A PAM-independent CRISPR-Cas12a-based method for detecting PPP1CA gene isoforms and its application

Abstract

This invention relates to the field of molecular biology, specifically disclosing a method for detecting specific splice isoforms of PPP1CA based on the CRISPR-Cas12a system. The CRISPR-Cas12a detection system includes: a specific crRNA targeting the PPP1CA gene isoform PPP1CA-SV1, a Cas12a protein, and a reporter molecule; wherein the crRNA contains a scaffold sequence that binds to the Cas12a protein and a targeting sequence that specifically binds to the exon-exon junction region unique to the PPP1CA-SV1 isoform; the DNA to be detected in the detection system is ssDNA, and detection is achieved by utilizing the characteristic of Cas12a to recognize ssDNA without relying on the PAM sequence.

Description

Technical Field

[0001] This invention relates to the field of molecular biology, and specifically discloses a method and application for detecting PPP1CA gene isoforms based on PAM-independent CRISPR-Cas12a. Background Technology

[0002] Hepatocellular carcinoma (HCC) is the most common pathological type of primary liver cancer, accounting for 75%–85% of all liver cancers. According to the 2022 Global Cancer Statistics, liver cancer ranks sixth in incidence and third in mortality worldwide, with approximately 865,000 new cases and over 750,000 deaths annually. Therefore, developing highly sensitive and specific diagnostic biomarkers for early diagnosis of HCC is of significant clinical importance.

[0003] PPP1CA (Protein Phosphatase 1 Catalytic Subunit Alpha) encodes the α isoform of the catalytic subunit of protein phosphatase 1 (PP1). PP1 is one of the most important serine / threonine protein phosphatases in eukaryotic cells. It participates extensively in core biological processes such as cell cycle regulation, gene transcription, protein synthesis, glycogen metabolism, and apoptosis by binding to different regulatory subunits to form a holoenzyme complex. Recent studies have found that the gene and protein expression levels of PPP1CA in hepatocellular carcinoma (HCC) tissues are significantly higher than in non-cancerous tissues, and there are significant differences in blood PPP1CA levels between HCC and non-HCC patients, supporting its potential as a biomarker for the diagnosis and prognosis of HCC.

[0004] The PPP1CA gene has multiple splice variants. Existing data show that the exon-deleted isoform (PPP1CA-SV1) is expressed at higher levels in HCC tissues than in normal tissues, suggesting it may serve as a novel molecular marker for HCC diagnosis. However, current detection methods for PPP1CA splice variants mainly rely on real-time quantitative PCR (qPCR) and sequencing technologies. These methods have limitations such as strong equipment dependence, complex operation, long detection cycles, and high costs, making it difficult to meet the needs of rapid clinical diagnosis and point-of-care testing (POCT). Therefore, developing a rapid, sensitive, and convenient PPP1CA-SV1 detection method is of great significance for the early diagnosis and prognostic assessment of HCC. Summary of the Invention

[0005] The present invention aims to overcome the shortcomings of the prior art and provide a method for detecting PPP1CA specific splice isomers based on the CRISPR-Cas12a system.

[0006] The core of the CRISPR-Cas12a diagnostic system lies in the trans-cleavage activity activated after the Cas12a enzyme recognizes the target, cleaving a large number of reporter molecules to amplify the signal. Its high specificity relies on the precise pairing of crRNA and target and the dual recognition mechanism of PAM sequences. To achieve clinical-grade sensitivity, it is usually combined with isothermal amplification techniques such as RPA or LAMP to form a dual amplification strategy. In isoform detection, CRISPR-Cas12a has shown significant potential. For example, in the study of ovarian cancer circRNA, integrating RT-RCA with a dual Cas12a protein / multiplex crRNA strategy to detect hsa_circ_0049101 achieved a detection limit of 0.5 fM and a sensitivity 4–11 times higher than that of a single crRNA system. In miRNA multiplexing, a split crRNA-based amplification-free CRISPR / Cas12a biosensor combined with the SPR effect of gold nanoparticles has achieved sensitive multiplexing of breast cancer-related miRNAs. The split crRNA strategy significantly enhances the ability to resolve single-base mismatches. In the field of targeted detection of hepatocellular carcinoma biomarkers, studies have developed electrochemical sensors integrating DCHA-DNA walker cascade amplification with CRISPR / Cas12a, achieving detection limits of 18.89 fg / mL for AFP and 34.02 aM for miRNA-122. Furthermore, by engineering the 5′ end of the Cas12a activator strand, CRISPR / Cas12a activation behavior can be significantly regulated, providing a universal platform for the analysis of various biomarkers and the identification of clinical cancer tissues.

