Rapid and sensitive detection of circulating tumor DNA via a CRISPR/Cas12a-based catalytic hairpin assembly

Abstract

Cancer is one of the major diseases that endanger the human health. Circulating tumor DNA (ctDNA) is an ideal biomarker for the real-time monitoring of cancer. In the present work, a rapid and sensitive assay coupled with CRISPR/Cas12a and CHA (Cas12a-CHA) was constructed for the detection of ctDNA. We designed and prepared a trigger, which was the substrate of Cas12a. On the addition of ctDNA, crRNA-guided ctDNA activated the trans-endonuclease activity of Cas12a. After being activated, Cas12a exhibited a high trans-cleavage activity on the trigger, which resulted in a decrease in fluorescence. Owing to this design, the Cas12a-CHA assay enabled the sensitive detection of ctDNA with a linear range of 10 fM to 50 pM. Furthermore, a limit-of-detection of 5.8 fM was achieved within 40 min. Besides, the proposed assay had an excellent base mismatch recognition ability and worked well in human serum samples. Conclusively, this detection platform holds significant potential for application in early cancer diagnosis.

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1. Introduction

Cancer has become one of the leading causes of human mortality worldwide, and early detection and treatment can increase the probability of cure and overall survival of patients. Therefore, the development of technologies for early cancer detection can reduce cancer-related mortality. Tissue biopsy is the most commonly used means for cancer diagnosis, which is also considered the gold standard. It involves removing the tumor tissue from the body of the patient via professional clinical techniques like incision, forceps and puncture for diagnosis.¹ Despite its significant clinical research value, the tissue biopsy-based tumor diagnostic method has some limitations including (i) its inability to be used for the early diagnosis of disease, (ii) invasive tissue acquisition, resulting in the difficulty to repeat sampling at multiple time points, and (iii) the heterogeneity and variability of tumors, leading to the acquired tissue not being representative.²

Liquid biopsy is an emerging detection technology that has become an important advancement in the field of solid tumor research in recent years. It is a non-invasive test, which is more patient-friendly. Liquid biopsy refers to the detection of circulating tumor cells, circulating tumor DNA (ctDNA), circulating tumor RNA, and tumor-associated proteins in a variety of body fluids (such as blood, urine, and saliva) to diagnose and evaluate the disease.^3–5 Among them, ctDNA is a class of cell-free DNA (cfDNA), which is released into the bloodstream by tumor cells through apoptosis, necrosis and secretion. Therefore, ctDNA contains the same genetic alterations as the tumor DNA from which it is derived, including point mutations, rearrangements, methylation and DNA copy number variations.^6–9 These intrinsic characteristics make ctDNA a promising clinical biomarker for the early diagnosis of cancer.¹⁰ Nonetheless, the detection of ctDNA is technically challenging because of its very short half-life (15 min to several hours) and low fraction (∼1.0%).^11–13

Conventional technologies like real-time quantitative polymerase chain reaction (PCR), digital PCR and next-generation sequencing (NGS) are well established and widely utilized for the detection of ctDNA.^14–19 However, these technologies still have some shortcomings. For example, PCR-based techniques are simple and easy to use with high sensitivity and specificity. However, these methods can only analyze a limited number of loci with low throughput. NGS technology provides high-throughput analyses with high sensitivity, but it is time-consuming and expensive to test, limiting its use in clinical practice. Therefore, there is a need to develop low-cost, real-time, rapid and portable systems to detect ctDNA for cancer screening and diagnosis.

