Beta thalassemia detection kit based on multiple crisper-lba cas12a platform

Abstract

The application discloses a kit and a detection method for beta thalassemia detection based on multiple CRISPR-LbaCas12a, wherein the kit comprises crRNA specific recognition sequences, and the crRNA specific recognition sequences at least comprise SEQ ID NO: 11 for betaCD41-42 / betaN type, SEQ ID NO: 12 and SEQ ID NO: 13 for betaIVS-II-654 / betaN type, SEQ ID NO: 17 for beta-28 / betaN type, SEQ ID NO: 18 for betaCD17 / betaN type, and SEQ ID NO: 19 for betaCD26 / betaN type. The detection kit and the detection method can realize rapid and portable detection of common mutation types of thalassemia.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a β-thalassemia detection kit and a rapid diagnosis method based on a multiple CRISPR-Cas12a platform. Background Art

[0002] Thalassemia is a common autosomal recessive genetic disorder and a highly prevalent single-gene genetic disease worldwide. It is caused by mutations in the globin gene and is primarily categorized into α-thalassemia and β-thalassemia, depending on the mutated gene. Thalassemia is difficult to treat but preventable. The three-tiered prevention strategy for thalassemia is premarital and prenatal prevention for women of childbearing age, as well as early diagnosis and treatment of children with thalassemia. This is currently recognized internationally as the primary and most effective measure for the prevention and control of thalassemia. Currently, laboratory diagnostic techniques for thalassemia fall into two main categories: screening and genetic diagnosis. Common screening methods include complete blood count analysis, red blood cell morphology, and hemoglobin electrophoresis. These methods provide preliminary screening for anemia, red blood cell morphology, hemoglobin ratio, and structural abnormalities. These methods are simple to use and relatively affordable, but they can miss quiescent thalassemia.

[0003] Genetic diagnostic methods are more accurate but also relatively complex, technically demanding, and expensive. Currently, the commonly used thalassemia genetic testing methods in clinical laboratories are gap-PCR combined with agarose gel electrophoresis to diagnose large deletion mutations in the three α-thalassemia genes, or PCR amplification and reverse dot blot hybridization to diagnose point mutations in the three non-deletion α-thalassemia genes and 17 β-thalassemia genes. Both methods are cumbersome and require specialized laboratory and testing equipment. Multiple ligation probe amplification involves hybridization of probes to target DNA sequences. Deletions or duplications within the target range are detected through ligation, PCR amplification, and next-generation sequencing. First-generation Sanger sequencing is the gold standard for verifying point mutations, enabling direct and comprehensive detection of the location and type of point mutations in PCR products. These two methods are suitable for analyzing rare mutations, deletions, or duplications but are not suitable for routine testing of large samples. Second-generation sequencing (NGS) and third-generation sequencing can detect all known and unknown mutations in target sequences within the globin gene cluster as well as regulatory and modifier genes. However, these methods are costly and time-consuming, requiring expensive equipment and specialized bioinformatics analysis. The above-mentioned genetic diagnosis methods for thalassemia based on PCR amplification and product analysis rely on strictly partitioned PCR laboratories or high-throughput sequencing laboratories to avoid nucleic acid contamination. In addition, the equipment is not portable and therefore cannot meet the methodological requirements for large-scale molecular diagnostic screening of populations in medically backward areas.

[0004] In addition, technologies such as real-time fluorescence quantitative PCR and high-performance liquid chromatography (HPLC) have also been used for genetic diagnosis of thalassemia. Real-time fluorescence quantitative PCR allows for automated, closed-tube detection of thalassemia deletions, duplications, and point mutations, but it places high demands on sample pretreatment, experimental procedures, and instrument performance. There have also been reports of instrumenting amplification product analysis using high-resolution melting curve analysis or HPLC to detect thalassemia point mutations, but interpretation of the results requires specialized expertise and specialized instrumentation. Other technologies, such as matrix-assisted laser desorption ionization time-of-flight mass spectrometry, PCR-flow cytometry hybridization, and gene chips, each offer advantages for detecting thalassemia gene mutations, but all rely on specialized instrumentation. These methods, due to limitations such as high testing costs, expensive instrumentation, and high personnel requirements, are not suitable for remote areas with limited medical equipment, technology, and personnel.

[0005] Existing thalassemia genetic diagnostic technologies have certain limitations when used in remote areas. Therefore, it is necessary to develop a detection method that combines the advantages of simple operation and low cost of screening technology with the advantages of good specificity and high sensitivity of diagnostic technology, while also having the characteristics of rapidity and portability. This will make up for the methodological deficiencies in thalassemia genetic screening at the grassroots level and in underdeveloped medical areas, and further improve the large-scale population prevention and control system for thalassemia.