[0007] However, traditional CRISPR-Cas12a detection systems rely heavily on the presence of the Protospacer Adjacent Motif (PAM) when recognizing double-stranded DNA targets. Specifically, they require the presence of a specific 5′-TTTV-3′ (V=A / G / C) PAM sequence near the target sequence. This PAM dependency severely limits the application of CRISPR-Cas12a systems, particularly for detecting gene regions lacking PAM sites or specific splice variant junctions, often rendering them unusable.

[0008] This application addresses two core technical issues: first, the traditional CRISPR-Cas12a system cannot directly detect the PPP1CA-SV1 isoform-specific sequence due to its reliance on PAM sequences; second, existing detection methods (such as qPCR and sequencing) are cumbersome, equipment-dependent, have long detection cycles, and are costly, making it difficult to achieve rapid, sensitive, and convenient clinical testing. By solving these problems, this invention aims to achieve a highly sensitive and specific detection method suitable for early diagnosis, prognostic assessment, and treatment monitoring of hepatocellular carcinoma. Therefore, this invention proposes a PAM-independent CRISPR-Cas12a-based method for detecting PPP1CA gene isoforms and its application.

[0009] In a first aspect, this invention discloses a PAM-independent CRISPR-Cas12a-based PPP1CA gene isoform detection system, employing the following technical solution: A PAM-independent CRISPR-Cas12a-based detection system for PPP1CA gene isoforms, comprising: a specific crRNA targeting the PPP1CA-SV1 gene isoform, a Cas12a protein, and a reporter molecule; wherein the crRNA contains a scaffold sequence that binds to the Cas12a protein and a targeting sequence that specifically binds to the exon-exon junction region unique to the PPP1CA-SV1 isoform; the DNA to be detected in the detection system is ssDNA, and the detection is achieved by utilizing the PAM-sequence-independent recognition of ssDNA by Cas12a.

[0010] Preferably, the target sequence (i.e., the spacer region sequence) of the specific crRNA is selected from the sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:5.

[0011] Preferably, the Cas12a protein is the LbCas12a protein.

[0012] Preferably, the reporter molecule is a single-stranded DNA oligonucleotide probe, with a fluorescent reporter group labeled at its 5′ end and a fluorescent quencher group labeled at its 3′ end; the fluorescent reporter group is FAM and the fluorescent quencher group is BHQ1.

[0013] Secondly, the present invention discloses a kit for detecting the PPP1CA gene isoform PPP1CA-SV1, which includes the above-mentioned PAM-independent CRISPR-Cas12a-based PPP1CA gene isoform detection system.

[0014] Preferably, the DNA to be detected is ssDNA formed by alkaline denaturation and unwinding, and the kit further includes an alkaline denaturing reagent. The alkaline denaturing reagent includes an alkaline solution for capturing dsDNA and an acidic solution for neutralizing the system, preferably a 100 mM-500 mM NaOH solution and a 100 mM-500 mM HCl solution, more preferably a 100 mM NaOH solution and a 100 mM HCl solution.

[0015] Preferably, the sample further includes primer pairs for amplifying target nucleic acids in the sample to be tested; the primer pairs are PCR primer pairs or RPA primer pairs; the sequences of the PCR primer pairs are selected from those shown in SEQ ID NO:12 or SEQ ID NO:13; the sequences of the RPA primer pairs are selected from those shown in SEQ ID NO:14 or SEQ ID NO:15.