The CRISPR/Cas system is a prokaryotic adaptive immune system that binds and cleaves exogenous nucleic acids.^20,21 The emergence of the CRISPR/Cas system has accelerated the development of gene editing technology, which is widely used in the field of life science, such as biosensing,^22–25 gene modification,²⁶ and gene therapy.²⁷ Particularly, the application of the CRISPR/Cas system in sensing methodologies for DNA/RNA detection has been developing rapidly, which is attributed to the discovery of its trans-cleavage activity.^28,29 Cas12a is an RNA-guided DNase. When Cas12a protease binds to CRISPR RNA (crRNA), forming the Cas12a-crRNA complex, this complex can recognize the target sequence, inducing specific cleavage of the target DNA sequence and nonspecific trans cleavage of its nearby single-stranded DNA. Once activated, Cas12a protease can continuously cleave single-stranded DNA non-specifically, which generates a strong signal amplification effect. However, when the target concentration in the detection system is low, the number of activated Cas12a protease is limited, and thus it is difficult to generate a large number of detectable signals in a short period of time, which cannot meet the needs of rapid and sensitive detection. Hence, the amplification ability of the simple CRISPR/Cas system is restrained for trace DNA detection, requiring an enhancement in its sensitivity.

Some enzyme-based isothermal amplification technologies, such as loop-mediated isothermal amplification (LAMP)^30,31 and rolling circle amplification (RCA)^32,33 have been introduced in the CRISPR/Cas12a system to increase its detection sensitivity. However, these amplification technologies heavily rely on expensive enzymes for amplification, restricting their further application in the field of molecular diagnosis. Recently, catalytic hairpin assembly (CHA) has emerged as alternative cost-effective isothermal amplification technology due to its high enzyme-free signal amplification capability.³⁴ A typical CHA reaction consists of two complementary DNA hairpins and a target input strand. The complementary sequences of the two DNA hairpins are enclosed in the stem-loop, and there is a dynamic hindrance to spontaneous hybridization between the two hairpins in the absence of the target strand. Once triggered by the target, the hairpin sequences automatically hybridize to form thermodynamically stable duplexes. Research has been devoted to utilizing CHA for the detection of ctDNA.³⁵ Tan et al. combined locked nucleic acid and CHA circuit for the detection of ctDNA in the detection range from 10 pM to 5 nM. Also, a detection limit of 3.3 pM was achieved. Nevertheless, its detection limit and linear range were not particularly considerable.

Taking full advantage of the trans-cleavage ability of CRISPR/Cas12a and high amplification efficiency of CHA, we propose a cascade signal amplification system (called ‘Cas12a-CHA’) for the rapid and sensitive detection of ctDNA. This system composed two parts, Cas12a cleavage and CHA amplification. In the CRISPR/Cas12a assay, the presence of the target ctDNA could be specifically recognized by the Cas12a/crRNA complex via the complementarity between crRNA and the target. Then, Cas12a was activated, resulting in the destruction of the trigger, which served as the initiator for the subsequent isothermal amplification. This process is the first signal amplification. Later, the residual trigger induced a CHA reaction, followed by generating fluorescence signals and realizing amplification of the second signal. This synergistic amplification strategy achieved the ultra-sensitive and specific detection of ctDNA, which provides a new idea for the application of CRISPR/Cas technology in the early diagnosis of cancer.

2. Experimental

2.1 Reagents and apparatus

Lba Cas12a and 10× Cas12a reaction buffer (1 M Tris–HCl, pH = 8.0, 5 M NaCl, 1 M MgCl₂, 100 × 10⁻³ M DTT) was available from Meige Biological Technology Co., Ltd (Guangzhou, China). All the sequences used in this study are shown in Table S1 and synthesized by Shanghai Sangon Biological Engineering Technology & Services Co., Ltd (Shanghai, China). Native PAGE preparation kit, DEPC-treated water, and 4SGelred nucleic acid dye were purchased from Sangon Biological Co., Ltd (Shanghai, China). 10 bp DNA ladder was supplied by Plansee Biotechnology Co., Ltd (Beijing, China). 1 M Tris–HCl buffer with different pH (RNase free), 0.5 M EDTA solution (RNase free, pH = 8.0), and 5 M NaCl solution (RNase free) were obtained from Yuanye Biotechnology Co., Ltd (Shanghai, China). Serum Circulating DNA Kit was purchased from Tiangen Biotech Co., Ltd (Beijing, China). The apparatus used in this experiment is supplied in the SI.

