Beta thalassemia detection kit based on multiple CRISPR-LbaCas12a platforms

Abstract

The invention discloses a beta thalassemia detection kit based on multiple CRISPR-LbaCas12a and a detection method, the kit comprises crRNA specific recognition sequences, and the crRNA specific recognition sequences at least comprise SEQ ID NO: 11 aiming at a beta CD41-42 / beta N type, SEQ ID NO: 12 and SEQ ID NO: 13 aiming at a beta IVS-II-654 / beta N type, SEQ ID NO: 17 aiming at a beta-28 / beta N type, SEQ ID NO: 18 aiming at a beta CD17 / beta N type and SEQ ID NO: 19 aiming at a beta CD26 / beta N type. According to the detection kit and the detection method disclosed by the invention, rapid and portable detection of common mutation types of thalassemia can be realized, and the detection kit and the detection method have important practical significance and wide clinical application value for molecular screening of thalassemia genes of crowds in regions with relatively poor medical facility conditions, such as grass-roots, villages and towns, remote mountainous areas and the like.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a beta-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 disease and a highly prevalent single-gene genetic disease worldwide. Its etiology is due to mutations in the globin gene. Depending on the mutated gene, it is mainly divided into α-thalassemia and β-thalassemia. Thalassemia is a difficult but preventable disease. The three-level prevention strategy for thalassemia is to take pre-marital and pre-pregnancy prevention, prenatal prevention, and early diagnosis and treatment of children with thalassemia for the childbearing population. It is also the primary measure and important strategy for the effective prevention and control of thalassemia recognized internationally. At present, the laboratory diagnostic techniques used for thalassemia are mainly divided into two categories: screening and genetic diagnosis. Common screening methods include complete blood cell analysis, red blood cell morphology examination, hemoglobin electrophoresis detection, etc., which can perform preliminary screening of anemia, red blood cell morphology, hemoglobin ratio and abnormal structure. It has the advantages of simple operation and low economic burden, but there is a certain rate of missed diagnosis for the screening of static thalassemia.

[0003] Genetic diagnosis is more accurate, but also relatively complex, with high technical requirements and high costs. The commonly used thalassemia gene detection methods in clinical laboratories are Gap-PCR combined with agarose gel electrophoresis to diagnose large deletion mutations in three α-thalassemia genes, or PCR amplification and reverse dot hybridization to diagnose point mutations in three non-deletion α-thalassemia genes and 17 β-thalassemia genes. The operation process of these two methods is cumbersome and requires corresponding laboratories and detection equipment. Multiple ligation probe amplification is the hybridization of probes with target DNA sequences. Through ligation, PCR amplification and first-generation sequencing, deletions or duplications within the target range are detected. First-generation Sanger sequencing is the gold standard for verifying point mutations. It can directly and comprehensively detect the location and type of point mutations in PCR products. These two methods are suitable for analyzing relatively rare mutations, deletions or duplications, but are not suitable as routine detection methods for large-scale samples. Next-generation sequencing (NGS) and third-generation sequencing can detect all known and unknown mutations in the target sequence of the globin gene cluster and regulatory and modifying genes, but the detection cost is high, time-consuming, and requires expensive instruments and professional bioinformatics analysis. The above-mentioned genetic diagnosis method for thalassemia based on PCR amplification and product analysis relies on strictly partitioned PCR laboratories or high-throughput sequencing laboratories to avoid nucleic acid contamination, and the equipment is not portable, so it 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 are also used for genetic diagnosis of thalassemia. Real-time fluorescence quantitative PCR can detect thalassemia deletions, duplications and point mutations in a closed tube at one time, but it has high requirements for sample pretreatment, experimental operation and instrument performance. There are also reports of using high-resolution melting curve or high-performance liquid chromatography technology to detect thalassemia point mutations and realize the instrumentation of amplification product analysis, but the interpretation of the results requires professional experience and specific instruments. In addition, there are matrix-assisted laser desorption ionization time-of-flight mass spectrometry, PCR-flow fluorescence hybridization, gene chips and other technologies, which have their own advantages for the detection of thalassemia gene mutations, but all rely on special instruments and equipment. Due to the limitations of high detection cost, expensive instruments and high personnel requirements, the above methods are not suitable for remote areas with backward medical equipment, technology and personnel.