[0006] CRISPR-Cas12a (also known as Cpf1) is an endonuclease in the CRISPR-Cas system, which has important applications in gene editing and molecular diagnostics. CRISPR-Cas12a belongs to the Class 2, Type V, CRISPR-Cas system and is an RNA-guided endonuclease. The most commonly used Cas12a proteins are AsCas12a from the Acidaminococcus BV3L6 strain and LbaCas12a from the Lachnospiraceae family.

[0007] The application of the CRISPR-Cas12a system in pathogen or disease detection involves two steps: 1. Nucleic acid amplification of the sample to be tested; 2. The CRISPR system's cleavage reaction. Nucleic acid molecules are amplified using techniques such as PCR or isothermal amplification, while the CRISPR system's specific recognition, rapid response, and signal amplification enhance detection efficiency.

[0008] Cas12a can specifically recognize and cut double-stranded DNA (dsDNA) targets with a PAM sequence (5'-TTN-3' or 5'-TTTN-3), generating sticky ends. The recognition and cutting of single-stranded DNA (ssDNA) targets do not depend on the PAM sequence. The crRNA of Cas12a is significantly shorter than that of other types of CRISPR systems, and the secondary structure is also simpler, which means that the design, synthesis, and transcription of crRNA will be more economical and convenient. The CRISPR-Cas12a system is widely used in nucleic acid detection, including the detection of new coronaviruses, HPV viruses or other bacteria, mycoplasmas and other pathogens. It has the advantages of being fast and accurate, and can improve detection efficiency. Although the CRISPR-Cas12a system has the above-mentioned advantages in application detection, its application in multiple detection is still very rare, and designers need to make corresponding design countermeasures to truly realize its advantages. Summary of the Invention

[0009] Based on this, the purpose of the present invention is to provide a detection kit and detection method based on multiple RPA-multiple CRISPR-Cas12a, which can be well used for rapid screening and diagnosis of thalassemia.

[0010] The first aspect of the present invention is to provide a kit for detecting beta-thalassemia based on multiple CRISPR-LbaCas12a, which includes a crRNA-specific recognition sequence, wherein the crRNA-specific recognition sequence includes the following sequence for at least one mutant:

[0011] SEQ ID NO: 11 for βCD41-42 / βN type,

[0012] SEQ ID NO: 12 and SEQ ID NO: 13 for βIVS-Ⅱ-654 / βN type,

[0013] SEQ ID NO: 17 for β-28 / βN type,

[0014] SEQ ID NO: 18 for βCD17 / βN type,

[0015] SEQ ID NO: 19 for βCD26 / βN.

[0016] In some embodiments, the crRNA-specific recognition sequence includes the following five mutant sequences: SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19.

[0017] In some embodiments, PCR primers are further included, and the sequences of the PCR primers are shown in SEQ ID NO: 1 and SEQ ID NO: 2.

[0018] In some embodiments, multiple RPA amplification reaction primers are also included, and the multiple RPA amplification reaction primers include those shown in SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and SEQ ID NO: 8.

[0019] In some embodiments, the usage ratio of SEQ ID NO: 12 to SEQ ID NO: 13 is 1:(2-5), more preferably 1:2.

[0020] In some embodiments, the usage ratio of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19 is (3±0.1):(1±0.1):(2±0.1):(3±0.1):(3±0.1):(3±0.1), more preferably: 3:1:2:3:3:3.

[0021] In some embodiments, the signal output of the detection kit is through real-time fluorescence quantitative detection.

[0022] In some embodiments, the signal output of the detection kit is a portable smart phone detecting the end point fluorescence signal intensity.

[0023] In some embodiments, the signal output mode of the detection kit is chromatography test strip detection.

[0024] A second aspect of the present invention is to provide a method for detecting β-thalassemia, which method uses any of the above-mentioned kits and comprises the following steps:

[0025] S1: Obtain the DNA genome of the sample to be tested;

[0026] S2: Amplify the DNA genome of the sample to be tested to obtain an amplified product;

[0027] S3: Using the crRNA specific recognition sequence, LbaCas12a protein, amplified product as a template, FQ6C probe or FN6C probe in a reaction system to perform CRISPR-Cas12a cis-cleavage and trans-cleavage reactions;

[0028] S4: Perform signal detection.

[0029] In some embodiments, the amplified product is obtained by amplification using PCR primers or multiplex RPA amplification reaction primers.

[0030] In some embodiments, the reaction system of the multiplex RPA amplification reaction includes: 4.8 μL of 10 μM upstream and downstream primers, 13.2 μL of water, 2 μL of DNA genome, 5 μL of magnesium acetate I, and a total volume of 50 μL. The reaction procedure of the multiplex RPA amplification reaction is 37-39° C. for 20-30 min.

