CrRNA containing double differential basic groups and application thereof

Abstract

The invention relates to the technical field of gene detection, in particular to crRNA containing double differential bases and application of the crRNA. The crRNA containing the double differential bases is crRNA having two differential bases with a seed sequence or a sequence containing the seed sequence, and the positions of the two differential bases are any one of the second base, the fourth base, the seventh base, the ninth base, the twelfth base, the fourteenth base, or the seventeenth base and the nineteenth base which are away from the fifth end of the seed sequence. A large number of experiments prove that the crRNA introduced with two differential bases can greatly improve the detection sensitivity in one-step reaction, more importantly, the difference of a single base can be effectively distinguished, and wild type crRNA and single differential base modified crRNA cannot be effectively distinguished.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of gene detection, and particularly relates to a crRNA containing double differential bases and application thereof. BACKGROUND

[0002] Molecular detection technology is a key tool for analyzing nucleic acids in samples in the field of life sciences. Isothermal amplification techniques, such as recombinase polymerase amplification (RPA) and loop-mediated isothermal amplification (LAMP), have occupied an important position in the field of molecular detection due to their simple operation, low cost, strong portability and fast detection speed. These techniques are particularly suitable for point-of-care test (POCT) and can effectively make up for the deficiencies of traditional PCR techniques, including quantitative PCR (qPCR), reverse transcription quantitative PCR (RT-qPCR) and digital droplet PCR (ddPCR) in terms of operational convenience and cost-effectiveness. However, isothermal amplification techniques also have certain limitations, and their non-specific amplification easily produces false positive results, affecting the accuracy of detection.

[0003] The CRISPR / Cas nucleic acid detection method exhibits the characteristics of rapidness, high sensitivity, portability and low cost, and greatly promotes the development of POCT detection methods. However, the CRISPR / Cas detection technology, especially CRISPR / Cas12, is not satisfactory in terms of detection specificity, especially the ability to distinguish single bases.

[0004] The prior art (Genome-wide specificities of CRISPR-Cas Cpf1 nucleases in human cells; Genome-wide analysis reveals specificities of Cpf1 endonucleases in human cells) shows that the closer the distance to the PAM sequence, the more the mismatch can significantly affect the editing efficiency, and the introduction of two mismatches completely cannot edit.

[0005] Based on this, some studies have proposed to distinguish the SARS-CoV-2 G614 / D164 sites by introducing a single mismatched base near the mismatched base and close to the PAM position. However, this technology introduces a mismatched base in the detection substrate (target sequence), and adopts a method of first RT-PCR for amplification and then detection. On the one hand, the time is more than 2 hours, which is too long and has no advantage compared with qPCR. On the other hand, the process of amplification and detection needs to be opened, which is cumbersome and easy to cause aerosol cross contamination and false positives.

[0006] The prior art ([2021-biosensors] Cas12a and Lateral Flow Strip-Based Test for Rapid and Ultrasensitive Detection of Spinal Muscular Atrophy) also shows that a single base cannot be distinguished, and specific amplification primers must be used to distinguish the two templates to achieve accurate detection of the two templates. SUMMARY

[0007] The purpose of the present application is to provide a crRNA containing double difference bases which can distinguish and quickly distinguish single base difference and its application.

[0008] In order to achieve the above purpose, the technical scheme adopted by the present application is as follows:

[0009] A crRNA containing double difference bases, comprising an inherent sequence and a guide sequence, the guide sequence being used to bind to a target gene; the guide sequence has two difference bases with a seed sequence or a sequence containing a seed sequence; the positions of the two difference bases are any one group of the second, fourth, seventh, ninth, twelfth, fourteenth, or seventeenth, nineteenth bases away from the 5' end of the seed sequence.

[0010] The seed sequence is a sequence completely complementary to the target sequence, and the seed sequence is a part of the crRNA.

[0011] The crRNA with a seed sequence completely complementary to the target sequence is crRNA-WT.

[0012] In one preferred embodiment, the seed sequence is any of the following:

[0013] 5'-UUUAUUUUAAACAUGAACAAUCA-3'(SEQ ID NO.146);

[0014] 5'-GUCUAGUGUGUGCCACUUGUGAA-3'(SEQ ID NO.147);

[0015] 5'-GCCCGCCCAAAAUCUGUGAUCUU-3' (SEQ ID NO. 148);

[0016] 5'-CCCCCAGCGCUUCAGCGUUCUUC-3' (SEQ ID NO. 149);

[0017] 5'-AUUUUGUCUGAAACCCUGUAAGG-3' (SEQ ID NO. 150);

[0018] 5'-UGAAGUAGAUAUGGCAGCACAUA-3' (SEQ ID NO. 151);

[0019] 5'-ACAAUAUGUGCUUCUACACAGUC-3' (SEQ ID NO. 152).

[0020] In one preferred embodiment, the inherent sequence is UAAUUUCUACUCUUGUAGAU (SEQ ID NO. 153).

[0021] In one preferred embodiment, the rule of difference is that the C or G base at the original position is mutated to a U base, and the A or U base is mutated to a C base.

[0022] Through a large number of experimental verifications, it is found that although the crRNA with two difference bases introduced can activate the Cas12a cis cleavage activity lower than the unmodified crRNA fragment, it can greatly improve the detection sensitivity in the one-step reaction. More importantly, the double difference base modified crRNA can effectively distinguish single base difference in the one-step reaction, while the wild type crRNA and the single difference base modified crRNA cannot effectively distinguish.

[0023] In one preferred embodiment, when the sequence of the seed sequence is the sequence shown in SEQ ID NO. 146 or SEQ ID NO. 149, the positions of the two difference bases are any one group of the second, fourth, seventh, ninth, twelfth, fourteenth, or seventeenth, nineteenth bases from the 5' end of the seed sequence; preferably the twelfth and fourteenth bases.

[0024] In one preferred embodiment, when the sequence of the seed sequence is the sequence shown in SEQ ID NO. 147, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 151 or SEQ ID NO. 152, the positions of the two difference bases are the twelfth and fourteenth bases from the 5' end of the seed sequence.

[0025] In one preferred embodiment thereof, the target sequence is from a cell, an infectious bacteria or a virus, wherein the cell is any one of a spinal muscular atrophy pathogenic cell, a beta thalassemia pathogenic cell or a tumor cell; the bacteria is any one of a group B streptococcus, Staphylococcus aureus, Mycobacterium tuberculosis, Salmonella, Vibrio cholera, Bacillus anthracis; the virus is any one of a human papillomavirus, SARS-COV-2, hepatitis B virus, Epstein-Barr virus, cytomegalovirus, herpes virus.