[0005] The existing genetic diagnosis technology for thalassemia has 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, to make up for the methodological deficiencies in thalassemia genetic screening at the grassroots level and in medically underdeveloped areas, and to further improve the large-scale population prevention and control system for thalassemia.

[0006] CRISPR-Cas12a (also known as Cpf1) is a nuclease belonging to the CRISPR-Cas system, which has important application value in the fields of gene editing and molecular diagnosis. CRISPR-Cas12a belongs to the Class 2 V type of the CRISPR-Cas system and is an RNA-guided nuclease. The most commonly used Cas12a proteins are AsCas12a from the BV3L6 strain of the genus Acidaminococcus and LbaCas12a from the family Lachnospiraceae.

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

[0008] Cas12a can specifically recognize and shear double-stranded DNA (dsDNA) targets with PAM sequences (5'-TTN-3' or 5'-TTTN-3) to generate sticky ends. The recognition and shearing of single-stranded DNA (ssDNA) targets do not depend on the PAM sequence. The crRNA of Cas12a is significantly shorter than 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 coronavirus, HPV virus or other bacteria, mycoplasma 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 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 also included, and the sequences of the PCR primers are shown in SEQ ID NO:1 and SEQ ID NO:2.

[0018] In some of the 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), and more preferably is: 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 of the embodiments, the signal output mode of the detection kit is the intensity of the fluorescent signal at the end point detected by a portable smart phone.

[0023] In some of the 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, amplification 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 of the embodiments, the amplified product is obtained by amplification using PCR primers or multiplex RPA amplification reaction primers.

[0030] In some of the embodiments, the reaction system of the multiple 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 multiple 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 2μL, 5μM FQ6C probe or 5μM FN6C probe 0.5-1μL, 5μM LbaCas12a protein 0.2-0.8μL, 5μM crRNA 0.2-0.8μL, template DNA 0.5-8μL, and a total volume of 20μL; the reaction procedure of the CRISPR-Cas12a cis-cleavage is: 37° C., 30-60min.

[0032] The present invention can design appropriate crRNA specific recognition sequences, and can multiplex and once mix six crRNA combinations of cr41-2, cr654-4, cr654-4-U3G, cr28-2, cr17-5, and cr26-1 to obtain stronger fluorescence signals and high signal-to-noise ratios when cutting mutant samples (βCD41-42 / βN, βIVS-Ⅱ-654 / βN, β-28 / βN, βCD17 / βN, βCD26 / βN). Moreover, through further appropriate PCR primers or multiple RPA amplifications and optimized crRNA combinations, the nonspecific recognition signals generated by wild-type samples are 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 distinction between wild-type and β-thalassemia mutant samples is achieved.

[0033] The detection kit and detection method described in the present invention can realize the rapid and portable detection of common mutation types of thalassemia. By adopting RPA isothermal amplification technology, CRISPR-Cas12a diagnosis, intelligent device detection and other technical means, this has important practical significance and broad clinical application value for the molecular screening of thalassemia genes for 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 CRISPR-Cas12a trans-cleavage fluorescence curve based on PCR amplification and single crRNA. The upper column is the trans-cleavage fluorescence curve of CRISPR reaction for 60 minutes, and the lower column is the fluorescence signal-to-noise ratio at 30 minutes of reaction.

[0036] Figure 3 Fluorescence curves of CRISPR-Cas12a diagnosis based on PCR amplification and introduction of mismatched crRNA. The three figures on the left are trans-cleavage fluorescence curves after 60 minutes of CRISPR reaction, and the figure on the right is the fluorescence signal-to-noise ratio after 30 minutes of reaction.

[0037] Figure 4 Fluorescence curves of PCR-based quadruple and quintuple CRISPR-Cas12a simultaneous diagnosis of thalassemia. The information above shows the composition and ratio of different crRNA combinations. The figure below shows the trans-cleavage fluorescence curve of the CRISPR reaction for 60 minutes.

[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 triple RPA amplification products. M represents the molecular weight of DNA.

