What this is
- Phosphorothioate () modifications enhance the CRISPR/Cas system's sensitivity for molecular diagnostics.
- This work presents a Scattered Nucleic Acid-driven Cas (SACA) that allows for exponential amplification.
- SACA demonstrates improved detection capabilities for HPV16 and HPV18 mRNA in cervical cancer cells.
Essence
- The Scattered Nucleic Acid-driven Cas (SACA) achieves significant sensitivity improvements in detecting low-abundance biomarkers, specifically HPV16 and HPV18 mRNA, by enabling exponential amplification without external enzymes.
Key takeaways
- SACA enhances Cas12a and Cas13a sensitivity by 50,000-fold and 10,000-fold, respectively. This improvement allows for the detection of low-abundance nucleic acids, addressing the limitations of conventional methods.
- The tailored modification strategy effectively decouples Cas enzyme activation from substrate trans-cleavage resistance, allowing for robust and precise in situ imaging of cervical cancer markers.
Caveats
- The system's complexity may increase due to the dual-crRNA strategy, which could limit multiplexing capabilities in practical applications.
Definitions
- phosphorothioate (PS) modifications: Chemical alterations to nucleic acids that confer resistance to degradation by nucleases, enhancing stability.
- autocatalytic system: A system in which a product of a reaction catalyzes further reactions, leading to exponential amplification.
Simplified
Introduction
The CRISPR/Cas (Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins) system serves as a gene-editing tool, demonstrating transformative potential in molecular diagnostics [1, 2]. Among its variants, CRISPR/Cas12a and CRISPR/Cas13a are particularly notable for their ability to perform both cis-cleavage and trans-cleavage activities. This dual catalytic capacity, combined with high programmability, enables homogeneous detection and facilitates in situ analysis [3–7]. However, conventional CRISPR/Cas diagnostic platforms are inherently constrained by a “one-target, one-activation” turnover mechanism. This linear signal amplification restricts sensitivity to the picomolar (pM) range, which is often insufficient for the trace analysis of low-abundance biomarkers [8–10]. To surmount this limitation, prevalent approaches often rely on coupling CRISPR/Cas with external enzymatic amplification methods [11–13]. Such integrated approaches, however, increase system complexity and introduce challenges such as poor compatibility and target competition [14, 15], restricting their applicability in complex biological matrices, especially within the intracellular environment. Thus, there is a compelling need to engineer intrinsic CRISPR/Cas systems capable of “one-target, numerous-activation” exponential amplification to meet the demands of sensitive and reliable in-cell analysis.
Engineering autocatalytic feedback loops mediated by nucleic acid substrates represents a promising strategy for achieving CRISPR/Cas exponential amplification [16]. This is typically realized by designing a multifunctional DNA probe as both an activator for the Cas enzyme and a substrate for trans-cleavage [17, 18]. Upon trans-cleavage, the probe releases the activator, which in turn activates other Cas enzymes, creating a self-sustaining, substrate-driven signal amplification cascade. To this end, current strategies often employ engineered DNA secondary structures to physically mask the activator, shielding it from both nonspecific activation and accidental trans-cleavage by Cas enzymes. For instance, Goldys E. et al. developed circular DNA probes that use steric hindrance to block double-stranded DNA (dsDNA) activator access, achieving attomolar (aM)-level sensitivity [19, 20, 21]. However, the synthetic complexity and low cyclization efficiency of such probes present significant challenges. Alternatively, hairpin DNA probes have been explored to encapsulate the single-stranded DNA (ssDNA) activator [22–26]. A key limitation emerges after ssDNA activator release: the free activator can be scavenged by Cas12a trans-cleavage. Moreover, such structurally complex probes are often prone to thermodynamic instability in physiological environments. Therefore, it is imperative to design simpler, more robust substrates that ensure stable and efficient Cas exponential amplification.
Phosphorothioate nucleic acids (PS DNA/RNA) are chemically modified molecules characterized by their strong resistance to nuclease degradation, rendering them highly durable tools for biological applications [27]. In this work, we revealed that fully PS-modified activators can recruit Cas12a into a “closed” (inactive) conformation by hydrophobic anchoring. By adjusting the PS modification sites, we found the “scattered” modification pattern attenuates this hydrophobic effect, effectively decoupling enzymatic activation from nuclease resistance and ultimately restoring the high activity of Cas12a. Inspired by this finding, we engineered a coordinator using “scattered” PS activators, achieving both site-directed control of Cas12a trans-cleavage and enhanced overall activation capacity. Building on this design, we successfully constructed a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). Remarkably, SACA achieved an ultralow limit of detection (LOD): 500 aM for the Cas12a-based assay and 5 fM for the Cas13a-mediated assay, significantly surpassing conventional methods. Compared to complex secondary structures, the PS-modified probe efficiently masks activators while maintaining a single-stranded linear state, featuring simpler design, better biostability, and stronger anti-interference. Moreover, SACA exhibits excellent compatibility for biological applications, enabling efficient multiplex in situ detection of HPV16 and HPV18 in cervical cancer cells. This discovery pioneers the application of PS nucleic acid probes in Cas enzyme autocatalytic amplification and advances precise in situ analysis in complex cellular environments.