[0016] Thirdly, this invention discloses a PAM-independent CRISPR-Cas12a-based method for detecting PPP1CA gene isoforms, which employs the aforementioned CRISPR-Cas12a detection system. The method includes the following steps: (1) Extract nucleic acid from the sample to be tested to obtain double-stranded DNA; (2) The double-stranded DNA obtained in step (1) is subjected to alkaline denaturation to unwind it into single-stranded DNA; (3) Add the single-stranded DNA obtained in step (2) to the CRISPR-Cas12a detection system of the kit, incubate and monitor the fluorescence signal.

[0017] Preferably, the alkaline denaturation treatment in step (2) is performed by treating with NaOH solution and then neutralizing with HCl solution. More preferably, 2 μL of the amplification product of the sample to be tested is taken, 1 μL of 100 mM ~ 500 mM NaOH solution is added, incubated at room temperature for 1-5 min, and then 1 μL of 100 mM ~ 500 mM HCl solution is added to neutralize to neutral.

[0018] Preferably, the incubation conditions for step (3) are room temperature incubation for 1 to 5 minutes.

[0019] Fourthly, this invention discloses the application of a PAM-independent CRISPR-Cas12a-based method for detecting PPP1CA gene isoforms, which can be applied to the early diagnosis, prognostic assessment, and treatment monitoring of hepatocellular carcinoma.

[0020] Compared with the prior art, the present invention has at least the following beneficial effects: This invention, by designing crRNA at the exon-exon junction specific to the exon 2 deletion isoform of PPP1CA, can accurately distinguish the PPP1CA-SV1 isoform from other PPP1CA splice variants, avoiding false positives due to sequence similarity. Its specificity is significantly superior to detection methods based on shared sequences. Furthermore, utilizing the trans-cleavage activity of Cas12a to achieve signal cascade amplification, the detection sensitivity reaches the pM level, which is superior to traditional qRT-PCR methods and suitable for the detection of low-abundance RNA isoforms. Establishing a rapid CRISPR-Cas12a detection method for the hepatocellular carcinoma-related biomarker PPP1CA-SV1 can provide a new technical means for the early diagnosis, molecular subtyping, treatment monitoring, and prognostic assessment of hepatocellular carcinoma. Attached Figure Description

[0021] Figure 1 This is a flowchart outlining the experimental method of the present invention; Figure 2 The fluorescence signal results of the design and screening of specific crRNAs in Example 1 are shown in the figure. a) The fluorescence intensity of the PPP1CA-SV1 isoform was detected using 5 designed crRNAs, in the order from left to right: crRNA1, crRNA2, crRNA3, crRNA4, crRNA5, and negative control; b) The fluorescence intensity of the PPP1CA-SV2 isoform was detected using 5 designed crRNAs, in the order from left to right: crRNA1, crRNA2, crRNA3, crRNA4, and crRNA5; c) The fluorescence intensity of the two isoforms, PPP1CA-SV1 and PPP1CA-SV2, was detected using 4 designed crRNAs, respectively. Figure 3 The results of fluorescence signal determination for the detection limit in Example 1 are shown in the figure; a) Dose-response curve plotted with target concentration (logarithmic coordinates, x-axis) as independent variable and fluorescence intensity (y-axis) as dependent variable; b) Comparison of fluorescence intensity between different target concentrations and negative control, with the x-axis representing different target concentrations and negative control, and the y-axis representing the endpoint fluorescence intensity. Figure 4 This is a graph showing the fluorescence signal results of the PPP1CA-SV1 isoform detected in liver cancer cell lines and normal cells in Example 2. Detailed Implementation

[0022] To make the objectives, technical solutions, and advantages of this invention clearer, the specific embodiments and accompanying drawings are described in further detail below. The following embodiments are for illustrative purposes only and are not intended to limit the scope of protection of this invention.