[0031] In some embodiments, the reaction system of the CRISPR-Cas12a cleavage reaction includes: 10×reaction buffer 2 μL, 5 μM FQ6C probe or 5 μM FN6C probe 0.5-1 μL, 5 μM LbaCas12a protein 0.2-0.8 μL, 5 μM crRNA 0.2-0.8 μL, template DNA 0.5-8 μL, and a total volume of 20 μL; the reaction procedure of the CRISPR-Cas12a cis cleavage is: 37° C., 30-60 min.

[0032] By designing appropriate crRNA-specific recognition sequences, the present invention can multiplex and simultaneously mix six crRNA combinations of cr41-2, cr654-4, cr654-4-U3G, cr28-2, cr17-5, and cr26-1 to cut mutant samples (βCD41-42 / βN, βIVS-Ⅱ-654 / βN, β-28 / βN, βCD17 / βN, and βCD26 / βN) to obtain a strong fluorescence signal and a high signal-to-noise ratio. Moreover, through further appropriate PCR primers or multiple RPA amplification, as well as optimized crRNA combinations, the nonspecific recognition signal generated by wild-type samples is significantly reduced, the signal difference between mutant and wild-type samples is increased, the sensitivity and specificity of the detection are greatly improved, and highly accurate differentiation between wild-type and β-thalassemia mutation samples is achieved.

[0033] The detection kit and detection method described in the present invention can achieve rapid and portable detection of common mutation types of thalassemia. By adopting technical means such as RPA isothermal amplification technology, CRISPR-Cas12a diagnosis, and intelligent device detection, this has important practical significance and broad clinical application value for molecular screening of thalassemia genes in people in areas with poor medical facilities such as grassroots, townships, and remote mountainous areas, as well as scientific research on thalassemia, helping the country to reduce birth defects and improve the quality of the population. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1Agarose gel electrophoresis of PCR amplification products, M represents the molecular weight of DNA.

[0035] Figure 2 Fluorescence curves of CRISPR-Cas12a trans-cleavage based on PCR amplification and a single crRNA. The top column shows the trans-cleavage fluorescence curve after a 60-minute CRISPR reaction, and the bottom column shows the fluorescence signal-to-noise ratio after a 30-minute reaction.

[0036] Figure 3 Fluorescence curves of CRISPR-Cas12a diagnostics based on PCR amplification and introduction of mismatched crRNA. The three graphs on the left show the fluorescence curves of trans-cleavage after a 60-minute CRISPR reaction, and the graph on the right shows the fluorescence signal-to-noise ratio after a 30-minute reaction.

[0037] Figure 4 Fluorescence curves for PCR-based quadruple and quintuple CRISPR-Cas12a simultaneous diagnosis of thalassemia. The top image shows the composition and ratio of different crRNA combinations. The bottom image shows the trans-cleavage fluorescence curve after a 60-minute CRISPR reaction.

[0038] Figure 5 Statistical graph of the fluorescence signal-to-noise ratio of PCR-based quadruplex and quintuplex CRISPR-Cas12a diagnostics, showing the results after 30 minutes of reaction.

[0039] Figure 6 Agarose gel electrophoresis of triplex RPA amplification products. M represents the DNA molecular weight.

[0040] Figure 7 Fluorescence curves of CRISPR-Cas12a trans-cleavage based on triple RPA amplification and single crRNA. The upper figure shows the trans-cleavage fluorescence curve after 60 minutes of CRISPR reaction, and the lower figure shows the fluorescence signal-to-noise ratio after 30 minutes of reaction.

[0041] Figure 8 Fluorescence curves of quintuple CRISPR-Cas12a diagnostics based on triple RPA. The upper figure shows the composition and ratio of different crRNA combinations, and the lower figure shows the trans-cleavage fluorescence curve of a 60-minute CRISPR reaction.

[0042] Figure 9 Statistical graph of the fluorescence signal-to-noise ratio of the triple-RPA-based quintuple CRISPR-Cas12a synchronous diagnosis of thalassemia, showing the results after 30 minutes of reaction.

[0043] Figure 10Validation results of the PCR-CRISPR / crSix platform combined with real-time fluorescence quantitative simultaneous diagnosis of five types of β-thalassemia mutations in clinical samples. The upper left figure shows the trans-cleavage fluorescence curve of CRISPR / crSix detecting different types of thalassemia mutations; the upper right figure shows the fluorescence signal-to-noise ratio after 30 minutes of CRISPR reaction; the lower left figure shows the fluorescence intensity after 30 minutes of CRISPR reaction, with each point representing a sample; the lower right figure shows the ROC working curve after 30 minutes of CRISPR reaction. Figure 11 The PCR-CRISPR / crSix platform combined with test strips was used to simultaneously diagnose clinical samples of five β-thalassemia mutation types. The color development of only the C line indicated that no mutation sequence was detected, while the color development of the T line indicated that a mutation sequence was detected.

