and Figure S4). Furthermore, the introduced multi-Cas12a-crRNA system was demonstrated to specifically degrade contaminants without affecting the target template (Figure S5).

Similarly, because the on-target and trans-cleavage mechanisms of Cas12a are also dependent on Mg²⁺, we investigated the concentration of Mg²⁺ in the reaction [23]. Reducing the Mg²⁺ concentration significantly attenuated non-target ssDNA (primer) degradation (Figure 2b). At 1.5 mM Mg²⁺, little primer degradation was observed in the PCR. Although target cleavage may have been slower when using lower Mg²⁺ concentrations, all target-contaminant cleavage was still achieved at 1.5 mM Mg²⁺ after 10 min (Figure S6). This suggested that a concentration of 1.5 mM Mg²⁺ may help minimize trans-cleavage activity of Cas12a while maintaining its cis-cleavage activity. Subsequently, we also compared the dependence of CRISPR/Cas12a cleavage on buffers 1 and 2, which were Cas12a and PCR buffers, respectively. As shown in Figure S7, the cleavage efficiency of Cas12a was comparable under both buffering conditions. In addition, we evaluated the ratio of crRNA to Cas12a according to the degradation efficiency of the contaminants. As shown in Figure S8, the optimal ratio of crRNA to Cas12a was 1:1.

Generally, a very small amount of residual contamination in the air in a routine work environment can lead to false-positive results. Therefore, a series of simulated contaminants from L. monocytogenes was used to investigate the capacity of Cas12a to overcome this cross-contamination. As shown in Figure 2c, in the system without Cas12a, we observed amplification as low as 10¹ copies of the contaminant. In contrast, the system with Cas12a prevented the amplification of up to 10⁷ copies of carryover contaminant DNA (Figure 2c, lower panel). These results clearly demonstrated that the Cas12a pre-incubation step effectively eliminated PCR contaminants under experimental conditions. This finding is particularly important, as these results demonstrated that even minuscule amounts of contaminant nucleic acids can produce unwanted amplification. We also showed that the Cas12a-crRNA system reduced false-positive results caused by contamination. This was confirmed by performing Cas12a-free PCR reactions and Cas12a-treated PCR reactions in 10 copies of target DNA and simulating a range of carryover contaminant amplicons in the samples that mimicked a type of field contamination (e.g., a situation in which target DNA is scarce and background contamination is present). The Cas12a-free reactions amplified the nontargeted negative control DNA, generating false-positive amplification signals indistinguishable from real DNA signals. In contrast, PAM-PCR eliminated these false positives, and we were able to clearly distinguish the target (10 copies) from samples carrying contaminants of up to 10⁷ copies/μL (Figure S9). These results demonstrate the robustness of the CRISPR/Cas12a system, which efficiently removed DNA contaminants even when the target DNA was at very low concentrations, ensuring accurate results.

Based on these results, we further investigated the practical application of Cas12a for degrading contaminants containing PAM sites. We observed that negative control samples, in the absence of PCR cross-contamination, did not produce significant false-positive amplifications. However, in the presence of PCR cross-contamination, the negative control samples exhibited a significant amplification curve, leading to false-positive results. In contrast, when an additional pre-incubation step was performed before the PCR, the false positive results in the system disappeared, indicating that the contamination-cleavage ability of Cas12a was effective and that positive amplification was able to proceed without any adverse effects (Figure 2d).

To minimize carryover contamination that may occur when opening the tube, a Cas12a-detection complex (comprising Cas12a, crRNA3, trehalose, and the fluorophore–quencher-labeled ssDNA probe) present in the tube cover was used to detect the amplification products. As shown in Figure 3a, only the reaction mixture that contained the target DNA, Cas12a, and crRNA3 produced an ultra-bright fluorescent signal that was directly visualized under blue LED light, indicating that fluorescence generation depended on target-triggered activation of the trans-cleavage activity of Cas12a, which enabled nonspecific cleavage of the ssDNA probe. In addition, pronounced levels of cleavage fragments were visible (Figure 3b).

Generally, the Cas12a protein, as a thermolabile enzyme that typically operates at 37 °C, cannot tolerate a high PCR temperature of 95 °C. Some researchers have demonstrated that certain carbohydrates can be used as excipients to protect protein molecules from thermal denaturation. For example, the addition of trehalose was shown to maintain the normal activity of enzymes at high temperatures and may have improved the activity of these enzymes [27,28,29,30]. Therefore, to test the influence of different sugar solutions on the thermal stability of the Cas12a protein at various reaction temperatures, the Cas12a-crRNA complex was incubated in NEBuffer 2.1 for 10 min at temperatures ranging from 37 °C to 70 °C, with trehalose, sucrose, sorbitol, or pullulan, or without a sugar solution (Figure 3c). When incubated at 50 °C, the addition of three sugars had a heat-stabilizing effect on Cas12a, with trehalose and sucrose exhibiting the best performance. However, within the test temperature range (37–70 °C), the fluorescence intensity of Cas12a with pullulan solution was similar to that without sugar addition, indicating that the addition of pullulan solution did not have any marked thermostabilizing effect. This may be because pullulan typically exerts its protective effects in solid states, such as tablet or film forms, to highly restrict the molecular movement of proteins [31,32,33,34]. The thermal stability of this enzyme varied considerably with the addition of sugar solution at 54 °C. Although some sugars effectively improved the thermal stability of Cas12a, all activity was lost after reacting at 54 °C for more than 1 h (Figure S10). Despite this limitation, the addition of these sugar solutions reduced the rate of enzyme thermal inactivation to some extent. To further investigate the thermal protection of the Cas enzyme by trehalose solution, we also optimized the concentration of trehalose solution, as a concentration that was too low would result in insufficient hydrogen bond substitutions (due to water removal), while a concentration that was too high would result in insufficient hydrogen bonding on the protein surface (due to crystallization) [35]. At 37 °C, with increasing trehalose concentrations, the trans-cleavage fluorescent signal tended to be stable. This result confirmed that the addition of trehalose to the assay system did not interfere with the fluorescence signal or cleavage efficiency, ensuring that the enhanced signals observed at higher temperatures were solely due to improved enzyme stability. At 54 °C, with increasing trehalose concentrations, the fluorescent signal was gradually enhanced (Figure 3d). This was consistent with prior reports that trehalose stabilizes enzymes at high temperatures, reducing thermal inactivation [27]. Meanwhile, at a trehalose concentration of 0.6 M, the fluorescent signal was close to that at 37 °C, which was comparable to the maximum concentration documented for yeast cells in the literature [36].