[0023] The key and innovative aspect of this invention lies in the establishment of a novel, highly sensitive, and rapid detection method for the PPP1CA protein, which is highly expressed in hepatocellular carcinoma. Since the protein products of splice isoforms are unstable and difficult to detect, this invention bypasses the unstable protein and detects it at the upstream nucleic acid level, targeting the mRNA encoding the protein. A specific crRNA sequence is designed, with the crRNA targeting the exon-exon junction region unique to the PPP1CA-SV1 isoform. When the crRNA specifically recognizes the target isoform, it activates the trans-cleavage activity of Cas12a, cleaving the fluorescent reporter molecule and releasing a detectable fluorescent signal. Traditional CRISPR-Cas12a detection systems strictly rely on the PAM sequence (5′-TTTV-3′) when recognizing double-stranded DNA targets, which severely limits their application in detecting gene regions lacking PAM sites or specific splice variant junctions. This invention converts double-stranded DNA in the test sample into single-stranded DNA through alkaline denaturation. Utilizing the characteristic of the Cas12a enzyme that recognizes and cleaves single-stranded DNA independently of the PAM sequence, it successfully bypasses PAM restrictions. Through these techniques, highly sensitive and specific detection of the PPP1CA-SV1 isoform is achieved.

[0024] The present invention will be further described in detail below with reference to specific embodiments.

[0025] Example 1: Construction of a PAM-independent CRISPR-Cas12a-based PPP1CA gene isoform detection system 1.1 Oligonucleotide Fragment Synthesis 1.1.1 Synthesis of partial sequences of PPP1CA-specific splice isomer Bioinformatics analysis was used to obtain the gene sequence of the PPP1CA-SV1 isoform, with a focus on its unique exon-exon junction region (SEQ ID NO. 1). The PPP1CA-SV1 isoform partially deletes exon 2, resulting in a unique base sequence in its exon-1 and exon-2 junction region. This sequence differs significantly from the full-length PPP1CA isoform and other splicing variants. The base sequence of the junction region was synthesized using chemical methods, yielding only single-stranded DNA that can bind complementary to crRNA.

[0026] The sequence at the junction is: ATGTCCGACAGCGAGAAGCTCAACCTGGACTCGATCATCGGGCGCCTGCTGGAAG(exon 1) / / (exon 2)TGCAGGGCTCGCGGCCTGGCAAGAATGTACAGAGAACGAGATCCGCGGTCTGTGCCTGAAATCCCGGGAGATTTTTCTGAGCCAGCCCATTCTTCTGGAGCTGGAGGCACCCCTCAAGATCTGCGG.

[0027] 1.1.2 crRNA Synthesis Based on the Cas12a crRNA design principles, the crRNA targeting sequence was designed into the exon-exon linkage region specific to PPP1CA-SV1, ensuring that the crRNA can specifically recognize this isoform while not recognizing other isoforms. The crRNA was prepared using chemical synthesis methods, purified by HPLC, and stored at -80℃ for later use. The crRNA sequence is shown in Table 1 below.

[0028] Table 1 crRNA Sequence

[0029] *The table only shows the spacer region sequence of crRNA. The fixed scaffold region sequence is automatically added during the synthesis by the biotechnology company, specifically 5'-TAATTTCTACTAAGTGTAGAT -3'. According to WIPO ST.26 standards, uracil in RNA molecules is represented by the letter 't' in the submitted sequence listing. Therefore, in the RNA sequences listed, all 't' characters represent uracil.