[0044] Figure 12 Validation results of clinical samples for the simultaneous diagnosis of five β-thalassemia mutation types using the multiplex RPA-CRISPR platform combined with real-time fluorescence quantification. The upper left figure shows the trans-cleavage fluorescence curve of CRISPR / crSix detecting different types of thalassemia mutation samples; the upper middle figure shows the trans-cleavage fluorescence intensity statistical graph of different types of mutation samples; the upper right figure shows the fluorescence signal-to-noise ratio after 30 minutes of CRISPR reaction; the lower left figure shows the fluorescence intensity (each point represents a sample) and the ROC working curve after 30 minutes of CRISPR reaction; and the lower right figure shows the fluorescence intensity and ROC working curve after 60 minutes of CRISPR reaction.

[0045] Figure 13 Methodological comparison of the multiplex RPA-CRISPR platform and traditional PCR-RBD in diagnosing different types of thalassemia mutation samples.

[0046] Figure 14 Validation results of clinical samples using a multiplex RPA-CRISPR platform combined with smartphone photography for simultaneous diagnosis of five β-thalassemia mutation types.

[0047] Figure 15 Statistical analysis results of clinical sample validation using a multiplexed RPA-CRISPR platform combined with smartphone photography for simultaneous diagnosis of five β-thalassemia mutation types. The left figure shows the fluorescence intensity statistics for each sample after 60 minutes of CRISPR reaction, and the right figure shows the receiver operating characteristic (ROC) curve.

[0048] Figure 16 The multiplex RPA-CRISPR platform combined with test strips was used to simultaneously diagnose clinical samples of five β-thalassemia mutation types. The color development of only the C line indicated that no mutation sequence was detected, while the color development of the T line indicated that a mutation sequence was detected.

[0049] Figure 17Comparative results of PCR-based CRISPR detection technology screening crRNA that recognizes single-base mutations. DETAILED DESCRIPTION

[0050] To facilitate understanding of the present invention, the present invention will be described more fully below. The present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the understanding of the present disclosure more thorough and comprehensive.

[0051] Experimental procedures in the following examples, where specific conditions are not specified, generally followed conventional conditions, such as those in Molecular Cloning: A Laboratory Manual (4th edition, edited by Green and Sambrook, published in 2013), or according to manufacturer recommendations. All commonly used chemical reagents used in the examples were commercially available.

[0052] Unless otherwise defined, all technical and scientific terms used herein have the same meanings as commonly understood by those skilled in the art to which this invention pertains. The terms used in this specification are for the purpose of describing specific embodiments only and are not intended to limit the invention. The term "and / or" as used herein includes any and all combinations of one or more of the associated listed items.

[0053] Definitions To facilitate understanding of this technology, certain terms and phrases are defined below.

[0054] Recombinase Polymerase Amplification (RPA) is an isothermal nucleic acid amplification technology that rapidly amplifies specific DNA or RNA nucleic acid sequences under constant temperature conditions of 37-42°C through the synergistic action of three enzymes: recombinase, single-strand binding protein, and DNA polymerase. The principle is: first, the recombinase protein forms a complex with the primer and searches for homologous sequences in the double-stranded DNA. The primer is then inserted into the homologous site through the chain displacement reaction of the recombinase, and the single-strand binding protein stably displaces the DNA chain. Subsequently, the recombinase is decomposed, which causes the 3' end of the primer to be chain-displaced and bind to the polymerase, causing it to extend the primer. By repeating this process cyclically, exponential amplification of the target region on the template is achieved.

[0055] The present invention first adopts two different technologies for pre-amplification of nucleic acids, including PCR technology and multiplex RPA amplification technology. The two amplification technologies can be flexibly selected in different scenarios.

[0056] Thalassemia is mostly caused by single-base mutations. In CRISPR-Cas12a diagnostics, the use of a suboptimal protospacer adjacent motif (PAM) can slow the reaction kinetics of Cas12-mediated cis-cleavage of the DNA substrate and trans-cleavage of the fluorescent reporter DNA. We have exploited the properties of the suboptimal PAM to improve the specificity of Cas12 in recognizing single-base mutations. In addition, introducing mismatched bases during crRNA design can also increase the differential signal of single-base recognition, thereby achieving specific and accurate recognition of single-base mutations.

[0057] In addition, we achieved the simultaneous detection of multiple mutation sites in thalassemia using multiple crRNA mixtures without reducing the sensitivity and specificity of the detection, and achieved good compatibility between the multiplex CRISPR diagnostic platform and PCR amplification and RPA amplification technologies.