Materials and methods
Reagents and materials
All oligonucleotide sequences used in this study were synthesized by Sangon Biotechnology Co. Ltd. (Shanghai, China) and are listed in. LbCas12a Nuclease (product #M0653T), 10x NEBuffer r2.1 (10x Buffer 1) were obtained from New England Biolabs (Ipswich, MA, USA). LwaCas13a (Cat. No. E381) and 10 × reaction buffer (10x Buffer 2) were purchased from Novoprotein (China). Recombinant DNase I, S1 nuclease, RNase inhibitor, and PrimeScript™ FAST RT reagent Kit were purchased from Takara Biomedical Technology Co. Ltd. (Beijing, China). Gibco Trypsin-EDTA (0.25%), DiI Cell Marked Solution, and Invitrogen™ Platinum™ Taq DNA Polymerase were purchased from Thermo Fisher Scientific (China). Minimum Essential Medium (MEM) and Dulbecco’s modified Eagle’s medium (DMEM) were purchased from Procell system (Wuhan, China). Fetal bovine serum (FBS) was purchased from ExCell (Australia). 20x phosphate-buffered saline was obtained from Sangon Biotechnology Co. Ltd. (Shanghai, China). Triton X-100 was obtained from Biosharp Life Science (Beijing, China). 4′, 6-Diamidino-2-phenylindole (DAPI) was purchased from Beyotime Institute of Biotechnology (Shanghai, China). RNA-Quick Purification Kit was purchased from Shanghai Yishan Biotechnology Co. Ltd. (Shanghai, China). All chemical reagents were of analytical grade and used directly without further purification. Supplementary Table S4
Regulation of Cas12a activity by PS activator
Inhibition assay of PS DNA on Cas12a activation
The reaction system consisted of 10 nM Cas12a/S-crRNA, 500 nM D-FQ, 10 nM various PS DNA, 0.8 U/μl RNase inhibitor, and 1 × Buffer 1 in a total volume of 100 μl. The mixture was incubated at 37°C for 60 min.
Tolerance assay of PS DNA to Cas12a trans-cleavage
The reaction system consisted of 1 nM pre-activated Cas12a/crRNA/target complex, varying concentrations of PS DNA, and 1 × Buffer 1 in a total volume of 20 μL. The mixture was incubated at 37°C for 60 min.
The development of SACA system
Cas12a-based SACA system
The total reaction volume was 20 μl. First, 25 nM Cas12a/crRNA, 1nM Target (HPV16 DNA), 80 nM ST10-10, 0.8 U/μl RNase inhibitor, and 1x Buffer 1 were incubated at 37°C for 10 min. Then, 80 nM Cas12a/S-crRNA and 2.5 μM D-FQ were added and incubated at 37°C for 40 min.
Cas13a-based SACA system
The procedure was similar to that of the Cas12a-based SACA system. First, 25 nM Cas13a/’crRNA, 1nM ‘Target (HPV16 RNA), 40 nM ’ST14-14, 0.8 U/μl RNase inhibitor and 1x Buffer 2 were incubated at 37°C for 10 min. Then, 40 nM Cas13a/’S-crRNA and 2.5 μM R-FQ were added and incubated at 37°C for 40 min.
Cas12a/Cas13a-based multiplex SACA system
The procedure is identical to that of single-system detection; simply mix and react the components of both systems in 1x Buffer 1.
Polyacrylamide gel electrophoresis (PAGE) analysis
Reaction products (10 μl) were analyzed by 12% native PAGE using a BioRad electrophoresis analyzer (Bio-Rad, USA) at a constant voltage of 110 V for 40 min. After staining with GelRed for 20 min, the gel was imaged using a gel imaging system (Bio-Rad Laboratories, USA).
Fluorescence intensity measurement
Fluorescence intensity was measured by the FS5 fluorescence spectrophotometer (Edinburgh Instruments, UK). Excitation/emission wavelengths were set to 490/517 nm for FAM and 630/664 nm for Cy5, with slit widths of 1 nm (excitation) and 1.5 nm (emission). A 7500 Real-Time PCR System (Thermo Fisher Scientific, China) was used for real-time fluorescence signal collection.
Cell culture and pretreatment
Human cervical carcinoma cell lines Siha (product #CL-0210), Hela (product #CL-0101), and C33A (product #CL-0045) were purchased from Procell Life Science & Technology Co. Ltd. (Wuhan, China). They were cultured in designated media at 37°C in a humidified atmosphere with 5% CO₂. The cells were grown on 14-mm glass slides placed in 24-well plates until reaching 40–50% density. For subsequent processing, the cells were fixed at room temperature using a methanol: acetic acid solution (3:1) for 20 min, washed with 1 × PBS at 37°C for 5 min, and permeabilized with 0.5% Triton X-100 in 1 × PBS at 37 °C for 5 min.
RT-PCR analysis of HPV 16/18 mRNA in cervical cancer cells
Cellular total RNA extraction
Total RNA was extracted from 1 × 106 cells (Siha and Hela, respectively) using the RNA-Quick Purification Kit according to the manufacturer’s protocol, and the concentration was determined by NanoDrop One (Thermo Fisher Scientific, USA).
RT-PCR analysis of HPV16 mRNA in Siha cells and HPV18 mRNA in Hela cells
According to the protocol of the PrimeScript™ FAST RT reagent Kit, 1 μg of total RNA extract was first treated with gDNA Eraser to remove potentially contaminated DNA, and then reverse-transcribed into cDNA. The resulting cDNA was diluted 1000-fold and used for subsequent PCR amplification. The PCR system was performed on QuantStudio (Thermo Fisher Scientific, USA). Especially, the β2M as the RNA reference gene and the Globin as the DNA reference gene.