[0026] In one preferred embodiment thereof, the crRNA comprising double-difference bases is any one of:

[0027] UAAUUUCUACUCUUGUAGAU UCUCUUUUAAACAUGAACAAUCA (SEQ ID NO. 125);

[0028] UAAUUUCUACUCUUGUAGAU UUUAUUCUCAACAUGAACAAUCA (SEQ ID NO. 126);

[0029] UAAUUUCUACUCUUGUAGAU UUUAUUUUAAAUACGAACAAUCA (SEQ ID NO. 127);

[0030] UAAUUUCUACUCUUGUAGAU UUUAUUUUAAACAUGACCCAUCA (SEQ ID NO. 128);

[0031] UAAUUUCUACUCUUGUAGAU GUCUAGUGUGUuCuACUUGUGAA (SEQ ID NO. 131);

[0032] UAAUUUCUACUCUUGUAGAU GCCCGCCCAAAcUuUGUGAUCUU (SEQ ID NO. 137);

[0033] UAAUUUCUACUCUUGUAGAU CUCUCAGCGCUUCAGCGUUCUUC (SEQ ID NO. 139);

[0034] UAAUUUCUACUCUUGUAGAU CCCCCAUCUCUUCAGCGUUCUUC (SEQ ID NO. 140);

[0035] UAAUUUCUACUCUUGUAGAU CCCCCAGCGCUCCCGCGUUCUUC (SEQ ID NO. 141);

[0036] UAAUUUCUACUCUUGUAGAUCCCCCAGCGCUUCAGCUUCCUUC (SEQ ID NO. 142);

[0037] UAAUUUCUACUCUUGUAGAU AUUUUGUCU G ACAUCCUGUAAGG (SEQ ID NO. 2);

[0038] UAAUUUCUACUCUUGUAGAU UGAAGUAGAUACGUCAGCACAUA (SEQ ID NO. 4);

[0039] UAAUUUCUACUCUUGUAGAU ACAUCCUGUAAGG (SEQ ID NO. 2);

[0040] Based on the same inventive concept, the present application also claims a detection system comprising the crRNA comprising the double differential base.

[0041] In one preferred embodiment thereof, the detection system further comprises RPA amplification reagent, AsCas12a, Cas12a Reaction Buffer, nuclease-free water, FAM-BHQ1 probe and non-specific primer.

[0042] Among them, the RPA amplification reagent is a common commercially available product. For example, DNA constant temperature rapid amplification kit (containing A buffer, B buffer), manufacturer: Ampure Future, product number: WLB8201KIT.

[0043] AsCas12a is a common Cas12 protein, such as disclosed in prior art (Zhou M, Hu Z, Qiu L, et al. Seamless genetic conversion of SMN2 to SMN1 via CRISPR / Cpf1 and single-stranded oligodeoxynucleotides in spinal muscular atrophy patient-specific iPSCs. [J]. Human Gene Therapy, 2018: hum.2017.255. DOI:10.1089 / hum.2017.255; Zeng Q, Zhou M, Hu Z, et al. Rapid and sensitive Cas12a-based one-step nucleic acid detection with ssDNA-modified crRNA [J]. Analytica chimica acta, 2023: 1276. DOI:10.1016 / j.aca.2023.341622; Zeng Q, Zhou M, Deng W, et al. Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA [J]. Analytica Chimica Acta, 2024, 1309(000): 10. DOI:10.1016 / j.aca.2024.342693).

[0044] The Cas12a Reaction Buffer is a commercially available reagent, for example, in the present application, the AsCas12a and the Cas12a Reaction Buffer are both purchased from: Yizhibio, item number: CAS-12A-100A.

[0045] In one preferred embodiment thereof, the sequence of the FAM-BHQ1 fluorescent probe is: FAM-TTATT-BHQ1.

[0046] In one preferred embodiment thereof, the non-specific primer is any one of the following groups:

[0047] S. aureus-RPA-F: CACAGCAATAATAATTGCTTTAGAAAGTCG (SEQ ID NO. 129),

[0048] S. aureus-RPA-R: GTTAGCCATTTGTCTGTGAGACCGATGC (SEQ ID NO. 130);

[0049] influenzaA-RPA-F: GCCAAGGAGGTGTCATTGAGCTATTCAACTG (SEQ ID NO. 132),

[0050] influenzaA-RPA-R: GTAGTAGCCATTTGTCTGTGAGACCGATGC (SEQ ID NO. 133);

[0051] EGFR-RPA-F: gaccctgaattcggatgcagagcttcttcc (SEQ ID NO. 134),

[0052] EGFR-RPA-R: cagctgctgcgagctcacccag (SEQ ID NO. 135);

[0053] COV-RPA-F: AAGGAAATTTTGGGGACCAGGAACTAATCAGA (SEQ ID NO. 143), COV-RPA-R: ATCCAATTTGATGGCACCTGTGTAGGTCAAC (SEQ ID NO. 144);

[0054] AF2: TAAGTAAAATGTCTTGTGAAACAAAATG (SEQ ID NO. 105),

[0055] AR1: TTCACTTTCATAATGCTGGCAGACTTACTC (SEQ ID NO. 104);

[0056] SF2: AAAATGTCTTGTGAAACAAAATGC (SEQ ID NO. 108),

[0057] AR1: TTCACTTTCATAATGCTGGCAGACTTACTC (SEQ ID NO. 104);

[0058] HPV16-RPA-F: TTGTTGGGGTAACCAACTATTTGTTACTGTT (SEQ ID NO. 111), HPV16-RPA-R: CCTCCCCATGTCGTAGGTACTCCTTAAAG (SEQ ID NO. 112);

[0059] HPV18-RPA-F: GCATAATCAATTATTTGTTACTGTGGTAGATACCACT (SEQ ID NO. 117),

[0060] HPV18-RPA-R: GCTATACTGCTTAAATTTGGTAGCATCATATTGC (SEQ ID NO. 118).

[0061] In one preferred embodiment, the detection system further comprises a target sequence from a cell, an infectious bacterium or a virus, wherein the cell is any one of a spinal muscular atrophy pathogenic cell, a beta thalassemia pathogenic cell or a tumor cell; the bacterium is any one of a group B streptococcus, Staphylococcus aureus, Mycobacterium tuberculosis, Salmonella, Vibrio cholerae, Bacillus anthracis; and the virus is any one of a human papilloma virus, SARS-COV-2, hepatitis B virus, Epstein-Barr virus, cytomegalovirus, herpes virus.

[0062] Based on the same inventive concept, the present application also claims a kit comprising the crRNA comprising two differential bases.

[0063] In one preferred embodiment, the kit comprises the detection system.

[0064] The present application is further explained as follows:

[0065] The present application introduces two differential bases into the crRNA to establish a detection technology that is fast and can distinguish single base differences. This technology not only significantly increases the specificity of Cas detection and can effectively distinguish single base differences, but also greatly improves the detection sensitivity in a one-step reaction (compared to crRNA without differential bases or with one or three differential bases).

[0066] The CRISPR-Cas system combined with isothermal amplification can achieve nucleic acid detection at a temperature of 39℃. However, the current CRISPR-Cas system cannot effectively recognize and detect templates with single base differences, or needs to be carried out in two steps to complete the detection, which is easy to cause inaccurate CRISPR detection results or cross contamination risk, resulting in false positive or false negative.