[0058] Traditional PCR also presents challenges with nucleic acid contamination and aerosols, but uncapped nucleic acid diagnostics can fundamentally address these issues. We demonstrated the method's practicality by using a one-pot multiplexed RPA-CRISPR assay for samples carrying five common β-thalassemia mutations, completing the assay in approximately one hour. Based on this platform, we have also expanded three signal output methods: real-time fluorescence quantitative detection, portable smartphone detection, and test strip visualization.

[0059] Our strategy provides a rapid, portable, and accurate solution for multi-locus simultaneous detection of genetic diseases such as thalassemia and may further advance CRISPR-based diagnostics.

[0060] The present invention is further described in detail below with reference to specific embodiments.

[0061] Example 1 Establishment of Multiple RPA-Multiple CRISPR-Cas12a Detection Kit

[0062] 1. Experimental Procedure

[0063] 1.1 Sample collection and genomic DNA extraction

[0064] A total of 64 samples from patients with confirmed thalassemia genes were collected, including 39 samples diagnosed as carrying β-thalassemia mutations (βCD41-42 / βN in 11 cases, βIVS-Ⅱ-654 / βN in 9 cases, β-28 / βN in 7 cases, βCD17 / βN in 8 cases, and βCD26 / βN in 4 cases), and 25 samples confirmed as not carrying the 17 common β-thalassemia genotypes. 2 mL of venous peripheral blood was collected from each case.

[0065] The genomic DNA of each of the above samples was extracted using a kit (OMEGA, Blood DNA Kit).

[0066] 1.2 PCR amplification

[0067] A PCR kit (TAKARA, Premix Taq) was used for amplification. Primers were synthesized by Suzhou Jinweizhi Co., Ltd. The primer sequences were: HBB-F: 5'-ACGGCTGTCATCACTTAGAC-3' (SEQ ID No: 1), HBB-R: 5'-CTCCCACATTCCCTTTTTAG-3' (SEQ ID No: 2): The PCR reaction procedure was: pre-denaturation at 98°C for 3 minutes; 98°C for 20 seconds, 51°C for 30 seconds, 72°C for 2 minutes, amplification for 35 cycles; and extension at 72°C for 7 minutes. The amplified products were detected and analyzed by 2% agarose gel electrophoresis ( Figure 1 ).

[0068] 1.3RPA and multiple RPA

[0069] RPA amplification reaction was performed using a kit (JIENNUO, DNA isothermal Amplification Kit; Qitian, RAA nucleic acid amplification kit). The RPA primer sequences were as follows: β2-F: 5'-CCAGTGCCAGAAGAGC CAAGGACAGGTAC-3' (SEQ ID No: 3), β2-R: 5'-AGTCTTCTCTGTCTCCACATGCCC AGTTTCTA-3' (SEQ ID No: 4), β3-F: 5'-AGAAGACTCTTGGGTTTCTGATAGGCACT G-3' (SEQ ID No: 5), β3-R: 5'-GAAAACATCAAGCGTCCCATAGACTCACC-3' (SEQ ID No: 6), β4-F: 5'-CCTAATCTCTTTCTTTCAGGGCAATAATGAT-3' (SEQ ID No: 7), β4-R: 5'-ACTCAGAATAATCCAGCCTTATCCCAACCAT-3' (SEQ ID No: 8). Reaction system: Take one tube of dry powder, add 25 μL of Vbuffer, 4.8 μL of 10 μM upstream and downstream primers, 13.2 μL of water, 2 μL of genomic DNA, and 5 μL of magnesium acetate I to a total volume of 50 μL. Mix thoroughly. RPA reaction program is 37°C for 20-30 minutes. Use a PCR instrument or a thermostatic metal bath or water bath to maintain the temperature. The amplified product is detected and analyzed by 2% agarose gel electrophoresis ( Figure 6 ). 1.4 Preparation of crRNA and probe

[0070] CrRNA is prepared by direct synthesis and synthesized by Suzhou Jinweizhi Company. The crRNA sequence includes a scaffold sequence (fixed part) and a spacer sequence (variable part), and the spacer sequence specifically recognizes the DNA nucleic acid target in a base complementary pairing manner and cuts the target. The crRNA sequence is shown in Table 1, wherein the crRNA scaffold sequence is crLb 5'-UAAUUUCUACUAAGUGUAGAU-3' (SEQ ID No: 9). After the Cas12 protein is activated, it randomly trans-cuts the surrounding single-stranded DNA reporter probes, FQ6C probes (5'-FAM-CCCCCC-BHQ-3') or FN6C probes (5'-FAM-CCCCCC-Biotin-3'), and the probes are directly synthesized by Suzhou Jinweizhi Company.