In situ imaging of HPV 16/18 mRNA in fixed cervical cancer cells
HPV 16 mRNA or HPV 18 mRNA imaging
For HPV 18 mRNA imaging, the prepared cells were incubated with a 20 μl mixture containing 100 nM I1-1/B1-1, 100 nM Cas12a/crRNA1, 200 nM ST10-10, 0.8 U/μl RNase inhibitor, and 1x Buffer 1 at 37 °C for 30 min. After washing, the cells were incubated with another 20 μl system (200 nM Cas12a/S-crRNA, 2.5 μM D-Cy5, 0.8 U/μl RNase inhibitor, and 1x Buffer 1) at 37 °C for 60 min. HPV 16 mRNA imaging was similarly processed; the first mixture includes 250 nM Cas13a/’crRNA, 120 nM ’ST14-14, 0.8 U/μl RNase inhibitor, and 1x Buffer 2. The other 20 μL system includes 120 nM Cas13a/’S-crRNA, 2.5 μM R-FQ, 0.8 U/μl RNase inhibitor, and 1x Buffer 2. Finally, the cells were stained with DAPI and imaged using fluorescence microscopy (Leica, Germany).
HPV 16/18 mRNA co-imaging
Hela cells were pre-stained with DiI Cell Marked Solution. Then Hela cells and Siha cells were digested, fixed with 4% paraformaldehyde, and resuspended to a concentration of 1 × 106 cells/mL, respectively. A 10 μl mixture of cell suspension was spread on slides, fixed at 60°C for 30 min, permeabilized with 0.5% Triton X-100 for 5 min, washed three times with preheated 1 × PBS, and air-dried. And then a 10 μl reaction system (500 nM Cas12a/crRNA1, 500 nM Cas13a/’crRNA, 100 nM In1-1/Bl1-1, 100 nM ST10-10, 100 nM ‘ST14-14, 0.8 U/μl RNase inhibitor, and 1x Buffer 1) was added, and incubated at 37°C for 30 min. After that, another reaction solution including 500 nM Cas12a/S-crRNA, 500 nM Cas13a/’S-crRNA, 2.5 μM D-Cy5, 2.5 μM R-FQ, 0.8 U/μl RNase inhibitor, and 1x Buffer 1 was also incubated at 37°C for 60 min. Finally, the cells were stained with DAPI and imaged.
In situ imaging of HPV 16/18 mRNA in cervical cancer FFPE sample
The FFPE tissue sections of human cervical cancer used in this study were provided by the Department of Pathology of the First Affiliated Hospital of Chongqing Medical University. The study protocol was approved by the hospital’s Ethics Committee (Approval No. 025–653-01). The sections were first baked at 69°C for 30 min, followed by dewaxing in fresh xylene and rehydration through a graded ethanol series. Antigen retrieval was performed in ultrapure water at 95°C for 15–20 min. Subsequently, the sections were digested with pepsin solution at 37°C for 20–25 min in a hybridizer (SH2000, China). After digestion, the sections were rinsed with 2 × SSC buffer at room temperature for 5 min, dehydrated through an ethanol series (75%, 85%, and 100%), and air-dried. Next, the sections were permeabilized with 5% Triton X-100 at 37°C for 5 min and then washed three times with 1 × PBS. Following complete air-drying, the aforementioned HPV 16/18 mRNA co-imaging method was applied for subtype identification.
Metadynamics simulation
Analysis of the conformation of Cas12a
Simulations were performed using GROMACS 2023.5 [28] with PLUMED 2.9.4 [29]. Both the Cas12a/crRNA/TDNA and Cas12a/crRNA/SDNA systems were solvated to mimic physiological conditions (150 mM NaCl, 10 mM MgCl₂, pH 7.5). Each system underwent identical pre-equilibration: energy minimization to resolve steric clashes, followed by 1 ns NVT and 2 ns NPT equilibration to stabilize temperature and pressure, respectively. A 100 ns NPT production run was conducted. Metadynamics simulations were performed to construct the conformational free energy landscape. Two collective variables (CVs) were defined: CV1 measured the distance between residues E925 and R386, CV2 measured the distance between residue D832 and nucleotide C41 of the crRNA. Gaussian hills (sigma = 0.15 nm, initial height = 1.0 kJ/mol) were deposited at regular intervals. Temperature was maintained using a velocity-rescaling thermostat (time constant = 0.1 ps) [30]. Pressure was controlled using Parrinello-Rahman coupling (pressure time constant = 2 ps, isothermal compressibility = 4.5 × 10⁻⁵ bar⁻¹) [31]. A 2 fs time step was used. Nonbonded interactions had a 10 Å cutoff; long-range electrostatics were handled with Particle Mesh Ewald [32]. Binding free energies for the open and closed conformations were calculated using the MM/GBSA method with gmx_MMPBSA [33].
Analysis of the channel geometry of Cas12a
First, all non-protein components (RNA and DNA) were removed from the original structures to generate protein-only models. A custom computational pipeline implemented in Python was used to characterize the channel geometry. For each structure, we calculated three key metrics: (1) channel volume, determined by calculating the convex hull of the complete protein structure; (2) minimum bottleneck diameter, identified by sampling cross-sectional areas along the channel axis; and (3) three-dimensional channel dimensions (length, width, height), derived by performing principal component analysis (PCA) on the atomic coordinates within the channel.
Results
Mechanism of PS-modified activator in suppressing Cas12a activity
To systematically evaluate the impact of phosphorothioate (PS) modifications on Cas12a kinetics, we synthesized a fully PS-modified 20-nt DNA activator (S20) (Fig. 1A). As expected, this modification conferred complete resistance to Cas12a-mediated trans-cleavage (Fig. 1C, Supplementary Fig. S1). However, this stability came at the cost of a significant reduction in activation efficiency to only 8% (Fig. 1B, Supplementary Fig. S2), thereby halting the Cas12a self-feedback loop. To resolve this trade-off and enable efficient PS DNA-driven exponential amplification, we sought to elucidate the precise mechanism of inhibition.
Given that sulfur substitution reduces the overall charge and increases the hydrophobicity of the DNA backbone [34], we investigated three potential inhibitory factors: complex assembly, suppression of cis-cleavage, and modulation of Cas12a conformation.