[0067] The application develops double-difference base modified crRNA for rapid and sensitive Cas12a one-step nucleic acid detection. The double-difference base modified crRNA exhibits 100-100000 times higher sensitivity than wild-type crRNA in one-step reaction, and the reason is that the double-difference base modified crRNA reduces the cis cleavage activity of Cas12a, reduces the cleavage of Cas12a protein to target DNA in one-step reaction, so that more target DNA templates can be amplified and accumulated in isothermal amplification, thereby improving the reaction sensitivity; more importantly, the double-difference base modified crRNA can effectively distinguish single base difference, realize high specificity and one-step detection of target nucleic acid.

[0068] The application has the advantages that:

[0069] The application develops double-difference base modified crRNA which exhibits 100-100000 times higher sensitivity than wild-type crRNA in one-step reaction, and can effectively distinguish single base difference, has the advantages of short detection time, high specificity, high sensitivity and simple operation. BRIEF DESCRIPTION OF DRAWINGS

[0070] UAAUUUCUACUCUUGUAGAU The double-difference base modified crRNA designed for the application is shown in the schematic diagram.

[0071] Figure 1 The fluorescence result diagram of crRNA detecting S. aureus substrates containing different double-difference bases in Example 1 of the application is shown in the figure; ****, P<0.0001.

[0072] Figure 2 The fluorescence result diagram of crRNA detecting influenza A virus substrates containing different double-difference bases in Example 1 of the application is shown in the figure; ****, P<0.0001.

[0073] Figure 3 The fluorescence result diagram of crRNA detecting EGFR gene substrates containing different double-difference bases in Example 1 of the application is shown in the figure; NC, negative control, ns, no significance, **, P<0.01, ****, P<0.0001.

[0074] Figure 4 The fluorescence result diagram of crRNA containing double-difference bases detecting S. aureus samples in Example 2 of the application is shown in the figure.

[0075] Figure 5The fluorescence difference value statistical result column chart of the crRNA containing double difference bases in Example 2 of the present application for detecting S. aureus samples for 20 minutes; NC, negative control, ns, no significance, **, P<0.05, **, P<0.01, ***, P<0.001, ****, P<0.0001.

[0076] Figure 6 The fluorescence result chart of the crRNA containing double difference bases in Example 2 of the present application for detecting EGFR samples; NC, negative control.

[0077] Figure 7 The fluorescence difference value result column chart of the crRNA containing double difference bases in Example 3 of the present application for detecting SARS-COV-2 samples for 20 minutes; NC, negative control.

[0078] Figure 8 The column chart of the detection sensitivity of the crRNA-12-14 containing double difference bases in Example 3 of the present application; NC, negative control, ns, no significance, **, P<0.05, **, P<0.01, ****, P<0.0001.

[0079] Figure 9 The column chart of the results of the crRNA-12-14 containing double difference bases in Example 3 of the present application for detecting 54 SARS-COV-2 clinical samples.

[0080] Figure 10 The fluorescence result chart of the crRNA containing double difference bases in Example 4 of the present application for detecting SMA.

[0081] Figure 11 The ultraviolet light chart of the crRNA containing double difference bases in Example 4 of the present application for detecting SMA by ultraviolet light method.

[0082] Figure 12 The fold line chart of the RPA primer optimization in Example 4 of the present application.

[0083] Figure 13 The ultraviolet light chart of the SMA clinical sample detected by the ultraviolet light method in Example 4 of the present application using the optimized primer and crRNA 10-12-14.

[0084] Figure 14 The fold line chart of the detection lower limit of the crRNA containing double difference bases and wild type crRNA in Example 5 of the present application for detecting HPV 16 and HPV 18.

[0085] Figure 15Bar chart of results of detecting single base difference of HPV16 and HPV18 by crRNA containing double difference bases in Example 5 of the present application; ns, no significance, **, P<0.01, ****, P<0.0001.

[0086] Figure 16 Bar chart of results of detecting HPV clinical samples by crRNA containing double difference bases in Example 5 of the present application; ns, no significance, ****, P<0.0001.

[0087] Figure 17 Graph of results of detecting double difference cis-cleavage activity in Example 6 of the present application.

[0088] Figure 18 Schematic diagram of the technical principle of the present application. DETAILED DESCRIPTION

[0089] The present application is not limited to the following specific embodiments, and those skilled in the art can implement the present application in other various specific embodiments according to the disclosed content of the present application, or any simple changes or modifications made by using the design structure and ideas of the present application, all fall within the protection scope of the present application. It should be noted that the embodiments in the present application and the features in the embodiments can be combined with each other without conflict.

[0090] Reagents involved in the present application:

[0091] 1. DNA isothermal rapid amplification kit (Changzhou Amp Future, WLB8201KIT): A Buffer, B Buffer, freeze-dried powder reagent;

[0092] 2. RNA isothermal rapid amplification kit (Changzhou Amp Future, WLRB8207KIT): A Buffer, B Buffer, freeze-dried powder reagent;

[0093] 3. Cas12a protease reagent (Shenzhen Yizhi Biology, CAS-12-100A): AsCas12a, 10x Cas12a Reaction Buffer;

[0094] 4. CRISPR nucleic acid detection test strip (Suzhou Geneno);

[0095] 5. dNTP 10mM Mixture (Shanghai Biotech, B500056-0500);

[0096] 6. Nuclease-free water (Thermo, AM9930);

[0097] 7. 10000×GelRed dye (US Everbright, S2001).

[0098] Example 1

[0099] crRNA bidifferential base position design

[0100] 1. Double-chain annealing experiment

[0101] (1) Design of annealed single-stranded deoxyribonucleic acid and crRNA

[0102] like Figure 19 As shown, based on the sequences of Staphylococcus aureus and Influenza A retrieved from the USCS Genome Browser, a sequence containing PAM (TTTV, where V represents the three bases other than T) was randomly selected. Wild-type (WT) and double-stranded deoxyribonucleic acid (DDNA) sequences were designed with mutations at positions 2 / 4, 7 / 9, 12 / 14, and 17 / 19 of the PAM sequence (complementary mutations between the two sequences, following the rule: C / GT; A / TC). Furthermore, differential bases (i.e., mutated bases, with complementary mutations between the two sequences, following the rule: C / GT; A / TC) were introduced into some double-differential templates at positions 5, 10, or 15 of the PAM sequence as simulated differential sites, which were then combined with double-differential sites to form 2 or 3 differential sites. Similarly, for the EGFR gene, wild-type (EGFR-WT) and double-stranded deoxyribonucleic acid (DNA) sequences were designed with mutations at positions 2 / 4 and 12 / 14 of the PAM sequence (complementary mutations between the two sequences, following the rule: C / GT; A / TC). Further, differential bases (i.e., mutated bases, with complementary mutations between the two sequences, following the rule: C / GT; A / TC) were introduced at positions 5, 7, 10, 15, or 15 of the PAM sequence in some double-differential templates as simulated differential sites. crRNAs targeting *S. aureus*, *Influenza A*, and EGFR were designed based on the WT sequences at each site, and all crRNAs were synthesized by Shanghai Sangon Biotech.