[0030] 1.1.3 Screening of crRNA The multiple crRNAs synthesized in 1.1.2 were added to the CRISPR-Cas12a reaction systems shown in Table 2. (See...) Figure 2 First, the fluorescence intensity of the PPP1CA-SV1 isoform was detected using five designed crRNAs, arranged from left to right as crRNA1, crRNA2, crRNA3, crRNA4, crRNA5, and the negative control. Figure 2As shown in a), crRNA1 to crRNA5 all produced significantly higher fluorescence signals than the negative control, indicating that these five crRNAs can effectively recognize the PPP1CA-SV1 isoform and activate the trans-cleavage activity of CRISPR-Cas12a. The fluorescence intensity of the PPP1CA-SV2 isoform was detected using the five designed crRNAs, and the order from left to right was crRNA1, crRNA2, crRNA3, crRNA4, and crRNA5. Figure 2 As shown in b), crRNA1 to crRNA4 produced low fluorescence signals to SV2, while crRNA5 produced significantly higher fluorescence signals to SV2 than the other four crRNAs. Preliminary analysis suggests that crRNA5 exhibits a strong cross-reactivity with the non-target SV2, indicating potentially low specificity. Finally, the fluorescence intensity of the two isoforms PPP1CA-SV1 and PPP1CA-SV2 was detected using the four designed crRNAs (crRNA1, crRNA2, crRNA3, and crRNA4). The results are as follows: Figure 2 As shown in c), all four designed crRNAs exhibited high fluorescence signals when detecting the SV1 isoform, and their fluorescence intensity differed significantly from that of the SV2 isoform. In contrast, the designed crRNA1 had a higher signal-to-noise ratio and lower background signal. Therefore, crRNA1 was selected as the optimal crRNA for subsequent experiments.

[0031] 1.1.4 Preparation of the CRISPR-Cas12a reaction system The CRISPR-cas12a reaction system was constructed according to Table 2, including the components of the synthesized crRNA mentioned above.

[0032] Table 2 CRISPR-cas12a reaction system

[0033] *HOLMES-FluorosDNA reporter 1 (FAM) is a single-stranded DNA oligonucleotide probe with the FAM fluorescent reporter group labeled at the 5′ end and the BHQ1 fluorescent quencher group labeled at the 3′ end.

[0034] 1.1.5 Sensitivity Test of the CRISPR-Cas12a PPP1CA Gene Isomer Detection System (1) The ssDNA synthesized in 1.1.1 was serially diluted with the following concentration gradients: 100 nM, 10 nM, 1 nM, 100 pM, 10 pM; (2) Take 2 μL and add it to the CRISPR-Cas12a reaction system in Table 2. Set up 3 technical replicates for each concentration. (3) Transfer the above reaction system to a qPCR instrument for monitoring, incubate at 37°C for 50 min; (4) Record the changes in fluorescence intensity in real time, read the fluorescence signal every 30 seconds, and detect and analyze the fluorescence intensity results after 50 minutes.

[0035] Test results are shown Figure 3 Using crRNA1 to detect different concentrations of the PPP1CA-SV1 isoform gene, the lowest detection concentration was initially found to be 10 pM. The designed crRNA1 also showed good specificity, and no obvious fluorescence signal was detected for higher concentrations of the PPP1CA-SV2 isoform.

[0036] Example 2: Validating the feasibility of the method using liver cancer cell lines This embodiment aims to verify the detection effect of the method of the present invention on the PPP1CA-SV1 isoform using actual cell samples.

[0037] 2.1 Cell Selection and Culture Three hepatocellular carcinoma (HCC) cancer lines were selected for the experiment: HepG2, Bel-7402, and HCC-LM3, with normal human umbilical cord mesenchymal stem cells (UC-MSCs-18) used as a negative control. The HepG2 HCC cancer line was purchased from the Shanghai Cell Bank of the Chinese Academy of Sciences; the Bel-7402 HCC cancer line was purchased from Jiangsu Kaiji Biotechnology Co., Ltd.; the HCC-LM3 highly metastatic HCC cancer line was purchased from Wuhan Pronosei Life Science Co., Ltd.; and the UC-MSCs-18 line was purchased from Zhongyuan Xiehe Cell & Gene Engineering Co., Ltd. (where 18 is the laboratory's internal code). HepG2 cells were cultured in MEM medium containing 10% fetal bovine serum, Bel-7402 cells were cultured in RPMI-1640 medium containing 10% fetal bovine serum, HCC-LM3 cells were cultured in high-glucose DMEM medium containing 20% ​​fetal bovine serum, and human umbilical cord mesenchymal stem cells (UC-MSCs) were cultured in DMEM / F-12 medium containing 20% ​​fetal bovine serum. All cells were cultured routinely at 37°C in a 5% CO2 incubator.

