Kit for detecting target nucleic acid by using Cas12 protein to cut unnatural sequence and application of kit

Abstract

The invention provides a method for detecting target nucleic acid by using Cas12 protein to cut an unnatural sequence, and belongs to the technical field of biology. The Cas12 protein belongs to a Cas12a / Cas12b protein family; the non-natural sequence comprises a chimeric sequence composed of ribonucleotide and deoxyribonucleotide, the sequence can be made into a probe for nucleic acid detection, the detection effect of the probe is better than that of a conventional ssRNA probe, and the detection effect of the probe is equivalent to that of an ssDNA probe and even superior to that of the ssDNA probe under specific conditions. In addition, the invention provides a system for detecting the target nucleic acid, and the system comprises a chimeric sequence, Cas12 protein and crRNA, the invention also proves that the pre-amplification can increase the detection limit of the system to reach the single molecule level. The invention further verifies that the Cas12-chimeric sequence detection system not only can be applied to a micro-fluidic chip, but also can be used for clinical examination. In conclusion, through the matched use of the Cas12 protein and the chimeric sequence, the CRISPR / Cas12 detection system is optimized, and the application of the Cas12a protein and the non-natural sequence is widened.

Description

[0001] This application claims the priority of the prior U.S. provisional application with the application number 63 / 633,180 and the filing date of April 12, 2024. Its claims, specification, accompanying drawings of the specification, and abstract are hereby incorporated by reference in their entirety as part of the present invention. Technical Field

[0002] The present invention belongs to the field of biotechnology. Specifically, it relates to a method and kit for detecting target nucleic acids by using Cas12 protein to cleave non-natural sequences. Technical Background

[0003] The CRISPR (Clustered regularly interspaced short palindromic repeats) gene editing technology has been widely used in various fields due to its outstanding editability and simple operation steps. Among them, the research on Cas proteins in the CRISPR / Cas editing system has brought breakthrough progress to the field of nucleic acid diagnosis and is considered a tool for the next generation of nucleic acid detection. The trans-cleavage activity of CRISPR Class 2 Cas proteins is the basis of protein diagnostic technology. The CRISPR Class 2 Cas proteins consist of two families, Cas12 and Cas13, and the trans-cleavage activity refers to the non-specific cleavage of nucleic acid sequences.

[0004] The principle of this technology is as follows: Under the guidance of CRISPR RNA (crRNA), Cas proteins can specifically recognize, bind to, and cleave target nucleic acids; once a Cas protein recognizes its target sequence, its trans-cleavage activity is immediately activated, that is, it can non-specifically cleave other adjacent nucleic acid sequences. According to this property of Cas proteins, the adjacent nucleic acid sequences are usually designed as oligonucleotide probes with a fluorescent group at one end and a fluorescence quenching group at the other end to reflect whether the Cas protein under the guidance of crRNA detects the presence of target nucleic acids; furthermore, due to the high programmability of crRNA, it can be used to guide Cas proteins to target any desired sequence. Under normal circumstances, the fluorescence emitted by the fluorescent group is quenched by the quenching group because they are close to each other; when the Cas protein recognizes the target nucleic acid under the guidance of crRNA, it trans-cleaves the probe, and the fluorescent group and the quenching group on the probe are separated, and the fluorescent group emits fluorescence normally, which is detected by the instrument, thereby achieving the purpose of indirectly detecting the target nucleic acid. In addition, any physical or chemical visualization method is applicable to detect the cleavage of the probe caused by the trans-cleavage activity of Cas proteins.

[0005] However, not all members of the Cas protein family possess trans-cleavage activity. Among them, Cas12 and Cas13 proteins in Class 2 of the Cas protein family have become hotspots for nucleic acid detection because they possess both cis-cleavage and trans-cleavage activities under the guidance of CRISPR RNA (crRNA). Interestingly, the cleavage modes of the Cas12 and Cas13 proteins are not exactly the same. Under the guidance of guide RNA (gRNA, or crRNA), Cas12 proteins (such as Cas12a and Cas12b proteins) cleave both double-strand DNA (dsDNA) with a specific sequence and non-specific single-strand DNA (ssDNA). The CRISPR / Cas12 gene editing system can specifically and highly sensitively detect target nucleic acids, so it is named HOLMES (one-HOur Low-cost Multipurpose highly Efficient System) or DETECTR (DNA Endonuclease-Targeted CRISPR Trans Reporter); Cas13 needs the assistance of gRNA to exercise its nucleic acid cleavage function, but it can non-specifically cleave single-strand RNA, and CRISPR / Cas13 can detect RNA molecules with extremely high precision and efficiency. It is named SHERLOCK (Specific High-sensitivity Enzymatic Reporterunlocking).