[0103] The detection template and crRNA used in this embodiment are as follows:

[0104] Table 1. Detection templates and crRNAs designed for Staphylococcus aureus.

[0105]

[0106]

[0107]

[0108]

[0109] Table 2 Detection templates and crRNAs designed for Influenza A

[0110]

[0111]

[0112]

[0113] Table 3 Detection templates and crRNAs designed for EGFR lung cancer

[0114]

[0115]

[0116]

[0117] (2) Double-stranded annealing

[0118] The centrifuged upstream and downstream ssODNs were dissolved with nuclease-free water. To avoid the activation of crRNA by binding with the complementary ssODN, the crRNA complementary strand was diluted to 10 mM, and the non-complementary ssODN was diluted to 100 mM. In the PCR tube, 18 mL of ddH2O, 1 mL of 100 mM non-complementary ssODN, and 1 mL of 10 mM complementary ssODN were added, mixed by blowing, and then spotted for annealing in the PCR instrument (95°C denaturation for 3 min, and then annealing to 25°C at a rate of 0.1°C / s).

[0119] 2. CRISPR / Cas12a detection of annealed double-stranded

[0120] (1) CRISPR / Cas12a pre-detection of annealed double-stranded

[0121] Because the concentration of the double-stranded complex formed after annealing is high, the fluorescence signal may reach the platform stage in a very short time after fluorescence detection, which is not conducive to analysis and exploration. Therefore, the concentration of the double-stranded complex after annealing was first diluted and then pre-detected to determine the optimal detection concentration.

[0122] The double-stranded annealed from WT ssODN was diluted with nuclease-free water to 1, 0.1, 0.01, 0.001 times. The detection system takes S. aureus as an example: each reaction is repeated 3 times, and the final detection result is the fluorescence average of the three wells. In each tube of the eight-tube, add 2 μL of 1 μM AsCas12a, 2 μL of 1 μM crRNA, 2 μL of 10x Cas12a Reaction Buffer and 13.5 μL of nuclease-free water, invert and mix, and then incubate at room temperature for 10 min after spotting. After incubation, add 0.5 μL of FAM-BHQ1 fluorescent probe (the sequence of FAM-BHQ1 probe is: FAM-TTATT-BHQ1) in the dark, gently blow and mix with the pipette, and then spot. In order to start the reaction at the same time, cover the PCR tube with annealed double-stranded product and nuclease-free water (instead of annealed double-stranded product) in the blank control, after the addition is completed, cover the eight-tube cover, invert and mix, spot, and quickly place in the CFX96 fluorescence PCR instrument, 39°C for 60 min, detect the fluorescence signal once every minute.

[0123] The reaction system of each well is as follows:

[0124]

[0125] After the detection is completed, select the concentration with moderate fluorescence signal response and rise according to the fluorescence signal trend chart for the next step detection.

[0126] (2) CRISPR / Cas12a detection of annealed double-stranded

[0127] According to the pre-detection of annealed double-stranded results, select the optimal detection concentration of S. aureus, Influenza A and EGFR, and detect the double-stranded without WT, double-difference base and three-difference base. The detection system is the same as (1).

[0128] The detection results of S. aureus, Influenza A and EGFR show that the introduction of double-difference sites can distinguish single base difference, especially the introduction of double-difference sites at a distance of 12 / 14 from the PAM sequence, which can effectively distinguish single base difference at a distance of 5-15 from the PAM sequence. Figure 1 The detection results show that the introduction of double-difference bases at a distance of 2 / 4, 7 / 9 and 17 / 19 from the PAM sequence can effectively distinguish single base difference at a distance of 5-15 from the PAM sequence in S. aureus and Influenza A, and the introduction of double-difference bases at a distance of 12 / 14 from the PAM sequence can not only effectively distinguish single base difference at a distance of 5-15 from the PAM sequence in S. aureus and Influenza A, but also effectively distinguish single base difference at a distance of 5-15 from the PAM sequence in EGFR. Figures 2-4 ​

[0129] Example 2

[0130] Double difference base crRNA detection of S.aureus, Influenza A and EGFR

[0131] 1. crRNA design of S.aureus

[0132] In Example 1, according to the sequences of S.aureus, Influenza A and EGFR, difference bases were introduced on the detection template. The results showed that double difference bases at 12 / 14 positions in the crRNA recognition region and distance from the PAM sequence could effectively distinguish single base differences at 5-15 positions from the PAM sequence. In order to further verify the detection effect of 12 / 14 double difference bases, the present application sets double difference on crRNA to detect S.aureus. According to the sequence of S.aureus, S.aureus-crRNA-2-4, S.aureus-crRNA-7-9, S.aureus-crRNA-12-14, S.aureus-crRNA-17-19 were designed in the sequence recognition region of S.aureus-crRNA-WT (bold sequence), wherein 2 / 4, 7 / 9, 12 / 14, 17 / 19 are the positions of introducing difference bases at the 5' end of the sequence recognition region of S.aureus-crRNA-WT (bold sequence).

[0133] Table 4 crRNA and primers of S.aureus, Influenza A and EGFR

[0134]

[0135]

[0136] 2. One-step detection of S.aureus (10 μL detection system)

[0137] Take one example of S.aureus clinical sputum sample and one example of control sputum sample, heat lysis at 95℃, and use S.aureus-crRNA-WT, S.aureus-crRNA-2-4, S.aureus-crRNA-7-9, S.aureus-crRNA-12-14, S.aureus-crRNA-17-19 for one-step detection, the detection system is as follows:

[0138] (1) RPA reaction system configuration

[0139] In the dry powder reaction tube, 29.4 μL of A buffer, 2 μL of 10 μM S-RPA-F, 2 μL of 10 μM S-RPA-R and 13.1 μL of nuclease-free water were sequentially added. The dry powder reaction tube was inverted several times to mix well and completely dissolve the freeze-dried powder in the tube. Centrifuge immediately and place on ice for standby. The reaction system table is as follows:

[0140]

[0141] (2) Cas12a one-step fluorescence detection of double-difference crRNA:

[0142] In the PCR tube, 0.3 μL of nuclease-free water, 0.2 μL of 10×Cas12a Reaction Buffer, 0.25 μL of 1 μM AsCas12a and 0.25 μL of 1 μM crRNA were sequentially added, mixed well, centrifuged immediately, and incubated at room temperature for 10 min. After incubation, 6 μL of RPA reaction system from (1) was added to the PCR tube, and 0.5 μL of 10 μM FAM-BHQ1 fluorescent probe was added in the dark. The PCR tube was inverted to mix well, and 7.5 μL was taken from it and added to an eight-tube. 2 μL of lysed sputum and 0.5 μL of B buffer were added to the eight-tube cap. After the eight-tube cap was closed, it was inverted to mix well, and centrifuged immediately. It was placed in a fluorescence instrument, set at 39°C, and the fluorescence signal was detected every minute. Each site was repeated three times, and the mean value of the three fluorescence signals was finally calculated.