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

[0041] Figure 8 Fluorescence curves of quintuple CRISPR-Cas12a diagnosis based on triple RPA, where the upper figure shows the composition and proportion of different crRNA combinations, and the lower figure shows the trans-cleavage fluorescence curve of the CRISPR reaction for 60 minutes.

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

[0043] Fig.10The clinical sample validation results of the PCR-CRISPR / crSix platform combined with real-time fluorescence quantitative simultaneous diagnosis of five types of β-thalassemia mutations. The upper left figure is the trans-cleavage fluorescence curve of CRISPR / crSix detecting different types of thalassemia mutation samples; the upper right figure is the fluorescence signal-to-noise ratio after 30 minutes of CRISPR reaction; the lower left figure is the fluorescence intensity after 30 minutes of CRISPR reaction, and each point represents a sample; the lower right figure is the ROC working curve after 30 minutes of CRISPR reaction. Fig.11 The PCR-CRISPR / crSix platform combined with the test strips was used to simultaneously diagnose clinical samples of five types of β-thalassemia mutations. The color development of 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] Fig.12 The clinical sample validation results of the multiplex RPA-CRISPR platform combined with real-time fluorescence quantitative simultaneous diagnosis of five β-thalassemia mutation types, among which, the upper left figure is the trans-cleavage fluorescence curve of CRISPR / crSix detecting different types of thalassemia mutation samples; the upper middle figure is the trans-cleavage fluorescence intensity statistical graph of different types of mutation samples; the upper right figure is the fluorescence signal-to-noise ratio after 30 minutes of CRISPR reaction; the lower left figure is the fluorescence intensity after 30 minutes of CRISPR reaction (each point represents a sample) and the ROC working curve; the lower right figure is the fluorescence intensity and ROC working curve after 60 minutes of CRISPR reaction.

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

[0046] Fig.14 The multiplex RPA-CRISPR platform combined with smartphone photography to simultaneously diagnose clinical samples of five β-thalassemia mutation types was validated.

[0047] Fig.15 Statistical analysis results of the validation of clinical samples for the multiplex RPA-CRISPR platform combined with smartphone photography for simultaneous diagnosis of five types of β-thalassemia mutations; the left figure shows the fluorescence intensity statistics of each sample after 60 minutes of CRISPR reaction, and the right figure shows the ROC working curve.

[0048] Fig.16 The multiplex RPA-CRISPR platform combined with the test strips was used to simultaneously diagnose clinical samples of five types of β-thalassemia mutations. Among them, only the C line coloring represented that the mutation sequence was not detected, and the T line coloring represented that the mutation sequence was detected.

[0049] Fig.17Comparison of PCR-based CRISPR detection technology screening crRNAs that recognize single-base mutations. DETAILED DESCRIPTION

[0050] In order to facilitate the 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 disclosure of the present invention more thorough and comprehensive.

[0051] The experimental methods in the following examples without specifying specific conditions are usually carried out according to conventional conditions, such as the fourth edition of Molecular Cloning: A Laboratory Manual edited by Green and Sambrook, published in 2013, or according to the conditions recommended by the manufacturer. The various commonly used chemical reagents used in the examples are all commercially available products.

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

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

[0054] Recombinase Polymerase Amplification (RPA): It 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 with the synergistic action of three enzymes: recombinase, single-stranded binding protein, and DNA polymerase. The principle is: first, the recombinase protein forms a complex with the primer and searches for homologous sequences in double-stranded DNA. Then, the primer is inserted into the homologous site through the chain displacement reaction of the recombinase, and the single-stranded 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 area on the template is achieved.

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

[0056] Most cases of thalassemia are single-base mutations. In CRISPR-Cas12a diagnosis, the use of suboptimal protospacer adjacent motifs (PAMs) can slow down the reaction kinetics of Cas12-mediated cis-cleavage of DNA substrates and trans-cleavage of fluorescent reporter DNA. We use the characteristics of suboptimal PAMs to improve the specificity of Cas12 in recognizing single-base mutations. In addition, the introduction of mismatched bases when designing crRNA 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 of thalassemia with 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 has problems with nucleic acid contamination and aerosols, but nucleic acid diagnosis without opening the lid can solve this problem from the root. We use a one-pot multiplex RPA-multiplex CRISPR method to detect samples carrying five common β-thalassemia mutations to prove the practicality of the method, which can be completed in about 1 hour. Based on this platform, we have also expanded three signal output methods, including 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 (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), 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 the above samples was extracted using a kit (OMEGA, Blood DNA Kit).