[0038] 2.2 Total RNA extraction from cells The specific steps are as follows: (1) Discard the culture medium from the 6-well plate and wash with PBS 2-3 times; (2) Add 500 μL of Trizol to each well, pipette the cells and collect them into an EP tube; (3) Let stand for 5 minutes; (4) Add 100 μL (1 / 5 of the volume of Trizol) of chloroform, shake vigorously up and down, and let stand at room temperature for 10 minutes to allow the extract to separate into layers; (5) Centrifuge at 4℃ (13000 rpm, 15 min), the upper layer is RNA; (6) Transfer the supernatant to a new EP tube (avoid touching the intermediate layer), add 250 μL (1 / 2 volume of Trizol) isopropanol (pre-cooled), gently tap to mix, and let stand for 15 min or at -80℃ for 5 min to promote precipitation. (7) Centrifuge at 4℃ (13000 rpm, 15 min). A white precipitate (i.e. RNA) will form at the bottom of the tube. Carefully aspirate the supernatant. (8) Add 500 μL of 75% ethanol (prepared with RNase-free water / DEPC water) and gently invert to wash the precipitate; (9) Centrifuge at 4℃ (13000 rpm, 15 min), discard the supernatant, remove the residual liquid with a pipette, and air dry in a clean bench for about 15 min; (10) Add 15 μL of RNase-free water, gently tap the tube wall to dissolve the RNA, and let stand for 10 min to promote complete dissolution; (11) Take 2 μL of each sample into an eight-tube strip and use Nanodrop to detect the RNA purity and concentration: the A260 / A280 ratio should be in the range of 1.8-2.2 (for pure RNA), and the A260 / A230 ratio should be greater than 1.7 (to exclude guanidine salt contamination).

[0039] (12) RNA is unstable and needs to be reverse transcribed into cDNA for preservation after extraction. The reaction system is prepared in an eight-tube strip: Table 3 Reverse transcription reaction system

[0040] (13) Gently rotate the eight-piece tube to mix it completely and allow it to settle fully, and to eliminate air bubbles; (14) Place the eight-tube strip into the PCR instrument and set the program: Table 4 Reverse Transcription Procedure

[0041] (15) Dilute the generated cDNA 10 times and store it at -20℃ for a short period of time.

[0042] 2.3 Nucleic Acid Amplification This example demonstrates two methods for nucleic acid amplification: polymerase chain reaction (PCR) and recombinase polymerase amplification (RPA).

[0043] 2.3.1 Polymerase Chain Reaction (PCR) (1) Design primers based on the target fragment sequence. The primer sequence is PPP1CA-PCR-FP1 (SEQ ID NO.12) or PPP1CA-PCR-RP1 (SEQ ID NO.13), see Table 5; Table 5 Primer Sequences

[0044] (2) Prepare the PCR reaction system, as shown in Table 6, where the DNA template is cDNA: Table 6 PCR Reaction System

[0045] (3) Place the prepared PCR tubes into the PCR instrument for amplification reaction: Table 7 PCR reaction procedure