[0143] The total reaction system is as follows:

[0144]

[0145] The results show that S. aureus-crRNA-WT, S. aureus-crRNA-2-4, S. aureus-crRNA-7-9, S. aureus-crRNA-12-14 and S. aureus-crRNA-17-19 can detect S. aureus, but the difference between the fluorescence values of S. aureus-crRNA-2-4, S. aureus-crRNA-7-9, S. aureus-crRNA-12-14, S. aureus-crRNA-17-19 patient sputum and normal sputum is significantly higher than that of S. aureus-crRNA-WT. S. aureus-crRNA-12-14 has the highest fluorescence value in detecting patient sputum, followed by S. aureus-crRNA-17-19, and the fluorescence values of normal sputum detected by the two are not significantly different from the blank control Figures 2-4 and Figure 5)。Similarly, the detection of influenza A showed that the difference in fluorescence value of influenza A-crRNA-12-14 was the highest in the detection of patient nasopharyngeal swabs.

[0146] For the lung cancer EGFR mutation site, the present application uses EGFR-crRNA-4-12-14 and EGFR-crRNA-WT detection, and the results show that EGFR-crRNA-4-12-14 with double mismatched bases can effectively distinguish patients carrying EGFR mutations from normal people, while EGFR-crRNA-WT cannot distinguish them. Figure 6 )。

[0147] Example 3

[0148] Double difference base crRNA detection of new coronavirus (SARS-CoV-2)

[0149] 1. crRNA design of SARS-CoV-2

[0150] Similarly, the present application sets double differences on crRNA to detect SARS-CoV-2. According to the sequence of SARS-CoV-2, COV-crRNA-WT targeting SARS-CoV-2 is designed. In the sequence recognition region (bold sequence) of COV-crRNA-WT, difference genes are introduced at specific positions to design COV-crRNA-2-4, COV-crRNA-7-9, COV-crRNA-12-14, COV-crRNA-17-19, wherein 2 / 4, 7 / 9, 12 / 14, 17 / 19 are the positions of introducing differences at the 5' end of the sequence recognition region (bold sequence) of COV-crRNA-WT.

[0151] Table 5 crRNA and primers of SARS-CoV-2

[0152]

[0153] 2. One-step detection of SARS-CoV-2 (10 μL detection system)

[0154] Take 1 case of SARS-CoV-2 clinical nasopharyngeal swab sample and 1 case of normal nasopharyngeal swab sample, and use COV-crRNA-WT, COV-crRNA-2-4, COV-crRNA-7-9, COV-crRNA-12-14, COV-crRNA-17-19 for one-step detection, and the detection system is as follows:

[0155] (1) RT-RPA reaction system configuration

[0156] In the dry powder reaction tube, 29.4 μL of A buffer, 2 μL of 10 μM COV-RPA-F, 2 μL of 10 μM COV-RPA-R and 13.1 μL of nuclease-free water were sequentially added. The dry powder reaction tube was inverted up and down several times to completely dissolve the freeze-dried powder in the tube. Centrifugation was performed instantly and the tube was placed on ice for standby. The reaction system table is as follows:

[0157]

[0158]

[0159] (2) Cas12a one-step fluorescence detection of double-difference crRNA (same as Example 2)

[0160] The results show that COV-crRNA-WT, COV-crRNA-2-4, COV-crRNA-7-9, COV-crRNA-12-14 and COV-crRNA-17-19 can effectively distinguish patients from normal people at 20 minutes Figure 7 ).

[0161] 3. Sensitivity of one-step detection of SARS-CoV-2

[0162] Continue to take 1 case of SARS-CoV-2 clinical nasopharyngeal swab sample and 1 case of normal nasopharyngeal swab sample, use nucleic acid extraction kit (Shengxiang Biological, S20025) to extract RNA, dilute the two nasopharyngeal swab samples to 40 ng / μL, 4 ng / μL, 4×10 -1 ng / μL, 4×10 -2 ng / μL, 4×10 -3 ng / μL, 4×10 -4 ng / μL, 4×10 -5 ng / μL, 4×10 -6 ng / μL, respectively. One-step detection was performed using COV-crRNA-WT and COV-crRNA-12-14, and the results showed that COV-crRNA-WT could detect 4×10 - 1 ng / μL, and COV-crRNA-12-14 could detect 4×10 -4 ng / μL, with a 1000-fold improvement in sensitivity Figure 8 ). The sensitivity of COV-crRNA-2-4, COV-crRNA-7-9 and COV-crRNA-17-19 was significantly improved compared with COV-crRNA-WT, but was inferior to COV-crRNA-12-14.

[0163] 4. Detection of SARS-CoV-2 clinical samples

[0164] 54 clinical samples were collected, and double-blind method was used to detect the samples by qPCR kit (2019-nCoV nucleic acid detection kit, Shanghai Biomedicine Technology Co., Ltd., item number: ZC-HX-201-2) and COV-crRNA-12-14 one-step method, respectively. The system of COV-crRNA-12-14 one-step method is as above. The results are shown in Table 6 and Table 7. Figure 9 The results show that the detection rate of COV-crRNA-12-14 is 100%, which is completely consistent with the accuracy of qPCR detection (Table 6). Figure 9

[0165] Table 6 qPCR and COV-crRNA-12-14 detection of new coronavirus clinical samples

[0166]

[0167] Example 4

[0168] Optimization of double-difference base crRNA for detecting spinal muscular atrophy (SMA)

[0169] 1. crRNA design for SMA

[0170] The detection results of S. aureus, Influenza A and EGFR site show that setting double-difference bases at 12 / 14 positions on the crRNA away from the PAM sequence can effectively distinguish the difference of single base at 5-15 positions away from the PAM sequence. Double-difference crRNA is set for the pathogenic gene SMN1 of genetic disease SMA. Similarly, according to the SMN1 gene sequence searched by USCS Genome Browser, crRNA-SMN1-WT targeting the 7th exon of SMN1 gene is designed, and then crRNA 10-12-14 and crRNA 10-2-4 are designed according to the recognition substrate region of crRNA-SMN1-WT, i.e. the bold region, wherein the 12 / 14 and 2 / 4 positions are double-difference bases introduced by the present application, and the position 10 is the difference base of SMN1 and SMN2 at the 7th exon (SMN1 c.840C, SMN2 c.840T, 6th base of the 7th exon), which is 10 bases away from the specific position (the specific position is the base corresponding to the PAM sequence TTTC of the template on the crRNA).