[0066] 1.2 PCR amplification

[0067] Amplification was performed using a PCR kit (TAKARA, Premix Taq). Primers were synthesized by Suzhou Jinweizhi Company, and 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 min; 98°C for 20 s, 51°C for 30 s, 72°C for 2 min, amplification for 35 cycles; 72°C for another 7 min. The amplified product was 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: 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 a tube of dry powder, add 25μL of Vbuffer, 4.8μL of 10μM upstream and downstream primers, 13.2μL of water, 2μL of genomic DNA, 5μL of magnesium acetate I, and a total volume of 50μL. Mix thoroughly. The RPA reaction program is 37℃ for 20-30min. Use a PCR instrument or a constant temperature metal bath or water bath to maintain the temperature. The amplified product was 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 and cuts the target in a base complementary pairing manner. 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 will randomly trans-cut 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 LbaCas12a protein from EZassay. 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 procedure is: 37°C, 60min, using a real-time fluorescence quantifier (detecting fluorescence signals once per minute) or other thermostats for reaction.

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

[0077] After the reaction is completed, a blue light meter is used to observe the fluorescence signal and a smartphone is used to take photos. The results are processed and analyzed using ImageJ software. The CRISPR test strip uses the FN6C probe. After the CRISPR reaction is completed, the test strip is tested. The steps are as follows: take 10 μL of the reaction product, add 40 μL of pure water, insert the lateral flow test strip (Warbio, CRISPR Cas12 / 13HybriDetect test strip), and observe the color development results within 10 minutes.

[0078] 1.7 One-tube RPA-CRISPR

[0079] Add the RPA amplification system to the top of the centrifuge tube lid with a volume of 4 μL, and add the CRISPR / Cas12 cutting system to the bottom of the centrifuge tube with a volume of 16 μL. After reacting at 37°C for 20 minutes, centrifuge to mix, and continue to react at 37°C for 30-60 minutes. Use three methods to output the signal: real-time fluorescence quantification instrument, blue light instrument or CRISPR test strips.

[0080] 1.8 Statistics

[0081] GraphPad Prism8 was used for statistical analysis and graphing, and 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 cut the PCR amplification product, and the fluorescence curve generated by the trans-cut reporter probe FQ6C was calculated, and the fluorescence intensity signal-to-noise ratio of the mutant and wild-type samples relative to the negative control was calculated.

[0085] During the design, we also used LbaCas12a / crRNA in Table 2 to specifically recognize and cut the PCR amplification products. According to the fluorescence curve generated by the trans-cutting reporter probe FQ6C, and calculated 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. Fig.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, CD26 are the sample results of βCD41-42 / βN, βIVS-Ⅱ-654 / βN, β-28 / βN, βCD17 / βN 8 cases, and βCD26 / βN 4 cases, respectively.

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

[0093] Since the specificity of multiple crRNAs designed for the IVS-Ⅱ-654 mutation site was not ideal, in order to further reduce the nonspecific 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 with the mutant type and a 2-base difference with the wild type. We designed 2 and 1 crRNA recognition sequences for cr654-4 and cr654-6 respectively (cr654-4-U3G, cr654-4-C6A, cr654-6-C7A). All three crRNAs significantly reduced the nonspecific 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 chose 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 (as shown in 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 stronger cleavage signals for IVS-Ⅱ-654 samples 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 IVS-Ⅱ-654 samples was weaker than that for other genotypes. We unexpectedly found that the combination of 6 crRNAs, especially the mixed use of cr654-4 and cr654-4-U3G, increased the fluorescence signal of IVS-Ⅱ-654 samples, 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 3 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), all 5 common loci of β-thalassemia can be amplified, and the amplified fragment sizes are 232bp, 318bp and 367bp respectively ( Figure 6 ).

[0100] Based on multiple RPA amplification, a single crRNA was first verified. The six crRNAs designed previously can accurately identify mutant and wild-type samples. The fluorescence signal and signal-to-noise ratio generated by cutting the RPA product are also similar to those of the PCR product ( Figure 7 ).