First, we assessed the binding integrity. Melting curve analysis showed that while the melting temperature (Tm) of the S20/S-crRNA complex decreased slightly, it remained robustly above 65 °C (Fig. 1D), a finding further confirmed by PAGE analysis (Supplementary Fig. S3). We next verified whether S20 could form a ternary complex with Cas12a/S-crRNA. As shown in Fig. 1E, S20 produced a distinct complex band (lane 4) comparable to the unmodified control T20 (lane 5), confirming successful assembly. Notably, prior studies suggest that PS-modified non-activators can non-specifically adsorb to the Cas12a activator-binding pocket via hydrophobic interactions [35]. To test if S20 binding is similarly driven, we incubated S20 with a non-matching Cas12a/T-crRNA complex. S20 exhibited a higher binding affinity for Cas12a than the non-matching T-crRNA (Supplementary Fig. S4) and disrupted the binary complex (Fig. 1F, lane 5). These data suggest a stepwise binding mechanism: PS activator is initially recruited to the binding site via non-specific hydrophobic interactions, followed by electrostatic stabilization upon crRNA hybridization.
Next, we investigated whether the inhibition resulted from blocked cis-cleavage, a process sensitive to PS modifications [36]. We designed two variants with partial PS modifications positioned either to cover (S5’-8) or distal to (S3’-8) the presumed cleavage site (Fig. 1G). Surprisingly, S5’-8 retained 75% activation efficiency, outperforming S3’-8 (Fig. 1H). This result indicates that blockage of the cis-cleavage site alone does not account for the profound suppression observed with S20.
Finally, we employed metadynamics (MD) simulations to uncover the structural basis of inhibition. We compared the conformational dynamics of the Cas12a/crRNA complex bound to either S20 (SDNA system) or T20 (TDNA system). The SDNA system displayed a restricted range of motion, failing to fully access the catalytically essential “open” conformation (Supplementary Fig. S5). Structurally, the distance between the Cα atoms of GLU354 and LYS1080 was significantly shorter in the SDNA system (21.2 Å) compared to the TDNA system (28.1 Å) (Fig. 1I), effectively constricting the catalytic pocket and limiting substrate accessibility. While the TDNA complex exhibited some instability in the closed state, the SDNA complex adopted a rigid closed conformation, stabilized by a specific hydrogen bond between R1076 and D367 (Fig. 1J). Binding free energy calculations revealed the thermodynamic basis for this “locking” effect: the SDNA system exhibits a strong energetic bias toward the closed conformation due to significantly enhanced protein-crRNA interactions (−427.79 kcal/mol versus −268.84 kcal/mol for TDNA). These simulation calculations were carried out by GROMACS 2023.5.
In summary, the PS-activator stably binds to the Cas12a/crRNA complex but acts as a molecular anchor driven by hydrophobic interactions. This binding stabilizes the closed conformation, altering the energy landscape and trapping the Cas12a enzyme in an inactive state, thereby preventing the transition required for trans-cleavage.
Mechanism analysis of Cas12a inhibition by PS activator. () Schematic representation of Cas12a inhibition by S20, and resistance to Cas12a trans-cleavage. () Fluorescence intensity of Cas12a activation assay with S20 and T20. T20 is a control sequence without PS modification. () PAGE assay of S20 and T20 after incubation with activated Cas12a (aCas12a). () The melting curve analysis of the S20–crRNA complex and T20–crRNA complex. () PAGE assay to evaluate the binding of S20 and T20 with the Cas12a/S-crRNA complex, respectively. The concentration of each reactant was 1 μM, and the 20 μl mixture was incubated at 37°C for 20 min. () PAGE assay to evaluate the binding of S20 and T20 with the Cas12a/T-crRNA complex, respectively. The concentration of each reactant was 1 μM, and the 20 μl mixture was incubated at 37°C for 20 min. () Schematic representation of S23 and its variants with partial PS modifications near or distal tocleavage sites. Thecleavage site for LbaCas12a is located between nucleotides 21 and 22 on the Target strand (TS), counting from the PAM-distal end. () The activation efficiency of the above S23 variants toward Cas12a. The reaction system consisted of 10 nM Cas12a/S-crRNA, 10 nM S23 variants, and 1x Buffer 1, and was incubated at 37°C for 60 min. () Quantification of the open conformation of SDNA complex and TDNA complex. Marking line showing the distance between the Cα atoms of residues GLU354 and LYS1080. () Close-up view of the hydrogen bond stabilizing the closed catalytic pocket in the SDNA complex. An enlarged view of the hydrogen bonding region is presented in the orange box. Data represent mean ± s.d. of three technical replicates. FI: Fluorescence Intensity. A B C D E F G H I J cis- cis-
Tailored PS activator can regulate Cas12a activity
To overcome the inhibition caused by the fully modified PS-activator and enable autocatalysis, we investigated the impact of PS modification sites on Cas12a activity (Fig. 2A). We first evaluated a “clustered” design, starting with the fully modified S20 and progressively inserting central unmodified spacers (S20-n, n: unmodified spacers) (Fig. 2B). As the unmodified spacer length increased, Cas12a activity was progressively restored, exceeding 90% when 10 unmodified bonds were present. This correlated with a reduction in nonspecific binding to Cas12a (Supplementary Fig. S6), confirming that alleviating the hydrophobic effect mitigates inhibition. However, this strategy severely compromised nuclease resistance: cleavage exceeding 50% occurred with just 4 unmodified bonds (S20-4) and approached completion with 12 bonds (S20-12) (Fig. 2C). Thus, the “clustered” design presents an unacceptable trade-off between activity and stability, rendering it unsuitable for autocatalytic systems.