2.2 CRISPR/Cas12a assay

Firstly, 40 nM Cas12a, 40 nM crRNA and various concentrations of ctDNA were mixed in 1× reaction buffer. Then, FQ-trigger was added to make a final concentration of 100 nM. After adding DEPC-treated water, 20 µL of the abovementioned reaction mixture was incubated at 37 °C for 15 min. Its fluorescence spectra were measured using an FL970 spectrometer at the excitation wavelength of 490 nm. The spectra were recorded in the wavelength range of 500 to 600 nm.

2.3 Cas12a-CHA assay

The detection of ctDNA based on the Cas12a-CHA system is divided into two procedures, i.e., CRISPR/Cas12a collateral cleavage assay and CHA amplification reaction. Firstly, 0.8 µL of Cas12a, 0.8 µL of crRNA, 2 µL of ctDNA, 4 µL of trigger, 2 µL of 10× Cas12a reaction buffer, and 10.4 µL of DEPC-treated water were mixed to form a system with the final volume of 20 µL. This mixture was incubated at 37 °C for 15 min. After the Cas12a collateral cleavage assay, 8 µL of M1, 16 µL of M2, 56 µL of CHA reaction buffer (5 × 10⁻² M Tris–HCl, 5 × 10⁻² M NaCl, and 1 × 10⁻³ M EDTA, pH = 9.0) were added to the abovementioned mixture to obtain 100 µL of the reaction solution. The reactants were kept at 37 °C for 25 min to realize CHA amplification.

2.4 Electrophoresis assay

Briefly, 6× loading buffer and the analyte samples were mixed in a volume ratio of 1 : 5. The above-mentioned solution was transferred into prepared 12% polyacrylamide gel. After that, the gel was immersed in 1× running buffer (5 × 10⁻³ M MgCl₂, 89 × 10⁻³ M Tris, 200 × 10⁻³ M boric acid, pH = 8.0). The electrophoresis analysis was carried out at a constant voltage of 110 V. After running at room temperature for 40 min, the gel was stained with 4SGelred nucleic acid dye. Subsequently, the gel pictures were recorded using a FluorChem E instrument.

2.5 ctDNA analysis in serum sample

A healthy human serum sample, collected by standard clinical operating procedures, was obtained from the Affiliated Hospital of North Sichuan Medical College Hospital. The pretreatment procedure of the obtained serum sample was performed as follows: firstly, the sample was centrifuged for 10 min at a rotational speed of 10 000 g and the supernatant was obtained. Afterwards, the supernatant was diluted with 1× Cas12a reaction buffer at the ratio of 1 : 200. After heat treatment (65 °C, 15 min), the supernatant was subjected to centrifugation at 10 000 g for 10 min. Then, different concentrations of ctDNA target were added to the serum sample. At last, the detection processes were carried out according to the above-mentioned technology.

2.6 Clinical samples detection

Clinical serum samples from normal and cancer cases were collected from the Affiliated Hospital of North Sichuan Medical College (Sichuan, Nanchong). All experiments were performed in accordance with the Guidelines of Clinical Sample Management Rules of the Affiliated Hospital of North Sichuan Medical College, which were reviewed and approved by the Ethics Committee at the Affiliated Hospital of North Sichuan Medical College (2024ER519-1). Informed consents were received from the blood donors in this project. Nucleic acids in serum samples were extracted by the Serum Circulating DNA Kit (Tiangen) following the manufacturer's instructions. The detection procedures were conducted as reported in Section of 2.3.