[0006] Currently, due to its complementarity with the CRISPR / Cas9 system, the CRISPR / Cas12 system is hailed as a rising star in the CRISPR / Cas system. Therefore, many studies have revealed exciting new functions of Cas12 proteins, which provide new possibilities for the development of the subsequent diagnostic field. Current studies have shown that the CRISPR / Cas12a system and the CRISPR / Cas12b system can recognize DNA sequences and trans-cleave single-strand DNA sequences modified with fluorescent labels, magnetism, color, etc.; Cas12a2 and Cas12g proteins can trans-cleave ssRNA, ssDNA, and dsDNA; in addition, some variants of Cas12 can directly target RNA with the assistance of DNA sequences; the LbCas12a protein has been found to possess RNase (RNase) activity, although its RNA trans-cleavage activity is much lower than its DNA activity. However, it is still unclear whether the weak RNase activity of Cas12 proteins can be applied to the field of nucleic acid detection.

[0007] This requires improvement in the detection of target nucleic acids using CRISPR or other methods to expand the specific diagnostic and detection applications of the system. Summary of the Invention

[0008] In view of the above traditional problems, the present invention provides a method, kit, and system for detecting the presence or quantity of target nucleic acids by using Cas12 protein to cleave unnatural sequences, belonging to the field of biotechnology.

[0009] On the one hand, the present invention provides a use of Cas12 protein for preparing a reagent for trans-cleaving an unnatural sequence to detect target nucleic acids, and the unnatural sequence includes any one or more of the following:

[0010] (1) A sequence composed of both deoxynucleotides and ribonucleotides;

[0011] (2) A sequence composed of natural or unnatural deoxynucleotides and / or ribonucleotides, but the deoxynucleotides and / or ribonucleotides carry artificially created modifications that do not exist under natural conditions;

[0012] (3) A sequence containing deoxynucleotides and / or ribonucleotides, and the backbone composed of the deoxynucleotides and / or ribonucleotides carries artificially created modifications that do not exist under natural conditions.

[0013] In the second aspect of the present invention, the present invention provides a kit for detecting target nucleic acids, and the kit includes: Cas12 protein for binding to target nucleic acids, and unnatural or non-natural nucleic acid sequences. The non-natural sequences include one or several of the following sequences:

[0014] (1) A sequence composed of both deoxynucleotides and ribonucleotides;

[0015] (2) A sequence composed of natural or unnatural deoxynucleotides and / or ribonucleotides, but the deoxynucleotides and / or ribonucleotides carry artificially created modifications that do not exist under natural conditions;

[0016] (3) A sequence containing deoxynucleotides and / or ribonucleotides, and the backbone composed of the deoxynucleotides and / or ribonucleotides carries artificially created modifications that do not exist under natural conditions.

[0017] In some embodiments, the kit further includes necessary reagents for amplifying the target nucleic acid, including enzymes for necessary amplification, inorganic salts, etc. In some embodiments, the methods for amplifying the target nucleic acid include temperature-variable PCR or isothermal amplification. Isothermal amplification includes methods such as LAMP, RPA, RAA, etc. All reagents or components capable of amplifying the target nucleic acid can be used as an embodiment of the present invention, such as primers, probe sequences that bind to the target nucleic acid, and the like.

[0018] Here, the target nucleic acid is the nucleic acid for detection or diagnosis purposes. The target nucleic acid is generally a natural nucleic acid sequence or a partial sequence generated by methods such as synthesis and nucleic acid amplification. For example, human tissues, microorganisms such as viruses, bacteria, fungi, and also human or mammalian cells, etc.

[0019] In some embodiments, the target nucleic acid is DNA or RNA.

[0020] In some embodiments, the DNA or RNA in the target nucleic acid includes double-stranded or single-stranded.

[0021] In some embodiments, the DNA in the target nucleic acid is double-stranded and the RNA is single-stranded.

[0022] In some embodiments, the artificially synthesized target nucleic acid includes the nicked target nucleic acid in the RAPID system.

[0023] In a third aspect of the present invention, the present invention provides a method for detecting the presence or quantity of a target nucleic acid. The method includes: binding the Cas12 protein to the target nucleic acid, and simultaneously allowing the Cas12 protein or an enzyme to trans-cleave a non-natural sequence, and detecting or identifying the presence or quantity of the target nucleic acid from the quantity of the cleaved non-natural sequence.

[0024] In some embodiments, the non-natural sequence includes one or more of the following sequences:

[0025] (1) A sequence composed of both deoxynucleotides and ribonucleotides;

[0026] (2) A sequence composed of natural or non-natural deoxynucleotides and / or ribonucleotides, but the deoxynucleotides and / or ribonucleotides carry artificial modifications that do not exist under natural conditions;

[0027] (3) A sequence containing deoxynucleotides and / or ribonucleotides, and the backbone composed of the deoxynucleotides and / or ribonucleotides carries artificial modifications that do not exist under natural conditions.