To harmonize the high activation efficiency with robust resistance, we devised a “scattered” PS modification strategy (SmTn, m: consecutive modified bonds. N: unmodified spacers) (Fig. 2D). Fixing n = 1, reducing the size of the PS blocks (m) progressively improved Cas12a activity and kinetics (Fig. 2D, Supplementary Fig. S7). Concurrently, the Tm of the activator-crRNA duplex increased (Supplementary Fig. S8), while nonspecific adsorption to Cas12a decreased (Supplementary Fig. S9). Notably, at m = 1 (S1T1), activity fully recovered to unmodified levels, indicating that the weakened hydrophobic effects no longer impede Cas12a’s transition to the catalytically active “open” conformation. Further increasing the spacer length (n, with m = 1) restored the Tm fully to control levels (Supplementary Fig. S10). Interestingly, several scattered variants exceeded 100% activation efficiency, with S1T4 reaching 111% (Fig. 2D, Supplementary Fig. S11). PAGE analysis confirmed weak residual Cas12a adsorption for these variants (Supplementary Fig. S9). To elucidate the mechanism by which scattered PS modification enhances Cas12a activity, we performed MD simulations on the most effective S1T4 variant (S1DNA). The results show that the S1DNA system adopts a distinct open conformation (Fig. 2E), while in the closed state, the constraining hydrogen bond between R1076 and D367 is lost (Fig. 2F), indicating structural relaxation toward the unmodified system. Although the distance between the Cα atoms of GLU354 and LYS1080 (25.3 Å) is slightly smaller than that in the TDNA system (28.1 Å), the catalytic pocket volume is significantly increased. Geometric analysis indicates that S1DNA achieves the highest conformational score (Fig. 2G). Therefore, scattered PS modifications likely enhance Cas12a activation by enlarging the catalytic pocket, thereby facilitating substrate access. Furthermore, the broad applicability of the scattered PS strategy was demonstrated by its extension to non-activator sequences, which caused minimal inhibition when m = 1 and n ≥ 2 (Supplementary Fig. S12).
We next assessed the trans-cleavage resistance of scattered PS activators using probes S1T1–S1T5, which do not inhibit Cas12a. Resistance remained above 90% when the unmodified segment length n ≤ 2, but dropped sharply when n ≥ 3 (Fig. 2H). This is because Cas12a’s intrinsic preference is for cleaving ssDNA into 2–4 nt fragments. Once ≥3 consecutive bonds are unmodified, PS-mediated protection weakens, making the sequence more susceptible to Cas12a trans-cleavage. To validate this mechanism, we employed two nucleases with distinct cleavage preferences: S1 nuclease, which targets single-bond sites, and DNase I, which prefers segments ≥2 nt and resembles Cas12a in its cutting behavior [37–39]. While S1 nuclease efficiently cleaved all activators (>80% cleavage even for S1T1; Supplementary Fig. S13), DNase I exhibited a cleavage profile similar to Cas12a, with strong resistance observed only for S1T1 and S1T2 (Supplementary Fig. S14). These findings confirm that when unmodified stretches are ≤2 nt, scattered PS modifications can create a “partitioning protection effect” that effectively shields the activator backbone from Cas12a trans-cleavage.
The regulation of tailored PS activators on Cas12a activity. () Schematic diagram of the activation and trans-cleavage tolerance of Cas12a by clustered and scattered PS activators. () Fluorescence assay of Cas12a activation efficiency by various clustered PS activators. () PAGE assay of various clustered PS activators after incubation with aCas12a. () Fluorescence assay of Cas12a activation efficiency by various scattered PS activators. () Quantification of the open conformation of the S1DNA complex. Marking line showing the distance between the Cα atoms of residues GLU354 and LYS1080. () The close-up conformation of the S1DNA complex. () The channel geometry analysis of three protein variants (TDNA, S1DNA, and SDNA). () PAGE assay of various scattered PS activators after incubation with aCas12a. Activation Efficiency (%)= FI/FI*100. Data represent mean ± s.d. of three technical replicates. Data represent mean ± s.d. of three technical replicates. FI: Fluorescence Intensity. A B C D E F G H (SmTn) (T20)
SACA-based Cas12a exponential amplification
Based on our observation that Cas12a efficiently cleaves spacers containing ≥3 unmodified bonds between PS modifications (Fig. 2H), we designed a bifunctional “coordinator” probe, termed STx-y (Fig. 3A). This probe was engineered to orchestrate a CRISPR/Cas autocatalytic reaction by functioning simultaneously as a trans-cleavage substrate and an activator. Specifically, we inserted an unmodified random sequence (the “z region”) into a PS-modified activator, effectively splitting it into two segments, x and y. The Z region serves a dual purpose: as a designated cleavage site for activated Cas12a, and as a steric blocker that, according to previous reports, prevents non-specific activation by impairing the thermodynamic binding with crRNA [40, 41]. Upon cleavage, the released x and y fragments (STx and STy) cooperatively bind to and activate other Cas12a enzymes. Using this design alongside a reporter probe, we established a Scattered PS Nucleic Acid-driven Cas Autocatalytic system (SACA). As illustrated in Fig. 3B, the cycle begins when target-activated Cas12a cleaves the z region of STx-y. The liberated split activators then trigger the nearby Cas12a enzyme, establishing an exponential amplification network. Concurrently, the system cleaves a FAM-BHQ1 reporter to generate a fluorescent signal.