3. Results and discussion

3.1 Working principle of the Cas12a-CHA strategy

The principle for the sensitive detection of ctDNA based on the CRISPR/Cas12a system and CHA with cascade signal amplification is displayed in Scheme 1. We designed a DNA oligo (named “trigger”), which not only served as the cleaved substrate of the Cas12a protein, but also as a triggering agent to induce the CHA reaction. In M1, the 5′-end of the sequence was labeled with a fluorescent dye fluorescein (FAM) and a quencher BHQ-1 in the other end. Cas12a protein bound to crRNA to form a nucleic acid–protein complex (Cas12a/crRNA). When the target was added to the detection system, the Cas12a/crRNA complex reacted with the target to assemble the Cas12a/crRNA/target ternary complex due to the complementarity between crRNA and the target. After that, the structure of the Cas12a protein changed, activating its single-stranded DNA cleavage activity for the surrounding triggers. When the trigger was cleaved by the activated Cas12a, both M1 and M2 maintained their hairpin structures in the solution. Therefore, no fluorescence signal was obtained at 519 nm. Relatively, Cas12a was inactivated when the target is absent. Hence, the trigger did not break by Cas12a, which effectively initiated subsequent CHA reaction. The intact trigger hybridized with the total loop region of M1 to form the trigger-M1 intermediate, opening the hairpin structure of M1 and exposing the sticky end, which possesses complementary sequences with M2. Subsequently, M2 is complementary to the unfolded M1, forming the trigger-M1–M2 intermediate. Due to the stand-displacement process, M2 substituted the trigger to form a more stable duplex of M1–M2. Moreover, the fluorophore FAM was removed from the quencher BHQ-1, leading to obvious fluorescence recovery. The released trigger again hybridized with another M1, inducing a new CHA cycle. Based on this mechanism, a large amount of M1–M2 duplex was generated. As the concentration of the target increased, the fluorescence signal at 519 nm was finally weakened, and thus the concentration of the target could be detected quantitatively.

Scheme 1 Illustration of the Cas12a-CHA assay for ctDNA detection. (A) Schematic of the Cas12a-CHA system with and without target and (B) detailed procedure of CHA.

3.2 The detection feasibility

An electrophoresis assay was conducted to demonstrate the procedures of the CRISPR/Cas12a assay and Cas12a-mediated CHA reaction (Fig. 1). The bands observed in lanes 1, 2 and 3 were crRNA, target ctDNA and trigger, respectively. Once assembled with Cas12a, the Cas12a/crRNA/target ternary complex was formed, which effectively activated the single-stranded DNA cleavage activity of the Cas12a protein. Thus, the trigger was cleaved into fragments. This phenomenon was presented in lane 4, validating the successful process of CRISPR/Cas12a. As shown in Fig. 1, the two obvious bands in lane 5 and 6 represented hairpin probe M1 and M2, respectively. Lane 7 and 8 displayed the gel bands of the control and experimental groups, respectively. Lane 7 shows that new bands were observed without the presence of the target ctDNA, which indicated that double-stranded DNA was formed. Higher electrophoretic mobility was shown by the M1–M2 duplex, while the trigger-M1–M2 triplex exhibited lower electrophoretic mobility. By contrast, after the addition of the target to the Cas12a-CHA system, M1 and M2 did not interact with each other, owing to the digestion of the trigger. Thus, there was no new band in lane 8.

Fig. 1 Electrophoresis analysis result of various DNA specimens (“+” and “−” represent the presence and absence of reactant, respectively). M is the nucleic acid marker.

To go a step further, fluorescence emission spectra were recorded under different conditions to evaluate the detection feasibility, as shown in Fig. S1. There was no fluorescence signal for the crDNA + Cas12a + crRNA + trigger, as well as ctDNA + Cas12a + crRNA + trigger + M2 systems. In the reaction system of ctDNA + Cas12a + crRNA + trigger + M1, a relatively low fluorescence signal was presented. The fluorescence signal showed high intensity when the reaction system was Cas12a + crRNA + trigger + M1 + M2. However, a decrease in fluorescence signal was obtained upon the addition of the target ctDNA to the system. These findings demonstrated that the proposed Cas12a-CHA assay is feasible for detecting ctDNA.