[0071] Table 1. crRNA sequences

[0072]

[0073]

[0074] 1.5 CRISPR-Cas12a cis- and trans-cleavage

[0075] Use EZassay's LbaCas12a protein. The reaction system is: 10×reaction buffer 2μL, 5μM FQ6C probe (5'-FAM-CCCCCC-BHQ-3') or 5μM FN6C probe (5'-FAM-CCCCCC-Biotin-3') 0.5-1μL, 5μM Cas12a protein 0.2-0.8μL, 5μM crRNA 0.2-0.8μL, template DNA 0.5-8μL, total volume 20μL. The reaction program is: 37°C, 60min, using a real-time fluorescence quantifier (detecting fluorescence signal once per minute) or other thermostat for reaction.

[0076] 1.6 Blue light meter and CRISPR test strip signal output

[0077] After the reaction is complete, the fluorescence signal is observed using a blue light meter and photographed with a smartphone. The results are processed and analyzed using ImageJ software. The CRISPR test strip uses the FN6C probe. After the CRISPR reaction is complete, the test strip is tested as follows: 10 μL of the reaction product is added to 40 μL of pure water, and the strip is inserted into a lateral flow strip (Warbio, CRISPR Cas12 / 13 HybriDetect test strip). The color development is observed within 10 minutes.

[0078] 1.7 One-tube RPA-CRISPR

[0079] Add the RPA amplification system to the top of the centrifuge tube lid in a volume of 4 μL, and the CRISPR / Cas12 cleavage system to the bottom of the centrifuge tube in a volume of 16 μL. Incubate at 37°C for 20 minutes, then centrifuge briefly to mix, and continue incubating at 37°C for 30-60 minutes. Signal output can be obtained using a real-time fluorescence quantification instrument, a blue light analyzer, or CRISPR test strips.

[0080] 1.8 Statistics

[0081] GraphPad Prism 8 was used for statistical analysis and plotting, and the t-test and ROC curve were used as statistical methods.

[0082] 2. Experimental Results

[0083] 2.1 Suboptimal PAM sequences improve the specificity of CRISPR-Cas12a in recognizing single-base mutations in dsDNA targets

[0084] We used PCR technology to specifically amplify genomic DNA from peripheral blood samples, and used CRISPR / Cas to trans-cleave the surrounding single-stranded DNA fluorescent reporter probes, and then used a real-time fluorescence quantitative PCR instrument to monitor the changes in fluorescence signals. The five mutation sites to be detected are all located on the HBB gene, of which CD41 / 42 is a 4-base deletion, and the other four genotypes are single-clip mutations or insertions. First, the target fragment was successfully amplified by PCR ( Figure 1 ). For each mutation site, a crRNA-specific recognition sequence was designed. Since the optimal PAM sequence canonical PAM (cPAM) "TTTN" or "TTN" around these mutation sites is very limited, we selected a suboptimal PAM sequence ( Figure 2 LbaCas12a / crRNA was used to specifically recognize and cleave the PCR amplification product, and the fluorescence curve generated by the trans-cleavage reporter probe FQ6C was used to calculate the fluorescence intensity signal-to-noise ratio of the mutant and wild-type samples relative to the negative control.

[0085] During the design process, we also used LbaCas12a / crRNA in Table 2 to specifically recognize and cleave PCR amplification products. Based on the fluorescence curve generated by the trans-cleavage reporter probe FQ6C and the calculation of the fluorescence intensity signal-to-noise ratio of mutant and wild-type samples relative to the negative control, we found that suboptimal PAM sequences, such as "CCCA", "TTGC", "TCTG", and "CCTG", mostly have better specificity when identifying single-base mutations, especially the "CCTG" sequence.

[0086] However, the crRNA sequences in Table 2 are difficult to accurately identify single-base mutations. Figure 17 Detection results of some crRNA sequences in .

[0087] Table 2. crRNA sequences

[0088]

[0089]

[0090] Figure 2 Among them, the crRNA corresponding to cr41-2 LbCas12a is cr41-2, the crRNA corresponding to cr654-4 LbCas12a is cr654-4, the crRNA corresponding to cr654-6 LbCas12a is cr654-6, the crRNA corresponding to cr28-2 LbCas12a is cr28-2, the crRNA corresponding to cr17-5 LbCas12a is cr17-5, and the crRNA corresponding to cr26-1 LbCas12a is cr26-1.

[0091] Blank is the blank control, NTC is the amplification control without template, CD41, IVS654, -28, CD17, and CD26 are the sample results of βCD41-42 / βN, βIVS-Ⅱ-654 / βN, β-28 / βN, βCD17 / βN in 8 cases, and βCD26 / βN in 4 cases, respectively.