[0171] Table 7 primers and crRNA designed for SMA

[0172]

[0173] 2. Fluorescence detection efficiency of crRNA 10-12-14​

[0174] Clinical samples of normal people and 1 case of SMA patient were detected by crRNA-SMN1-WT, crRNA 10-2-4 and crRNA 10-12-14, respectively. The detection system is as follows:

[0175] (1) RPA reaction system

[0176] Add 29.4 μL A buffer, 2 μL 10 μM AF1, 2 μL 10 μM AR2 and 13.1 μL nuclease-free water into the dry powder reaction tube in turn. Mix the dry powder reaction tube up and down several times to completely dissolve the freeze-dried powder in the tube. Centrifuge instantly and place on ice for standby. The reaction system table is as follows:

[0177]

[0178] (2) Cas12a one-step fluorescence detection of double-difference crRNA:

[0179] Add the following into the PCR tube in turn: 0.3 μL nuclease-free water, 0.2 μL 10x Cas12a Reaction Buffer, 0.25 μL 2 μM AsCas12a and 0.25 μL 1 μM crRNA, 6 μL RPA reaction system in (1), 0.5 μL 10 μM FAM-BHQ1 fluorescent probe and 0.5 μL B buffer in the dark. Mix well, centrifuge instantly, and incubate at room temperature for 10 min. After incubation, add 2 μL gDNA and cap the eight-tube cap. Mix well after capping the eight-tube cap, centrifuge instantly. Place in the fluorescence instrument, set 39℃, detect the fluorescence signal every minute. Repeat three times for each site, and finally calculate the mean value of the three fluorescence signals.

[0180] The total reaction system is as follows:

[0181]

[0182]

[0183] The results show that the detection efficiency of crRNA 10-12-14 is significantly higher than that of crRNA-SMN1-WT and crRNA 10-2-4. At 15 min, the fluorescence value of crRNA 10-12-14 for SMN1 is 3.56 times that of crRNA 10-2-4 ( Figure 10 ).

[0184] 3. Double-difference base crRNA combined with ultraviolet light detection

[0185] To further reduce the detection cost while realizing visualization, the present application combines double difference base crRNA with ultraviolet light for detection. The detection system is as follows:

[0186] (1) RPA reaction system configuration

[0187] Add 29.4 μL of A buffer, 2 μL of 10 μM AF1, 2 μL of 10 μM AR2 and 13.1 μL of nuclease-free water in the dry powder reaction tube in turn. Mix well by repeatedly inverting the dry powder reaction tube up and down to completely dissolve the freeze-dried powder in the tube. Spot and place on ice for standby. The reaction system is as follows:

[0188]

[0189] (2) Ultraviolet light detection

[0190] Dilute AsCas12a to 2 μM, dilute crRNA 10-12-14 to 1 μM, and dilute normal human and SMA clinical samples to 4 ng / μL. In the PCR tube, add 0.25 μL of 2 μM AsCas12a, 0.25 μL of 1 μM crRNA, 0.2 μL of 10×Cas12a Reaction Buffer and 0.3 μL of nuclease-free water in turn. After gentle mixing with a pipette, spot, incubate at room temperature for 10 min. Then add 0.5 μL of 10 μM FAM-BHQ1 fluorescent probe and 6 μL of RPA reaction system, and add 0.5 μL of B buffer and 2 μL of 4 ng / μL gDNA to the tube cover. Mix well by inverting up and down, spot, place under ultraviolet light to take pictures and save, and mark as 0 min. Then place the PCR tube in a metal bath at a constant temperature of 39℃, and set the incubation time to 1 h. Place the reaction tube under ultraviolet light to take pictures and save every 10 min.

[0191] At 50 minutes, crRNA 10-12-14 can effectively distinguish between normal people and SMA patients. It is shown that setting double difference bases at positions 12 / 14 can have detection Figure 11 ).

[0192] (3) Optimization of ultraviolet light detection system

[0193] In order to further improve the detection efficiency of the ultraviolet light method, 12 pairs of primers (AF1-AR1, AF2-AR2, AF1-AR2, AF2-AR1, AF1-AR3, AF2-AR3, SF2-SR2, SF2-AR1, SF2-AR2, SF2-AR3, AF1-SR2, AF2-SR2) are designed to amplify the 7th exon of the SMN1 gene, and compared with the primers used in the fluorescence detection and ultraviolet light detection described above, and the gDNA of normal people and SMA patients is subjected to fluorescence detection. The detection system is as described above.

[0194] The results show that the primers AF2-AR1 and SF2-AR1 have the earliest rising fluorescence signal Figure 12 , so one pair of primers is selected for further ultraviolet light one-step detection of SMA clinical samples.

[0195] The specific operation is as follows: the RPA reaction system is configured as in (1), and the primer AF2-AR1 is used. The ultraviolet light detection is as in (2), the metal bath is incubated at 39℃ for 35 min, and the photographing under ultraviolet light and saving are performed.

[0196] The results are shown in Figure 13 , among 22 SMA clinical samples, crRNA 10-12-14 can effectively detect SMA patients at 35 min, and the detection sensitivity and specificity reach 100%.

[0197] Example 5

[0198] crRNA 12-14 detects HPV16 / 18

[0199] 1. Design of crRNA and RPA primers

[0200] According to the sequences of HPV16 and HPV18, a sequence containing PAM (shown as SEQ ID NO. 115 and SEQ ID NO. 122) is selected, HPV16-crRNA-WT and HPV18-crRNA-WT targeting the sequence are designed, the recognition substrate in HPV16-crRNA-WT is different, that is, the bold bases, the 12th / 14th and 10th / 12th / 14th 5th from the bold bases are introduced into the difference bases, and are named as HPV16-crRNA 12-14 and HPV16-crRNA 10-12-14 and HPV18-crRNA 12-14 and HPV18-crRNA 10-12-14, a total of 6 crRNAs. At the same time, the RPA primers of HPV16 and HPV18 are designed, which are directly synthesized by Shanghai Shengong Bio.

[0201] The crRNAs and primers synthesized in this example and the target sequences detected are as follows:

[0202] Table 8 Primers, crRNA and detection templates designed for HPV16

[0203]

[0204]

[0205] Table 9 crRNA designed for HPV18

[0206]

[0207]

[0208] 2. HPV16 / 18 clinical sample collection

[0209] (1) With the patient's informed consent and signed informed consent form, the hospital collects the patient's sample. The hospital collects the female cervical mucus sample and divides it into 1.5 mL EP tubes. Centrifuge at 500g for 5 min at room temperature, discard the supernatant.

[0210] (2) Lysis at 95℃ for 3 min, ready for detection, the remaining sample is stored at -20℃ or -80℃

[0211] 3. Detection of HPV16 / 18 clinical samples

[0212] 1) Extraction of gDNA from HPV16 / 18 clinical samples

[0213] (1) Prepare lysis solution (40 mL): 50 mM Tris Buffer (pH = 8.3), 200 μL 0.5% TritonX-100, add ddH2O to 40 mL;

[0214] (2) Divide the clinically collected female cervical mucus sample into 1.5 mL EP tubes and label them. Centrifuge at 500g for 5 min at room temperature, discard the supernatant;

[0215] (3) Add 50 μL / tube of prepared lysis solution to the EP tube, mix well with a shaker after addition, point off, lysis at 95℃ for 5 min; After point off, the crude lysis sample is obtained and stored at -80℃.