To further mitigate contamination risk, we created a one-pot approach that integrated template amplification with the Cas12a-detection complex reaction. In our evaluation of the PCR-Cas12a-based single-tube assay, we found that trehalose alone was insufficient to preserve Cas enzyme activity, as no fluorescence was visible after amplification. To overcome the problem of temperature incompatibility, we took advantage of the ability of trehalose solution to improve the thermal stability of Cas12a, combined with mineral oil insulation and temperature control of the cap of the PCR instrument, thus preventing the inactivation of the Cas12a protein located within the tube cap. We found that the PCR-Cas12a-based one-pot method was able to produce a bright yellow-green fluorescent signal (Figure 3e). Similarly, we applied this one-pot method to the decontamination and visualization tests of the Cas12a system, confirming that the combination of cross-contamination removal, PCR amplification, and fluorescence visualization in the closed tube was successful (Figure 3f). As shown in Figure 3f, the high fluorescence intensity observed in both Tube 3 (positive) and Tube 4 (negative control under contaminated conditions) highlights the impact of carryover contamination, where residual amplicons act as false-positive. However, after Cas12a-mediated pre-incubation, the false-positive signal (Tube 6) was successfully eliminated, while the true target (Tube 5) remained detectable. This confirms that the fluorescence is strictly template-dependent and that our platform effectively distinguishes true targets from contamination.

The sensitivity of the Cas12a-PCR system was further tested by performing a 10-fold gradient dilution of L. monocytogenes. Compared with the traditional PCR method, this method was able to successfully detect down to 100 copies of L. monocytogenes samples (Figure S11). Importantly, the Cas12a-treated PCR method could identify genomic samples without producing off-target signals, demonstrating its high specificity even in the presence of other pathogenic bacteria, such as Staphylococcus aureus (S. aureus), Salmonella spp., Escherichia coli, and Enterobacter sakazakii (E. sakazakii). As a proof-of-concept, we further evaluated the contaminant cleavage ability of the Cas12a-crRNA assay in actual sample analysis by measuring L. monocytogenes spiked in an artificially contaminated milk sample. The results showed that our proposed method achieved comparable results to the UDG method, which is a widely used decontamination strategy in practice (Figure S12). Therefore, this method has demonstrated its potential for use with complex samples.

Similarly, Cas12a-based methods can also be used in LAMP detection to eliminate cross-contamination (Figure 4a). LAMP, as a convenient constant-temperature method suitable for field testing, requires test templates to be placed in closed tubes, thereby preventing cross-contamination. Because the inner primer in the LAMP reaction contains a TTT spacer, it can bind specifically to Cas12a without any special primer engineering design or modification, adding convenience without affecting specificity or amplification [37]. Because the Mg²⁺ concentration affected both the cleavage efficiency and the LAMP amplification efficiency, a CRISPR/Cas-LAMP assay was performed with 6 μM Mg²⁺ (Figure S13). Significant product degradation was only observed in the presence of contaminants (Figure 4b). Good degradation of residual contaminants stemming from prior amplification was achieved, which effectively avoided target amplification of up to 10⁶ copies of contaminant DNA (Figures S14 and S15). Moreover, the effects of Cas12a-LAMP assay specificity and sensitivity on the accuracy of the results were evaluated. The real-time fluorescence kinetic curve demonstrated that this method could detect as few as 100 copies/μL, and no non-targeted signals appeared even when other pathogenic bacteria were present, proving its robust specificity (Figure S16). Importantly, the target was easily visually identified when the Cas12a-crRNA3 complex was pre-positioned in the tube cap (Figure 4c). These results clearly show that CRISPR/Cas12a can be used to eliminate contamination in LAMP assays and demonstrate the potential of one-pot detection methods.

We have developed a novel one-pot decontamination strategy for PCR and LAMP, utilizing the dual activities of Cas12a. During the initial stage, the system maximizes cis-cleavage to degrade contaminants while minimizing trans-cleavage to prevent nonspecific degradation of single-stranded primers. After amplification, the trans-cleavage activity of the Cas12a-crRNA3 complex, released from the tube cap, is activated to enable sensitive visual detection. This strategy ensures both effective contamination removal and accurate detection. Compared to other methods that combine contamination removal with endpoint detection, our method has significant advantages in the types of contaminant amplicons, detection time, and ability to remove contaminants. Furthermore, it only requires a single Cas12a protein to perform two functions in one pot, namely contaminant removal and visual detection (Table S4) [9,13,14,18,19,20,38,39,40,41]. When the contaminant sequence is known, we can design specific crRNAs with appropriate PAM sites based on its specific DNA. However, a potential limitation of this approach is the reliance on sequencing for unknown sources of contamination before designing corresponding crRNAs and primers [42]. In short, our research has proposed a reliable decontamination strategy, demonstrating that a single Cas12a enzyme is sufficient to achieve contaminant removal and visual detection in a single tube.