3.3 Optimization of experimental conditions

The activation of Cas12a and the CHA reaction played crucial roles in the Cas12a-CHA reaction system. Therefore, several vital factors including Cas12a concentration, crRNA concentration, reaction temperature, and reaction time for the CRISPR/Cas12a system were examined. ΔF refers to the difference in fluorescence intensity between the experimental and control group under the same conditions. Fig. 2A shows that ΔF increased when the concentration of Cas12a ranged from 10 nM to 40 nM. With an increase in the concentration of Cas12a, the upward trend of ΔF was destroyed and started to decline. Hence, 40 nM was adopted as the optimal concentration for Cas12a. Then, the influence of crRNA concentration was monitored. Fig. 2B displays the changes in ΔF produced by a crRNA concentration ranging from 10 nM to 50 nM. It can be seen that ΔF was the maximum when the concentration of crRNA was 40 nM. Thus, 40 nM of crRNA was applied for the detection system. Afterwards, the reaction temperature was investigated. As shown in Fig. 2C, ΔF increased with an increase in the reaction temperature from 17 °C to 37 °C. This might be caused by the relatively low temperature, which affected the interaction between crRNA and the target. When the reaction temperature exceeded 37 °C, ΔF decreased, which was contributed to the influence of high temperature for the protein activity of Cas12a. The finding demonstrated that 37 °C was enough for the CRISPR/Cas12a system. It can be found in Fig. 2D that ΔF was enhanced with an increase in the reaction time and reached the maximum when the reaction time was 15 min. With the extension of the reaction time, ΔF maintained a relatively stable state. Therefore, 15 min was chosen as the optimal cleaved time for the following experiment.

Fig. 2 Optimization of experimental conditions for the CRISPR/Cas12a assay. (A) Cas12a concentration, (B) crRNA concentration, (C) reaction temperature, and (D) reaction time. The error bars are the standard deviation of three replicate measurements.

Next, the concentration of M1 and M2, the effect of pH, the incubation temperature, and the reaction time were optimized to realize a better detection performance for CHA reaction. The value of ΔF₁ is used as an index to exhibit the optimal conditions for the CHA reaction, where ΔF₁ is the difference in fluorescence intensity in the presence and absence of the trigger. Firstly, we studied the optimal concentration of M1. As exhibited in Fig. 3A, the highest ΔF₁ was observed when the concentration of M1 was 800 nM. Also, ΔF₁ remained stable after the concentration of M1 reached over 800 nM. Thus, the optimal concentration of M1 was 800 nM. The optimal concentration of M2 was also explored. As presented in Fig. 3B, the concentration of M2 was measured from 400 nM to 2000 nM. There was a maximum for ΔF₁ when the M2 concentration was 1600 nM. As the concentration augmented, ΔF₁ had an negligible change. Thus, an M2 concentration of 1600 nM was selected as the ideal condition for the CHA reaction. Next, we investigated the optimal pH for the CHA reaction. Fig. 3C depicts that ΔF₁ successively increased with an increase in pH beginning at 7.0 and reached the peak value at pH = 9.0. Moreover, the amplification of the pH value resulted in a reduction in ΔF₁. Therefore, pH = 9.0 was used in subsequent experiments. Regarding the incubation temperature, when the temperature increased from 27 °C to 37 °C, the ΔF₁ gradually increased. With an increase in the incubation temperature, ΔF₁ tended to decline. Hence, 37 °C was employed for further experiments. Additionally, the reaction time for the CHA reaction was also evaluated. According to Fig. S2, it was clear that ΔF₁ increased with an increase in the reaction time. After 25 min, ΔF₁ reached a steady state. Therefore, 25 min was chosen as the optimum CHA reaction time. Finally, the concentration of trigger for the Cas12a-CHA assay was investigated. Fig. S3 reveals that ΔF₂, which is defined as the difference in fluorescence when the target ctDNA is absent or present, rapidly increased when the concentration of trigger increased from 20 nM to 100 nM, but the rate of increase was not obvious after 100 nM. Hence, we chose 100 nM as the optimal concentration of trigger for the Cas12a-CHA assay.