[0046] (4) Nucleic acid gel electrophoresis: (4.1) Weigh agarose powder and prepare 1% agarose gel: Weigh 0.25 g of agarose and add it to a beaker. Measure 25 mL of 1×TAE solution and mix it with the agarose. Heat it in a microwave oven to dissolve it. After it is completely dissolved, add the nucleic acid dye Super Red. Insert the comb into the gel tank and pour in the prepared solution. Let it stand at room temperature for about 20 min to solidify. (4.2) Place the solidified nucleic acid gel in the electrophoresis tank and immerse it in 1×TAE; (4.3) After the PCR program is completed, take out the PCR product and select a DNA marker that can indicate the appropriate length according to the gene length. Depending on the situation, it may be necessary to add DNA loading buffer. (4.4) Close the electrophoresis tank cover. Black is the negative electrode and red is the positive electrode. Set the electrophoresis program (120 V / 30-40 min) and run the gel. Generally, stop electrophoresis when the bromophenol blue migrates to two-thirds of the gel. (4.5) Using an ultraviolet flashlight and a gel imaging system, compare the positions of the gene bands with those indicated by the markers, observe whether the band sizes are correct, and take photos; (4.6) After confirming that the band size is correct, the target band is extracted and recovered by gel extraction using a product purification kit and the concentration is measured to obtain the amplified gene solution.

[0047] 2.3.2 Recombinase polymerase amplification (RPA) (1) Design primers based on the target fragment sequence. The primer sequence is PPP1CA-RPA-FP1 (SEQ ID NO.14) or PPP1CA-RPA-RP1 (SEQ ID NO.15), see Table 5; (2) Prepare the RPA reaction system, see Table 8, where the template is total cellular RNA: Table 8 RPA Reaction System

[0048] (3) Incubate in a constant temperature water bath at 42℃ for 30 minutes.

[0049] 2.4 Fluorescence Detection Based on PAM-Independent CRISPR-Cas12a System (1) Take 2 μL of the above PCR / RPA amplification product, add 1 μL of 100 mM NaOH solution, incubate at room temperature for 1 min to establish an alkaline environment, so that dsDNA unwinds to form ssDNA; (2) Add 1 μL of 100 mM HCl solution to the above system to neutralize the system until the pH is neutral, to ensure that the activity of CRISPR-Cas12a enzyme is not affected by acid and alkali, and obtain the test solution containing ssDNA; (3) Add the test solution containing ssDNA to the CRISPR-Cas12a reaction system in Table 2; (4) Transfer the above reaction system to a qPCR instrument for monitoring, incubate at 37°C for 90 min; (5) Record the changes in fluorescence intensity in real time, read the fluorescence signal every 30 seconds, and detect and analyze the fluorescence intensity results after 90 minutes.

[0050] Test results are shown Figure 4 The method used in this study was used to determine the content of PPP1CA-SV1 isoforms in hepatocellular carcinoma cells (HepG2, Bel-7402, HCC-LM3) and normal umbilical cord mesenchymal stem cells (Uc-Mscs). After isothermal amplification by RPA, the normal cell samples produced a weak fluorescence signal, but it was significantly weaker than the detection signal in the hepatocellular carcinoma cell lines. This demonstrates the feasibility of this method for cell sample detection and its ability to distinguish between hepatocellular carcinoma and non-hepatocellular carcinoma cells.

[0051] Example 3: Clinical Sample Detection Methods See Figure 1 For blood samples from clinical patients, two different processing methods can be used: Method 1: Reverse transcription of RNA in blood samples to obtain cDNA, followed by PCR amplification or RPA amplification; Method 2: Direct RPA amplification of RNA samples.

[0052] After amplification, alkaline denaturation and CRISPR-Cas12a fluorescence detection were performed according to the method in Example 2. The expression level of the PPP1CA-SV1 isoform in the sample was determined by the intensity of the fluorescence signal, thereby assisting in the diagnosis, prognostic assessment or treatment monitoring of hepatocellular carcinoma.

[0053] In summary, this invention converts double-stranded DNA into single-stranded DNA through alkaline denaturation. Utilizing the PAM sequence-independent recognition of single-stranded DNA by Cas12a, and combining this with crRNA designed into the exon-exon linker region specific to the PPP1CA-SV1 isoform, a highly sensitive (detection limit down to 10 pM) and highly specific method for detecting PPP1CA gene isoforms has been successfully established. This method is simple to operate, has a short detection cycle, and can effectively distinguish between liver cancer cells and non-liver cancer cells, providing a new technical means for the early diagnosis, molecular subtyping, treatment monitoring, and prognostic assessment of hepatocellular carcinoma.