We first validated the design using PAGE analysis, which confirmed the site-specific cleavage of STx-y (Fig. 3C). The disappearance of the full-length band and the emergence of two lower-molecular-weight bands indicated the successful generation of split activators. Consistently, fluorescence assays verified that cleavage was confined to the Z region (Supplementary Fig. S15) and that the cleavage products activated Cas12a as effectively as the synthetic STx and STy controls (Fig. 3D). When integrated into a target-activated Cas12a reaction, the addition of STx-y significantly boosted the fluorescence signal (Fig. 3E), confirming its ability to drive exponential amplification. Notably, the cleavage products exhibited even stronger co-activation than unmodified Tx and Ty (Supplementary Fig. S16A), analogous to the enhancement seen with full-length scattered PS activators (Fig. 2D). This enhancement was more pronounced (123% vs. 111%, Supplementary Fig. S16B), likely due to the intrinsically weaker initial co-activation of the short (10 nt), low-Tm split activators. To probe the underlying mechanism, we tested a control probe (LTx-y) containing locked nucleic acid (LNA) modifications. Although LTx-y was cleaved specifically, its products failed to activate Cas12a (Supplementary Fig. S17). We attribute this to the increased negative charge of LNAs, which likely weakens the hydrophobic interactions necessary for activation. This finding strongly supports our hypothesis that scattered PS modifications enhance Cas12a activity primarily through hydrophobic effects.
To maximize the efficiency of the autocatalytic reaction, we systematically optimized the STx-y probe. We began with the structure of the z region. Contrary to reports suggesting that Cas12a prefers hairpin substrates, hairpin-structured z regions failed to support amplification in our system, likely due to inhibitory overhangs generated post-cleavage (stem lengths: 1–3 nt; Supplementary Fig. S18). Consequently, a linear design was adopted. We then optimized the z region length. A 5-nt length proved optimal, balancing signal strength against background noise (Fig. 3F; Supplementary Fig. S19), with the sequence TTATT yielding the highest performance (Supplementary Fig. S20). Next, we adjusted the lengths of the x and y segments (Fig. 3G). A symmetric design (10 nt each) with a central z region provided the lowest background and highest amplification (Fig. 3H; Supplementary Fig. S21), likely by ensuring thermodynamically balanced cooperative binding. Finally, we compared the probe orientation: forward (STx-y) versus reverse (STy-x). Both STx-y and STy-x were designed based on the same principle that a redundant structure (the Z region for STx-y, and the flanking sequences for STy-x) could introduce steric hindrance. The forward configuration exhibited significantly lower background noise (Fig. 3I). NUPACK simulations suggested that the reverse design (STy-x) tends to form higher-order complexes with S-crRNA, leading to non-specific activation (Supplementary Fig. S22). Thus, the optimized probe (ST10-10) features a linear 5-nt TTATT z region flanked by 10-nt x and y segments in a forward configuration.
We further optimized the concentration of ST10-10 (Supplementary Fig. S23), its ratio to S-crRNA (Supplementary Fig. S24), and the cleavage time (Supplementary Fig. S25, Supplementary Fig. S26) in the SACA system. Optimal amplification was achieved under the following conditions: ST10-10 concentration of 80 nM, a 1:1 ratio with S-crRNA, and a two-step cleavage with a total duration of 50 min (10 min + 40 min). Under these optimized conditions, we evaluated the SACA system using HPV16 DNA as a model target. The system achieved an ultralow limit of detection (LOD) at 500 aM (Fig. 3J), representing a 50 000-fold improvement over the traditional Cas12a detection system, demonstrating its potential for detecting low-abundance nucleic acid biomarkers. In addition, it also demonstrated good specificity in detecting HPV16 DNA from different HPV subtypes (Supplementary Fig. S27). To enhance its practicality for point-of-care diagnostics, we systematically compared the performance of one-step and two-step formats. The results demonstrate that the one-step method can also achieve efficient exponential amplification with a detection sensitivity of 1 fM. However, given the requirement for ultra-high sensitivity in detecting low-abundance biomarkers, the subsequent cellular imaging experiments were conducted using the two-step format (Supplementary Figs S28 and29).
The development of the SACA-based Cas12a exponential amplification strategy. () Schematic representation of the design of the STx-y probe, and the effect on Cas12a activation. () Schematic representation of the SACA-based Cas12a exponential amplification strategy. () PAGE assay of STx-y after incubation with aCas12a. () Fluorescence spectrum of the Cas12a activation assay using uncleaved STx-y and cleaved STx-y. The control is independently synthesized STx/STy probes (ST/ST). () Fluorescence spectrum of Cas12a trans-cleavage assay using SACA system (STx-y + Target) and conventional Cas12a system (Target). () Fluorescence intensity of the SACA system using various STx-y probes with varying lengths of the z region. () Diagram illustrating the design of STx-y probes featuring varying lengths of the STx/STy region. () Fluorescence intensity of the SACA system using various STx-y probes with varying lengths of the STx/STy region. () Fluorescence intensity of the SACA system using forward (STx-y) and reverse (STy-x) conformational probe. () Comparison of HPV16 DNA detection by the SACA-based Cas12a assay and conventional Cas12a assay. The ΔFI (FI– FI) of the SACA assay and conventional Cas12a for various concentrations of HPV16 DNA. Data represent mean ± s.d. of three technical replicates. A B C D E F G H I J 10-ΔTT -10-ΔATT HPV16 DNA Control
SACA-based Cas13a exponential amplification
Given that phosphorothioate (PS) modification confers nuclease resistance to RNA as well as DNA [42], we hypothesized that the scattered PS strategy could be adapted to modulate Cas13a activity (Fig. 4A and B). Consistent with our findings for Cas12a, a fully PS-modified RNA activator (‘S28) strongly inhibited Cas13a, whereas an activator with scattered PS modifications (‘S1T2) restored activation efficiency to levels comparable to unmodified RNA (‘T28) (Fig. 4D). Crucially, ‘S1T2 exhibited substantial resistance (∼90%) to Cas13a trans-cleavage (Fig. 4E, Supplementary Fig. S30). Leveraging the design principles established for the Cas12a probe (ST10-10), we engineered a Cas13a-specific coordinator, termed ’ST14-14, to establish an SACA-based Cas13a exponential amplification platform. As illustrated in Fig. 4C, activated Cas13a (aCas13a) cleaves the z region of ‘ST14-14, liberating the split activators (‘ST14- and ‘ST-14). These fragments synergistically trigger other Cas13a enzymes, creating a catalytic network that concurrently cleaves FAM-BHQ1 reporter probes to generate a fluorescent readout. Proof-of-concept fluorescence assays confirmed that the ‘ST14-14-mediated system produced significantly enhanced signals (Fig. 4F), demonstrating its robust capacity for autocatalytic amplification. Following systematic optimization of reaction parameters, including probe concentration (Supplementary Fig. S31), probe-to-’crRNA ratio (Supplementary Fig. S32), and cleavage time (Supplementary Fig. S33, Supplementary Fig. S34), we identified optimal conditions, including 40 nM ‘ST14-14, a 1:1 ratio with ‘S-crRNA, and a two-step cleavage with a total duration of 50 min (10 min + 40 min). Under these conditions, the system achieved a 10 000-fold improvement in sensitivity for HPV16 RNA detection compared to conventional Cas13a assays (Fig. 4G), and also showed good specificity in detecting HPV 16 RNA from different HPV subtypes (Supplementary Fig. S35), enabling the reliable quantification of low-abundance RNA targets.