Fig. 3 Optimization of experimental conditions for the Cas12a-CHA assay. (A) M1 concentration, (B) M2 concentration, (C) effect of pH, and (D) incubation temperature. The error bars are the standard deviation of three replicate measurements.

3.4 Detection of ctDNA

Firstly, under the optimum working conditions, a range of concentrations of ctDNA was tested using the CRISPR/Cas12a system. The trans-cleavage substrate in this part was the FQ-trigger, which has a FAM at its 5′-end and the other possesses a quencher BHQ-1 at its 3′-end. After the specific recognition, Cas12a was activated and showed non-specific cleavage capability toward the FQ-trigger. The FQ-trigger was cleaved and separated FAM from BHQ-1, resulting in an intense fluorescence enhancement. As illustrated in Fig. S4A, increasing the concentration of ctNDA induced the augmentation of fluorescence intensity. As shown in Fig. S4B, when the concentration of ctDNA was in the range of 0.1 nM⁻¹⁰ nM, ΔF showed a linear relationship with the logarithm of the concentration of ctDNA, and the correlation equation was expressed as ΔF = 14500 Ig C_ctDNA − 28 100 (R² = 0.9911). The actual detection limit was 0.1 nM.

Furthermore, the detection sensitivity of the Cas12a-CHA assay was measured by utilizing a variety of ctDNA concentrations. Fig. 4A displays the fluorescence spectroscopy results for different amounts of ctNDA. As seen, the fluorescence intensity declined gradually as the amount of ctDNA increased from 10 fM to 2 nM. This appearance was attributed to the fact that when more ctDNA was present, more trigger was cleaved, leading to a noneffective HCR reaction. Fig. 4B exhibits the variation in ΔF₂ under various amounts of ctDNA. In the insert of Fig. 4B, a linear relationship was observed between ΔF₂ and the logarithm of the concentration of ctDNA from 10 fM to 50 pM. The linear regression equation was calculated to be ΔF₂ = 1324 Ig C_ctDNA − 728.1 with a correlation coefficient of 0.9856. Meanwhile, the detection limit was 5.8 fM based on the S/N = 3 rule. This result verified that the analytical sensitivity of the Cas12a-CHA system was approximately four orders of magnitude higher than that of the CRISPR/Cas12a system. In comparison with the other systems reported in the literature (Table 1), the proposed assay exhibited a shorter detection time and higher sensitivity, indicating its excellent detection performance for ctDNA.

Fig. 4 (A) Fluorescence spectra of the Cas12a-CHA assay for various ctDNA concentrations and (B) linear correlation between ΔF₂ and the concentration of ctDNA. The error bars are the standard deviation of three replicate measurements.

Table 1 Analytical performance of the Cas12a-CHA assay compared with those of other assays for the detection of ctDNA

Method	Time	Linear range	LOD	Ref.
WCHA/FHCR	210 min	10 fM–5 nM	8.3 fM	36
Cas9-ESDR	90 min	10 fM–500 pM	0.13 pM	37
Cas12a-MOF	270 min	1 fM–5 nM	5.6 fM	38
Cas12a-HCR	150 min	1 pM–400 pM	316 fM	39
Cas12a-EXPAR	140 min	1 fM–1 nM	0.11 fM	40
Cas12a-CHA	40 min	10 fM–2 nM	5.8 fM	This work

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3.5 Analysis of specificity and anti-interference capability