[0216] 2) Detection limit

[0217] Take 1 case of crude lysis of HPV16 clinical sample, dilute the HPV16 clinical sample to 300 ng / μL, 30 ng / μL, 3 ng / μL, 3 x 10 -1 ng / μL, 3 x 10 -2 ng / μL, 3 x 10 -3 ng / μL, 3 x 10-4 ng / μL, 3×10 -5 ng / μL, 3×10 -6 Nine concentration gradients (ng / μL) were established. The targets at each of the nine concentration gradients were detected using HPV16-crRNA, HPV16-crRNA 12-14, and HPV16-crRNA 10-12-14. The detection system was a one-step fluorescence assay using double-differential-base crRNA binding to Cas12a, as described in point 2 of Example 4, with primers HPV16-RPA-F / R used.

[0218] Similarly, one crudely lysed HPV18 clinical sample was taken, diluted to the same concentration, and the target at nine concentration gradients was detected using HPV18-crRNA, HPV18-crRNA 12-14, and HPV18-crRNA 10-12-14. The detection system was a one-step fluorescence assay using double-differential-base crRNA bound to Cas12a, and the primers used were the same as in point 2 of Example 4, using HPV18-RPA-F / R.

[0219] The results are as follows Figure 14 As shown in the figure. The results showed that when the detection time was 20 min, the detection sensitivity of HPV16-crRNA 12-14 was 100 times that of HPV16-crRNA-WT, and the sensitivity of HPV18-crRNA 12-14 reached 1000 times that of HPV18-crRNA-WT; when the detection time was extended to 30 min, the sensitivity of HPV18-crRNA 12-14 reached 100,000 times that of HPV18-crRNA-WT. Figure 15 ).

[0220] At a sample concentration of 3×10 -1 At concentrations above 3 × 10⁻¹⁴ ng / μL, the fluorescence value of HPV16-crRNA 12-14 was significantly higher than that of HPV16-crRNA 10-12-14, indicating that the detection efficiency with two differential bases was much higher than with three differential bases. Similarly, at concentrations above 3 × 10⁻¹⁴ ng / μL, the fluorescence value of HPV16-crRNA 12-14 was significantly higher than that of HPV16-crRNA 10-12-14, indicating that the detection efficiency with two differential bases was much higher than that with three differential bases. - 2 The fluorescence value of HPV18-crRNA 12-14 was significantly higher than that of HPV18-crRNA 10-12-14 at ng / μL, indicating that the detection efficiency of introducing two differential bases was much higher than that of introducing three differential bases. Figure 15 ).

[0221] 3) HPV clinical sample testing

[0222] Ten clinical samples of HPV were collected, and one-step detection was performed using HPV16-crRNA 12-14 and HPV18-crRNA 12-14, respectively. The detection system was a double-difference base crRNA combined with Cas12a one-step fluorescence. The HPV16 primers used HPV16-RPA-F / R, and the HPV18 primers used HPV18-RPA-F / R. The results showed that the samples carrying HPV16 could be detected by HPV16-crRNA 12-14, the samples carrying HPV18 could be detected by HPV18-crRNA 12-14, and the clinical samples carrying HPV16 and HPV18 could be detected by HPV16-crRNA 12-14 and HPV18-crRNA 12-14 at the same time. The detection results were completely consistent with the detection results of the PCR-chip hybridization method (kit name: Beijing Bohui Innovative Biotechnology Group Co., Ltd., Human Papillomavirus Nucleic Acid Detection Kit (PCR-chip hybridization method), Medical Device Registration Certificate No.: Guoshengzhunqu 20163401108) used in clinical practice. Figure 16

[0223] Example 6

[0224] Mechanism of double-difference detection

[0225] 1. RPA amplification of HPV18

[0226] -20°C refrigerator to take out A buffer, placed on ice to melt. Add 29.4 μL A buffer to each dry powder tube, and mix well with a pipette. After the dry powder is completely dissolved, add 2 μL 10 μM HPV18-RPA-F and 2 μL 10 μM HPV18-RPA-R, respectively. Then add 1 μL nucleic acid template and 13.1 μL nuclease-free water to the reaction tube in turn. Then add 2.5 μL B buffer to the reaction tube and mix well. After centrifuging the reaction tube with a palm centrifuge, it is placed in a metal bath and reacted at 39°C for 30 min. The RPA reaction system is as follows:

[0227]

[0228] 2. cis cleavage experiment

[0229] ​After the super-clean bench was sterilized, 8 200 μL PCR tubes were taken, 16 μL of 2 μM AsCas12a and 16 μL of 1 μM HPV18-crRNA 12-14 were added to one of them, and then 16 μL of 10x Cas12a Reaction Buffer and 80 μL of nuclease-free water were added. The PCR tube was placed at room temperature for incubation, and the incubation time was 10 min. A group of HPV18-crRNA was configured in the same way. After incubation, the liquid in the PCR tube was mixed thoroughly, and then it was briefly centrifuged. 16 μL of the incubated reaction solution was taken and dispensed into the remaining 7 PCR tubes. 4 μL of RPA product in (1) was added to the caps of the 7 PCR tubes, which were mixed by inverting and then briefly centrifuged. Then they were quickly placed in a metal bath at 39℃ and incubated for 0 min, 0.5 min, 2 min, 5 min, 10 min, 20 min and 40 min, respectively. After incubation, the products were heated at 85℃ for 10 min to terminate the reaction, and the cis cleavage experiment products were obtained.

[0230] 3. Polyacrylamide gel electrophoresis

[0231] HPV18-crRNA, HPV18-crRNA 12-14 cis cleavage experiment products were subjected to polyacrylamide gel electrophoresis (step for reference existing technology [2024-ACA] Sensitive and visual detection of SARS-CoV-2 using RPA-Cas12a one-step assay with ssDNA-modified crRNA). The results are shown in Figure 17 . The results show that the RPA product using HPV18-crRNA without introducing differences is quickly cleaved, while HPV18-crRNA 12-14 is relatively slow Figure 18 ).

[0232] Therefore, this indicates that the introduction of double-difference crRNA 12-14 in the one-step method reduces the cis cleavage ability, and Cas12a reduces the cleavage of target DNA in the reaction, so that more products are used for RPA amplification, so that RPA can amplify and accumulate a large amount of target DNA, thereby improving the detection sensitivity. The schematic diagram of the assumed principle is shown in Figure 18 Figure 19 .

[0233] It should be noted that the above examples are only used to clearly illustrate the present application, and are not intended to limit the embodiments of the present application. For those skilled in the art, on the basis of the above description, other different forms of changes or variations can also be made. Here, all the embodiments cannot be exhausted. Any obvious changes or variations derived from the technical scheme of the present application are still within the scope of protection of the present application.

Claims

1. A crRNA comprising a double-discriminating base, characterized in that, The guide sequence is two different bases from the seed sequence or from a sequence comprising the seed sequence; the positions of the two different bases are any one of the following groups of bases from the 5' end of the seed sequence: the second, fourth, seventh, ninth, twelfth, fourteenth, or seventeenth, nineteenth bases; The seed sequence is a sequence completely complementary to the target sequence, and the seed sequence is a part of the crRNA.

2. The crRNA of claim 1, wherein, The rule of the difference is to mutate a C or G base at the original position into a U base, or to mutate an A or U base into a C base.