The development of the SACA-based Cas13a exponential amplification strategy. () Schematic representation of Cas13a inhibition by ‘S28. () Schematic diagram of the activation and trans-cleavage tolerance of Cas13a by ‘S1T2. () Schematic representation of the SACA-based Cas13a exponential amplification strategy. () Fluorescence intensity of Cas13a activation assay with ‘S28, ‘S1T2 and ‘T28. () PAGE assay of ‘S1T2 after incubation with aCas13a. () Fluorescence spectrum of Cas13a trans-cleavage assay using the SACA-based Cas13a assay (‘ST+ Target) and the conventional Cas13a system (Target). () Comparison of HPV16 RNA detection by the SACA-based Cas13a assay and the conventional Cas13a assay. The ΔFI (FI- FI) of the SACA-based Cas13a assay and conventional Cas12a for various concentrations of HPV16 RNA. Data represent mean ± s.d. of three technical replicates. A B C D E F G 14-14 HPV16 RNA Control
SACA-based multiplex in fixed-cell situ imaging analysis
We further evaluated the utility of SACA for multiplex in fixed-cell situ imaging, a technique that provides critical spatiotemporal information by directly visualizing pathogens within infected cells [43]. As high-risk HPV types 16 and 18 are the etiological agents for ∼70% of cervical cancers [44], we selected HPV16 and HPV18 mRNA as targets. mRNA detection was chosen over DNA as it better reflects the transcriptional activity of oncogenes [45, 46]. To achieve multiplexing, we overcame a key limitation: Cas12a recognizes DNA targets, whereas our analytes were mRNA. We therefore employed a Target-Replacement Component (TRC) strategy to convert HPV18 mRNA into a DNA activator for Cas12a. Specifically, hybridization of HPV18 mRNA with the TRC releases a specific DNA strand that activates Cas12a. After screening multiple designs (Supplementary Fig. S36) and optimizing the replacement time (Supplementary Fig. S37), we successfully integrated this module. Conversely, HPV16 mRNA was detected directly using the SACA-Cas13a system [47].
Feasibility was first assessed in a homogeneous reaction (Fig. 5A), using orthogonal reporters. The SACA-based Cas12a system employed a Cy5-BHQ3-labeled DNA (D-Cy5), whereas the SACA-based Cas13a system utilized a FAM-BHQ1-labeled RNA (R-FAM). Buffer compatibility was a prerequisite for simultaneous detection [48, 49]. We evaluated three buffer systems, including 10 × Buffer 1 (Cas12a companion buffer: 500 mM NaCl, 100 mM Tris–HCl, 100 mM MgCl₂, 1 mg/mL recombinant albumin, pH 7.9), 10 × Buffer 2 (Cas13a companion buffer: 400 mM Tris–HCl, 600 mM NaCl, 60 mM MgCl₂, pH 7.3), and 10 × Buffer 3 (in-house buffer: 50 mM MgCl₂, 200 mM KCl, 400 mM Tris–HCl, 10 mM DTT, 1000 µg/mL BSA, pH 8.8) [50]. We found that while both enzymes were active in 1 × Buffer 1 and 1 × Buffer 3 (Fig. 5B),1 × Buffer 3 induced nonspecific probe cleavage by Cas13a (Supplementary Fig. S38). In contrast, 1 × Buffer 1 (high-salt buffer) maintained high specificity and was selected for all multiplex experiments (Fig. 5C). The integrated system demonstrated specific cascade amplification in both single-target and dual-target assays (Fig. 5D). When combined with TRC, it also performed effectively in the multiplex analysis of HPV18 mRNA and HPV16 mRNA (Fig. 5E), further confirming its utility for detecting multiple nucleic acid targets.