To explore the selectivity of the detection system towards target ctDNA, single-base mismatched ctDNA, two-base mismatched ctDNA, three-base mismatched ctDNA and a random sequence were detected. The detail information for these sequences is listed in Table S1. As illustrated in Fig. 5, a strong fluorescence change was observed in the presence of ctDNA. It was clear that single-base mismatched ctDNA induced a notable change in the fluorescence signal, which was ascribed to the high sequence similarity between ctDNA and single-base mismatched ctDNA. Nevertheless, the fluorescence change of ctDNA was 1.2-fold higher than that of the single-base mismatched ctDNA. The change in the fluorescence signal of two-base mismatched ctDNA was not obvious compared with the single-base mismatched ctDNA. In addition, the change in the fluorescence signal of three-base mismatched ctDNA and random sequence was negligible compared to that of ctDNA. The obtained results indicate that the developed strategy has excellent capability for base discrimination, which relies on the critical match between ctDNA and crRNA. To evaluate the anti-interference capability of the Cas12a-CHA system, we measured its response to ctDNA in the presence of various interfering substances. As shown in Fig. 5B, the experimental group (containing both ctDNA and interferents) and the control group (containing interferents only) were tested. The Cas12a-CHA system successfully detected ctDNA despite the presence of these interferents. These results indicate that the system possesses excellent anti-interference capability and is suitable for the analysis of practical samples.

Fig. 5 (A) Specificity of the Cas12a-CHA assay for detecting ctDNA. (B) Evaluation of the robustness of Cas12a-CHA for ctDNA detection against various biomolecular interferents.

3.6 Application of the developed assay in serum samples

Finally, the Cas12a-CHA system was applied for the detection of ctDNA in real samples to evaluate its practical application potential. A series of concentrations of ctDNA (100 fM, 1 pM and 10 pM) were spiked into 200-diluted human serum samples and measured by the Cas12a-CHA detection system. As shown in Table 2, it can be found that the recovery rates of the detection system in the serum samples were in the range of 96.5–113.3% and the relative standard deviation (RSD) values were below 9.6%, confirming that the determination results were satisfactory. Furthermore, the Cas12a-CHA system was used to detect ctDNA in clinical serum samples, including five cancer patients and five healthy donors. As shown in Fig. 6, the fluorescence difference in the cancer patients was higher than that of the healthy donors. As a consequence, the results implied the validity of the developed Cas12a-CHA system for ctDNA detection in real applications.

Table 2 Performance of the Cas12a-CHA assay for ctDNA detection in serum samples

Added (fM)	Found (fM)	Recovery (%)	RSD (%)
100	113.39	113.3	9.6
1000	989.2	98.9	8.3
10 000	9651.2	96.5	7.6

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Fig. 6 Detection of ctDNA in clinical serum samples using the Cas12a-CHA system.

4. Conclusion

In summary, we provided a promising assay combining CRISPR/Cas12a with CHA (Cas12a-CHA assay) for the rapid and sensitive detection of ctDNA. A simple trigger was utilized as the cleaved substrate for Cas12a, which also served as a primer for the CHA reaction. Under the optimized conditions, a limit-of-detection of 5.8 fM was obtained. Compared with CRISPR/Cas12a, the sensitivity of the Cas12a-CHA system could be dramatically improved by 4 orders of magnitude, which was due to the high turnover efficiency of Cas12a and isothermal amplification ability of CHA. The specific detection results confirmed that the Cas12a-CHA assay presented high-performance discrimination and detection base mutation. The outstanding specificity was guaranteed by the high specificity recognition ability of the CRISPR/Cas12a system. More importantly, the application of the assay for the detection of ctDNA in human serum samples displayed satisfactory results. Thus, we believe that the proposed assay holds enormous promise for tumor diagnosis.

Conflicts of interest

The authors declare no competing interests.

Data availability

The data that support the findings of this study are available from the corresponding author, Yixia Yang, upon reasonable request.

Supplementary information (SI): apparatus, oligonucleotide sequences, fluorescence spectra of feasibility analysis, optimization results of experimental conditions, the detection results of ctDNA using the CRISPR/Cas12a system. See DOI: https://doi.org/10.1039/d5ay01624j.

Acknowledgements

The Scientific Research Development Plan Project of North Sichuan Medical College (CBY24-QDA27), Cultivating Project for Young Scholar at Hubei University of Medicine (2024QDJZR018), and the Natural Science Foundation of Hubei Provincial Department of Education (Q20242102) are greatly acknowledged.

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Footnote

† These authors made equal contributions to this work.

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