[0092] 2.2 Exploring the introduction of exogenous single-base mutations into crRNA to improve CRISPR diagnostic specificity

[0093] Since the specificity of multiple crRNAs designed for the IVS-Ⅱ-654 mutation site was not ideal, in order to further reduce the non-specific recognition of single-base differences, we introduced an exogenous single-base mismatch in the crRNA recognition sequence, so that the crRNA had a 1-base difference from the mutant type and a 2-base difference from the wild type. We designed 2 and 1 crRNA recognition sequences (cr654-4-U3G, cr654-4-C6A, cr654-6-C7A) for cr654-4 and cr654-6 respectively. All three crRNAs significantly reduced the non-specific recognition signal generated by the wild-type sample and increased the signal difference between the mutant and wild-type samples. Among them, cr654-4-U3G had the best effect. We selected this crRNA for subsequent multiple CRISPR diagnosis ( Figure 3 ).

[0094] 2.3 Multiple CRISPR strategies to screen for β-thalassemia gene mutations

[0095] We designed different crRNA combinations (see Table 3 below) for CRISPR quadruple and quintuple gene diagnosis ( Figure 4 ) and used a fluorescent quantitative PCR instrument to detect the fluorescent signal of trans-cleavage. In the quadruple and quintuple diagnoses, the combination containing cr654-4 had a stronger cleavage signal for the IVS-Ⅱ-654 sample than the cr654-4-U3G group, but its signal intensity and signal-to-noise ratio for recognizing -28 were significantly reduced. While cr654-4-U3G did not affect the recognition effect of -28, its fluorescence for the IVS-Ⅱ-654 sample was weaker than that of other genotypes. We unexpectedly found that the 6 crRNA combinations that mixed cr654-4 and cr654-4-U3G increased the fluorescence signal of the IVS-Ⅱ-654 sample and had less negative impact on other samples ( Figure 5 ), so we subsequently selected the crSix group for single-tube multiple screening diagnosis of thalassemia.

[0096] Table 3 crRNA combinations and molar ratios

[0097]

[0098] 2.4 CRISPR detection of β-thalassemia mutation sites based on multiplex RPA

[0099] Since PCR reaction time is long and requires a professional PCR instrument, in order to develop a more portable diagnostic system, RPA isothermal amplification technology was introduced to replace PCR technology. The fragment size amplified by RPA is generally short (no more than 500bp), and multiple RPA is limited. We successfully constructed a multiple RPA amplification system by specially designing primers and optimizing amplification conditions. Using three pairs of primers (SEQ ID NO: 3, SEQ ID NO: 4, SEQ ID NO: 5, SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8), we can amplify all five common sites of β-thalassemia, and the amplified fragment sizes are 232bp, 318bp, and 367bp respectively. Figure 6 ).

[0100] Based on multiplex RPA amplification, a single crRNA was first verified. The six crRNAs designed previously could accurately identify mutant and wild-type samples. The fluorescence signals and signal-to-noise ratios generated by the cleavage of RPA products were also similar to those of PCR products ( Figure 7 ).

[0101] Subsequently, different combinations of 5 and 6 crRNAs were designed for multiple CRISPR cutting. The 6 crRNA combination of cr654-4 and cr654-4-U3G produced stronger fluorescence and higher signal-to-noise ratio in cutting mutant samples ( Figure 8 In addition, we also verified that the addition ratio of cr654-4 and cr654-4-U3G was 1:2, which was better than 1:5. We subsequently selected the crSix-1:2 group for multiple RPA-CRISPR screening and diagnosis of thalassemia ( Figure 9 ).

[0102] Example 2 Clinical sample validation based on PCR-multiplex CRISPR method

[0103] We also collected 32 clinical samples for validation based on the PCR-multiplex CRISPR method (refer to the crSix-1:2 group in Example 1). The 32 samples included 4 normal controls, 5 α-thalassemia patients who did not carry β-thalassemia, and 23 patients with 5 different genotypes of β-thalassemia (5 cases of βCD41-42 / βN, 5 cases of βIVS-Ⅱ-654 / βN, 5 cases of β-28 / βN, 5 cases of βCD17 / βN, and 3 cases of βCD26 / βN). Two signal output methods, fluorescence quantitative PCR instrument and CRISPR test strips, were used. The results showed that there was a significant difference in fluorescence signals between thalassemia mutation samples and normal samples ( Figure 10 ), the fluorescence signal of mutant samples increased by more than 20 times compared with the negative control, and increased by more than 3 times compared with the wild-type samples ( Figure 10 ). The receiver operating characteristic (ROC) curves for wild-type and mutant samples were further plotted, and the area under the curve (AUC) was 1, indicating that the PCR-multiplex CRISPR-fluorescence method can accurately identify β-thalassemia mutations with 100% accuracy.