Then, we validated the system in fixed-cell lines (Fig. 6A). The SACA-Cas12a system (targeting HPV18) generated distinct red fluorescence exclusively in the cytoplasm of HPV18-positive Hela cells (Fig. 6B, Supplementary Fig. S39), while the SACA-Cas13a system (targeting HPV16) produced clear green fluorescence only in HPV16-positive SiHa cells (Fig. 6C, Supplementary Fig. S40). To validate the accuracy of the cell type-specific fluorescence, we performed RT-PCR analysis, which confirmed high expression of HPV16 mRNA in Siha cells and HPV18 mRNA in Hela cells, respectively (Supplementary Fig. S41, Supplementary Fig. S42). In addition, no signal was observed in negative controls. Notably, the SACA-based method yielded fluorescence signals approximately two-fold brighter than conventional Cas12a/Cas13a methods (Fig. 6D and E), significantly enhancing the visualization of positive cells. Furthermore, the combined multiplex system efficiently distinguished between HPV16- and HPV18-infected cells within mixed populations (Fig. 6F and G), highlighting the potential of SACA for precise, high-contrast multiplex in situ diagnosis.
Encouraged by the prominent ability of SACA to image intracellular mRNA, the developed multiplex SACA strategy was further extended to detect HPV16/18 mRNA in 20 clinical cervical cancer FFPE samples. The samples were categorized based on clinical pathological diagnosis: 8 samples showed high expression of p16 and Ki-67 (both are key indicators in cervical cancer pathology, with p16 expression directly linked to the carcinogenic effect of high-risk HPVs), while 12 samples were negative for these proteins (Supplementary Table S1). Using the multiplex SACA system, we detected HPV16 mRNA in 5 of the protein-positive samples and HPV18 mRNA in the remaining 3. Crucially, in the 12 protein-negative samples, neither HPV subtype was detected (Fig. 6H). All detection results were consistent with clinical reference standards (Supplementary Table S2), achieving 100% sensitivity and specificity (Supplementary Table S3). This demonstrates that our method is fully applicable for in situ nucleic acid imaging in clinical samples.
Development of a multiplex CRISPR detection platform based on the SACA system. () Schematic of dual-gene detection of HPV16 mRNA and HPV18 mRNA using the multiplex SACA assay. () Fluorescence intensity of Cas12a and Cas13a trans-cleavage activity across different buffer conditions. () Conventional Cas12a/Cas13a system for simultaneous detection of HPV16 DNA (Cas12a) and HPV16 mRNA (Cas13a). () SACA-enhanced Cas12a and Cas13a system for simultaneous detection HPV16 DNA (Cas12a) and HPV16 mRNA (Cas13a). () SACA-enhanced Cas12a and Cas13a system for simultaneous detection HPV18 mRNA (Cas12a-TRC) and HPV16 mRNA (Cas13a). A B C D E
Intracellular detection of HPV16/18 mRNA using a multiplex SACA-based CRISPR assay. () Workflow forimaging of HPV16 and HPV18 in fixed cells using the SACA system. () Fluorescence intensity in cells for the detection of HPV18 mRNA using SACA-based Cas12a. () Fluorescence intensity in cells for detection of HPV16 mRNA using SACA-based Cas13a assays. () Comparison of detection performance between the SACA-based Cas12a assay and conventional assays for HPV18 RNA. () Comparison of detection performance between the SACA-based Cas13a assay and conventional assays for HPV16 RNA. Orthogonal detection of HPV18 mRNA and HPV16 mRNA () using the multiplex SACA system. () Application of multiplex SACA system for the diagnosis of HPV18/16 mRNA in cervical cancer FFPE samples. A B C D E G H in situ
Discussion
Phosphorothioate (PS) -modified nucleic acids have been demonstrated to possess robust resistance to nuclease degradation [51–53]. This work systematically explores how PS modification affects CRISPR/Cas trans-cleavage and evaluates its capacity as an activator to initiate Cas activity. We unexpectedly observed that fully PS-modified activators retain the capacity to bind Cas12a and assemble into the ternary complex, yet they fail to trigger trans-cleavage. Dissecting this anomaly revealed a conformational bottleneck: the continuous PS backbone induces excessive non-specific hydrophobic interactions with the Cas12a protein. These interactions act as a molecular anchor, trapping the complex in a “closed” conformation that effectively silences catalytic ability despite successful binding. To break this deadlock, we proposed a “scattered” modification strategy. By rationally distributing PS modifications, we attenuated these hydrophobic constraints. This optimization preserves the necessary nuclease resistance while permitting the Cas12a/crRNA complex to undergo the essential conformational shift to a fully “open” state, thereby balancing the conflict between stability and activity.
Translating this mechanistic understanding into functional application, we engineered the “tailored coordinator” to drive the SACA system. The design logic exploits the Cas enzyme preference for cleaving phosphodiester bonds located within unmodified spacers of at least three nucleotides. By inserting a specific unmodified linker domain (z-region) within the PS backbone, we effectively partitioned the activator into a “pro-drug” like state. This z-region functions as a dual-purpose molecular gate: in the absence of a target, it exerts steric hindrance to suppress background noise; upon specific triggering, it acts as a separate site. The subsequent liberation of the PS-activator fragments initiates a robust Cas autocatalytic reaction, enabling the system to overcome the sensitivity limits inherent to linear signal accumulation. Unlike secondary structure probes that rely on intramolecular folding, such as hairpins or circular probes [19–26], which can be thermodynamically unstable in the crowded intracellular condition, the linear PS-probe ensures consistent performance, rapid synthesis, and superior biostability, making it uniquely suited for multiplexed in situ cellular imaging.
SACA offers high sensitivity but involves thermodynamic trade-offs. Scattered PS activators bind tightly to the Cas/crRNA complex, which, in a one–pot reaction, can compete with target DNA and reduce recognition efficiency at low concentrations. To address this, we used a dual–crRNA strategy, one for target binding and another for auto-catalysis. While effective, this increases system complexity and may limit multiplexing due to the need for multiple orthogonal crRNAs. Future work will aim to tune activator affinity through sequence design, enabling a simpler, integrated system for multiplexed detection without sacrificing sensitivity.