[0101] Subsequently, different combinations of 5 and 6 crRNAs were designed for multiple CRISPR cutting. The 6 crRNA combination of mixed 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 effect of 1:2 was better than 1:5, and we subsequently selected the crSix-1:2 group for multiple RPA-CRISPR screening and diagnosis of thalassemia ( Fig. 9 ).

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

[0103] We also collected 32 clinical samples for verification 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 were used: fluorescent quantitative PCR instrument and CRISPR test strips. The results showed that there was a significant difference in the fluorescence signal between thalassemia mutation samples and normal samples ( Fig.10 ), the fluorescence signal of mutant samples increased by about 20 times compared with the negative control, and increased by more than 3 times compared with the wild-type samples ( Fig.10 ). The ROC curves of wild-type samples and mutant samples were further drawn, 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%.

[0104] In addition, CRISPR test strips were 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 ( Fig.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 5 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 added the RPA amplification system to the top of the centrifuge tube and the CRISPR cutting system to the bottom of the centrifuge tube by physical isolation. After 20 minutes of RPA amplification (refer to Example 1), the amplification product was mixed with the cutting system by instant 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 for 30 minutes showed that there were very obvious differences between the wild-type and mutant samples ( Fig.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 the wild-type ( Fig.12 ), the genotype of the sample can be preliminarily determined based on the intensity of the fluorescence signal. We made a ROC working curve for the fluorescence values ​​of the CRISPR reaction at 30 minutes and 60 minutes, and AUC = 1, indicating that the RPA-CRISPR-fluorescence quantification method can accurately distinguish between wild-type and β-thalassemia mutant samples ( Figure 12-13 ). In addition, the portable visualized signal output was also used to detect clinical samples. There was a significant difference in the fluorescence signals between wild-type and mutant samples, and AUC = 1, proving that it is feasible to screen for β-thalassemia with 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 ( Fig.16 ). The portable platform and the equipment-free platform expand the application scenarios of the multiplex RPA-multiplex CRISPR method in the genetic diagnosis of thalassemia.

[0108] The above-mentioned embodiments only express several implementation methods of the present invention, and the descriptions thereof are relatively specific and detailed, but they cannot be understood as limiting the scope of the invention patent. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, and these all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A kit for detecting β-thalassemia based on multiple CRISPR-LbaCas12a, characterized in that: It includes a crRNA-specific recognition sequence, and the crRNA-specific recognition sequence includes the following sequence for at least one mutant type: 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, SEQ ID NO: 19 for βCD26 / βN.

2. The kit according to claim 1, characterized in that: 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.

3. The kit according to claim 1, characterized in that: It also includes multiple RPA amplification reaction primers, 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.

4. The kit according to claim 1, characterized in that: The usage ratio of SEQ ID NO: 12 to SEQ ID NO: 13 is 1:(2-5), more preferably 1:

2.

5. The kit according to any one of claims 2 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), preferably: 3:1:2:3:3:

3.

6. A method for detecting beta-thalassemia for non-diagnostic purposes, characterized in that: The detection method uses the kit according to any one of claims 1 to 5, comprising the following steps: S1: Obtain the DNA genome of the sample to be tested; S2: Amplify the DNA genome of the sample to be tested to obtain an amplified product; S3: Using the crRNA specific recognition sequence, LbaCas12a protein, amplification product as a template, FQ6C probe or FN6C probe in a reaction system to perform CRISPR-Cas12a cis-cleavage and trans-cleavage reactions; S4: Perform signal detection.

7. The detection method according to claim 6, characterized in that: The amplified product is obtained by amplification using PCR primers or multiplex RPA amplification reaction primers.

8. The detection method according to claim 6, characterized in that: The output mode of the signal detection is through real-time fluorescence quantification or end-point fluorescence intensity detection; or the output mode of the signal detection is chromatography test strip detection.

9. The detection method according to claim 6, characterized in that: The reaction system of the multiple 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 multiple RPA amplification reaction is 37-39° C. for 20-30 min.

10. The detection method according to claim 6, characterized in that: 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.

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