[0054] The technical solution provided by the invention has been described in detail above. Specific examples have been used to illustrate the principle and implementation of the invention. The description of the above embodiments is only for the purpose of helping to understand the method and core idea of ​​the invention. At the same time, for those skilled in the art, there will be changes in the specific implementation and application scope based on the idea of ​​the invention. Therefore, the content of this specification should not be construed as a limitation of the invention.

Claims

1. A PAM-independent CRISPR-Cas12a-based PPP1CA gene isoform detection system, characterized in that, The CRISPR-Cas12a detection system comprises: a specific crRNA targeting the PPP1CA gene isoform PPP1CA-SV1, a Cas12a protein, and a reporter molecule; wherein the crRNA contains a scaffold sequence that binds to the Cas12a protein and a targeting sequence that specifically binds to the exon-exon junction region unique to the PPP1CA-SV1 isoform; the DNA to be detected in the detection system is ssDNA, and the detection is achieved by utilizing the characteristic of Cas12a to recognize ssDNA without relying on the PAM sequence.

2. The PPP1CA gene isoform detection system based on PAM-independent CRISPR-Cas12a according to claim 1, characterized in that, The target sequence of the specific crRNA is selected from the sequences shown in SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4 and SEQ ID NO:

5.

3. The PPP1CA gene isoform detection system based on PAM-independent CRISPR-Cas12a according to claim 1, characterized in that, The Cas12a protein is the LbCas12a protein.

4. The PPP1CA gene isoform detection system based on PAM-independent CRISPR-Cas12a according to claim 1, characterized in that, The reporter molecule is a single-stranded DNA oligonucleotide probe, with a fluorescent reporter group labeled at its 5′ end and a fluorescent quencher group labeled at its 3′ end; the fluorescent reporter group is FAM and the fluorescent quencher group is BHQ1.

5. A kit for detecting the PPP1CA gene isoform PPP1CA-SV1, characterized in that, Includes the PPP1CA gene isoform detection system based on PAM-independent CRISPR-Cas12a as described in any one of claims 1-4.

6. The kit for detecting the PPP1CA gene isoform PPP1CA-SV1 according to claim 5, characterized in that, The DNA to be detected is ssDNA formed by alkaline denaturation treatment and unwinding. The kit also includes an alkaline denaturation reagent.

7. The kit for detecting the PPP1CA gene isoform PPP1CA-SV1 according to claim 5, characterized in that, It also includes primer pairs for amplifying target nucleic acids in the sample to be tested; the primer pairs are PCR primer pairs or RPA primer pairs; the sequences of the PCR primer pairs are selected from those shown in SEQ ID NO:12 or SEQ ID NO:13; the sequences of the RPA primer pairs are selected from those shown in SEQ ID NO:14 or SEQ ID NO:

15.

8. A method for detecting PPP1CA gene isoforms based on PAM-independent CRISPR-Cas12a, characterized in that, It employs the CRISPR-Cas12a detection system as described in any one of claims 1-4, and the method includes the following steps: (1) Extract nucleic acid from the sample to be tested to obtain double-stranded DNA; (2) The double-stranded DNA obtained in step (1) is subjected to alkaline denaturation to unwind it into single-stranded DNA; (3) Add the single-stranded DNA obtained in step (2) to the CRISPR-Cas12a detection system of the kit, incubate and monitor the fluorescence signal.

9. The method for detecting PPP1CA gene isoforms based on PAM-independent CRISPR-Cas12a according to claim 1, characterized in that, The alkaline denaturation treatment in step (2) is as follows: treatment with NaOH solution, followed by neutralization with HCl solution.

10. An application of the PAM-independent CRISPR-Cas12a-based PPP1CA gene isoform detection method as described in any one of claims 8-9, characterized in that, This method can be applied to the early diagnosis, prognostic assessment, and treatment monitoring of hepatocellular carcinoma.

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