3. The crRNA of claim 1, wherein, The sequence of the seed sequence is any one of the following: 5'-GUCUAGUGUGUGCCACUUGUGAA-3'(SEQ ID NO. 147); 5'-GCCCGCCCAAAAUCUGUGAUCUU-3'(SEQ ID NO. 148); 5'-CCCCCAGCGCUUCAGCGUUCUUC-3'(SEQ ID NO. 149); 5'-AUUUUGUCUGAAACCCUGUAAGG-3'(SEQ ID NO. 150); 5'-UGAAGUAGAUAUGGCAGCACAUA-3'(SEQ ID NO. 151); 5'-ACAAUAUGUGCUUCUACACAGUC-3'(SEQ ID NO. 152).

4. The crRNA of claim 1, wherein, When the sequence of the seed sequence is the sequence shown in SEQ ID NO. 146 or SEQ ID NO. 149, the positions of the two different bases are any one of the following groups of bases from the 5' end of the seed sequence: the second, fourth, seventh, ninth, twelfth, fourteenth, or seventeenth, nineteenth bases; preferably the twelfth, fourteenth bases; when the sequence of the seed sequence is the sequence shown in SEQ ID NO. 147, SEQ ID NO. 148, SEQ ID NO. 150, SEQ ID NO. 151, or SEQ ID NO. 152, the positions of the two different bases are the twelfth, fourteenth bases from the 5' end of the seed sequence.

5. The crRNA of claim 1, wherein, The crRNA comprising two different bases is any one of the following: UAAUUUCUACUCUUGUAGAU UCUCUUUUAAACAUGAACAAUCA (SEQ ID NO. 125); AAUGUAAAAGUCCUACCAAUGG (SEQ ID NO. 126); UGAAAAGUCC UAAUUUCUACUCUUGUAGAU UUUAUUCUCAACAUGAACAAUCA (SEQ ID NO. 126); UUUGAACAUGAACAAUCA (SEQ ID NO. 127); UUUGAACAUGAACAAU UAAUUUCUACUCUUGUAGAU UUUAUUUUAAAUACGAACAAUCA (SEQ ID NO. 127); UUUAUUUUAAAUACGAACAAUCA (SEQ ID NO. 127); and UUUAUUUU UAAUUUCUACUCUUGUAGAU UUUAUUUUAAACAUGACCCAUCA (SEQ ID NO. 128); UUUAUUUUAAACAUGACCCAUCA (SEQ ID NO. 128); UAAUUUCUACUCUUGUAGAU GUCUAGUGUGUuCuACUUGUGAA (SEQ ID NO. 131); GUGUuCuACUUGUGAA (SEQ ID NO. 132); GUGUuCuACUUGUGAA ( UAAUUUCUACUCUUGUAGAU GCCCGCCCAAAcUuUGUGAUCUU (SEQ ID NO. 137); GCCCGCCCAAAcUuUGUGAUUUG (SEQ ID NO. 138); GCCCGCCCAAAcU UAAUUUCUACUCUUGUAGAU CUCUCAGCGCUUCAGCGUUCUUC (SEQID NO.139); UAAUUUCUACUCUUGUAGAU CCCCCAUCUCUCUACGUCGUUCUUC(SEQID NO.140); UAAUUUCUACUCUUGUAGAU CCCCCAGCGCUCCCGCGUUCUUC(SEQID NO.141); UAAUUUCUACUCUUGUAGAU UAAUUUCUACUCUUGUAGAU CCCCCAGCGCUUCAGCUUCCUUC(SEQID NO.142); UAAUUUCUACUCUUGUAGAU AUUUUGUCU G ACAUCCUGUAAGG (SEQ ID NO. 2); UAAUUUCUACUCUUGUAGAU UGAAGUAGAUACGUCAGCACAUA (SEQ ID NO. 4); UAAUUUCUACUCUUGUAGAU ACAAUAUGUGCCUUUACACAGUC (SEQ ID NO. 120).

6. A detection system characterized in that, The crRNA comprising double differential bases according to any one of claims 1-5.

7. The detection system of claim 6, wherein, The detection system further comprises RPA amplification reagents, AsCas12a, Cas12a Reaction Buffer, nuclease-free water, FAM-BHQ1 probe and non-specific primers.

8. The assay system of claim 6, wherein the detection moiety is a fluorescent moiety. The non-specific primers are any one of the following groups: S. aureus-RPA-F: CACAGCAATAATAATTGCTTTAGAAAGTCG (SEQ ID NO. 129), S. aureus-RPA-R: GTTATTTCCGTTTTAGAATTTAACATTTACACG (SEQ ID NO. 130); influenzaA-RPA-F: GCCAAGGAGGTGTCATTGAGCTATTCAACTG (SEQ ID NO. 132), influenzaA-RPA-R: GTAGTAGCCATTTGTCTGTGAGACCGATGC (SEQ ID NO. 133); EGFR-RPA-F: gaccctgaattcggatgcagagcttcttcc (SEQ ID NO. 134), EGFR-RPA-R: cagctgctgcgagctcacccag (SEQ ID NO. 135); COV-RPA-F: AAGGAAATTTTGGGGACCAGGAACTAATCAGA (SEQ ID NO. 143), COV-RPA-R: ATCCAATTTGATGGCACCTGTGTAGGTCAAC (SEQ ID NO. 144); AF2: TAAGTAAAATGTCTTGTGAAACAAAATG (SEQ ID NO. 105), AR1: TTCACTTTCATAATGCTGGCAGACTTACTC (SEQ ID NO. 104); SF2: AAAATGTCTTGTGAAACAAAATGC (SEQ ID NO. 108), AR1: TTCACTTTCATAATGCTGGCAGACTTACTC (SEQ ID NO. 104); HPV16-RPA-F: TTGTTGGGGTAACCAACTATTTGTTACTGTT (SEQ ID NO. 111), HPV16-RPA-R: CCTCCCCATGTCGTAGGTACTCCTTAAAG (SEQ ID NO. 112); HPV18-RPA-R: GCTATACTGCTTAAATTTGGTAGCATCATATTGC (SEQ ID NO. 118). HPV16-RPA-R: CCTCCCCATGTCGTAGGTACTCCTTAAAG (SEQ ID NO. 112); HPV18-RPA-R: GCTATACTGCTTAAATTTGGTAGCATCATATTGC (SEQ ID NO. 118).

9. The assay system of claim 6, wherein the detection moiety is a fluorescent moiety. The detection system further comprises a target sequence from a cell, an infectious bacterium or a virus, wherein the cell is any one of a spinal muscular atrophy pathogenic cell, a beta thalassemia pathogenic cell or a tumor cell; the bacterium is any one of a group B streptococcus, a staphylococcus aureus, a mycobacterium tuberculosis, a salmonella, a vibrio cholera, a bacillus anthracis; the virus is any one of a human papillomavirus, a SARS-COV-2, a hepatitis B virus, an Epstein-Barr virus, a cytomegalovirus, a herpes virus.

10. A kit characterized in that, The crRNA comprising a double differential base according to any one of claims 1-5. The crRNA comprising a double differential base according to any one of claims 1-5.

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