[0104] In addition, CRISPR test strips were also used to verify 21 clinical samples, including 6 normal controls and 15 patients with 5 different genotypes of β-thalassemia (3 cases of βCD41-42 / βN, 3 cases of βIVS-Ⅱ-654 / βN, 3 cases of β-28 / βN, 3 cases of βCD17 / βN, and 3 cases of βCD26 / βN). The test strip method can also accurately distinguish mutant and wild-type samples with 100% accuracy ( Figure 11 ), which provides another optional signal output method for the screening diagnosis of β-thalassemia.

[0105] Example 3

[0106] One-tube method-multiple RPA-multiple CRISPR clinical sample validation in different scenarios

[0107] Finally, 64 clinical samples were collected to conduct a validation experiment on the multiplex RPA-multiplex CRISPR platform (refer to the crSix-1:2 group in Example 1). The 64 samples included 25 normal subjects and 39 patients with β-thalassemia carrying five types of gene mutations (11 cases of βCD41-42 / βN, 9 cases of βIVS-Ⅱ-654 / βN, 7 cases of β-28 / βN, 8 cases of βCD17 / βN, and 4 cases of βCD26 / βN). We used physical isolation to add the RPA amplification system to the top of the centrifuge tube and the CRISPR cutting system to the bottom of the centrifuge tube. After 20 minutes of RPA amplification (refer to Example 1), the amplification product was mixed with the cutting system by instantaneous centrifugation, and then reacted for 30-60 minutes (refer to the crSix-1:2 group in Example 1). The results of real-time monitoring of the fluorescence signal and the fluorescence intensity after 30 minutes showed that there were very obvious differences between the wild-type and mutant samples ( Figure 12 ), by calculating the signal-to-noise ratio of wild-type and mutant samples, the RPA-CRISPR method is higher than the PCR-CRISPR method, about 5 times higher, among which the signals of CD41 and CD17 samples are stronger, about 10 times higher than those of the wild type ( Figure 12 ), the genotype of the sample can be preliminarily determined based on the intensity of the fluorescence signal. We created an ROC curve for the fluorescence values ​​at 30 and 60 minutes of the CRISPR reaction, with AUC = 1, indicating that the RPA-CRISPR-fluorescence quantification method can accurately distinguish wild-type and β-thalassemia mutant samples with 100% accuracy ( Figure 12-13 In addition, the portable visual signal output was also used to detect clinical samples. The fluorescence signals of wild-type and mutant samples were significantly different, and the AUC was 1, proving that it is feasible to screen for β-thalassemia using small devices ( Figure 14-15 Finally, a device-free test strip was used for multiple diagnostics, and the test strip method was also able to accurately identify the β-thalassemia mutation ( Figure 16 The portable and device-free platforms expand the application scenarios of the multiplex RPA-multiple CRISPR method in the genetic diagnosis of thalassemia.

[0108] The above-described embodiments merely illustrate several implementations of the present invention, and while their descriptions are relatively specific and detailed, they should not be construed as limiting the scope of the patent. It should be noted that a person skilled in the art would be able to make numerous variations and improvements without departing from the spirit of the present invention, all of which fall within the scope of protection of the present invention. Therefore, the scope of protection of the patent for this invention shall be determined by the appended claims.

Claims

1. A kit for detecting β-thalassemia based on multiple CRISPR-LbaCas12a, characterized in that: It includes crRNA-specific recognition sequences for five mutant types, and the crRNA-specific recognition sequences are as follows: SEQ ID NO: 11 for βCD41-42 / βN type, SEQ ID NO: 12 and SEQ ID NO: 13 for βIVS-Ⅱ-654 / βN type, SEQ ID NO: 17 for β-28 / βN type, SEQ ID NO: 18 for βCD17 / βN type, and SEQ ID NO: 19 for βCD26 / βN.

2. The kit according to claim 1, wherein It also includes multiple RPA amplification reaction primers, which include primer pairs shown in SEQ ID NO: 3 and SEQ ID NO: 4, SEQ ID NO: 5 and SEQ ID NO: 6, and SEQ ID NO: 7 and SEQ ID NO:

8.

3. The kit according to claim 1, wherein The usage ratio of SEQ ID NO: 12 and SEQ ID NO: 13 is 1:(2-5).

4. The kit according to claim 3, wherein The usage ratio of SEQ ID NO: 12 and SEQ ID NO: 13 is 1:

2.

5. The kit according to any one of claims 1 to 4, characterized in that: The usage ratio of SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:17, SEQ ID NO:18, and SEQ ID NO:19 is (3±0.1):(1±0.1):(2±0.1):(3±0.1):(3±0.1):(3±0.1).

6. The kit according to claim 5, wherein The usage ratio of SEQ ID NO: 11, SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 17, SEQ ID NO: 18, and SEQ ID NO: 19 is 3:1:2:3:3:3.

Images