[0028] In some ways, the non-natural sequence includes a label, and the presence or quantity of the target nucleic acid is detected by detecting the amount or quantity of the label. The label includes fluorescence or any other labeling substance.

[0029] In some ways, the Cas12 protein belongs to the Cas12a / Cas12b protein family.

[0030] In some ways, the non-natural nucleic acid sequence contains a chimeric sequence composed of ribonucleotides and deoxyribonucleotides. Such sequences can be made into probes for nucleic acid detection. The detection effect of the chimeric sequence is better than that of conventional ssRNA probes, and in certain cases, it is comparable to that of ssDNA probes and even superior to ssDNA. Non-natural nucleic acids also include xeno nucleic acid (XNA), chimeric sequence, and hybridized sequences.

[0031] In some ways, the natural target nucleic acid can be amplified or not amplified. In some ways, the target nucleic acid has been pre-amplified and then tested by the method or system of the present invention. The present invention also proves that pre-amplifying the target nucleic acid can increase the detection limit of the system to the single-molecule level. The present invention further verifies that the Cas12-chimeric sequence detection system can not only be applied to microfluidic chips but also be used in clinical tests. In summary, the combination of the Cas12 protein and the chimeric sequence optimizes the CRISPR / Cas12 detection system and broadens the uses of the Cas12 protein and non-natural sequences.

[0032] In some embodiments, the non-natural or unnatural nucleic acids of the present invention include non-natural or unnatural sequences in a broad sense and also include non-natural or unnatural nucleic acid sequences in a narrow sense. In some embodiments, the non-natural nucleic acid sequences of the present invention in a broad sense. In some ways, the meanings of the "non-natural sequence" or "unnatural sequence" can be interchanged. They refer to nucleic acid sequences that cannot be produced during the long process of natural evolution or that cannot be stably inherited, that is, other sequences except natural or native DNA and RNA. Natural or native nucleic acid sequences (such as RNA or DNA) refer to nucleic acid sequences that can be produced during the long process of natural evolution or that can be stably inherited.

[0033] In some ways, a non-natural sequence refers to a sequence that is created by artificially changing the constituent elements or internal structure of natural DNA / RNA on the basis of existing natural DNA / RNA sequences, and is different from natural or native DNA / RNA, also known as xeno nucleic acid (XNA). In some ways, the artificial change of the constituent elements of DNA / RNA includes, but is not limited to, changing the combination method of the constituent elements (that is, deoxynucleotides and nucleotides can appear simultaneously in the same sequence), and performing unnatural and artificially created modifications on the internal components of nucleotides, such as pentose sugar, base or phosphate group. The artificial modification methods include, but are not limited to: changing the type of sugar group, introducing organic polymers and / or halogens, and combining several or multiple of multiple methyl or / and acetyl group modifications. In some ways, the artificial modification of bases can regulate the strength and specificity of base pairing, and the modification of sugar groups also has a significant impact on the properties of nucleic acids, such as double-strand formation ability, nuclease resistance, and toxicity to cells and animals. In some ways, the artificial change of the internal structure of DNA / RNA includes, but is not limited to, changing the nucleic acid backbone structure (such as phosphorothioate), introducing new artificial nucleosides (such as deoxyuridine), and modifying pentose sugar (such as the pentose sugar of glycol nucleic acid, deoxy and non-deoxy modifications, etc.). In some ways, the modification of the artificial phosphodiester backbone can improve nuclease resistance and pharmacokinetic properties. Narrowly speaking, a non-natural sequence refers to a sequence composed of deoxynucleotides and ribonucleotides, or a nucleic acid sequence containing deoxynucleotides or / and ribonucleotides or / sugar-phosphate backbone with modification types artificially created under non-natural conditions. The meaning of the "non-natural sequence" or "non-native sequence" described in the present invention includes the broad sense of "non-natural sequence" or "non-native sequence", and also includes the narrow sense of "non-natural sequence" or "non-native sequence" nucleic acid sequence.

[0034] In some ways, xeno nucleic acid (XNA) can store genetic information, replicate, and even evolve like natural DNA and RNA, but it is not produced by natural evolution, but is artificially created or generated. The "synthesis" in the present invention means artificial synthesis, rather than being produced in the process of natural evolution. The so-called "artificial synthesis" includes the subjective synthesis participated by humans, such as humans using machines and artificial intelligence (AI) to synthesize the "non-natural sequence" or "non-native sequence" defined in the present invention.

[0035] In some ways, non-natural nucleic acid sequences include (rUA) nNucleic acid sequences, where n is any natural number integer. For example, n can be any number from 1 to 100 million, such as 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, etc., with a natural length. In some ways, for example, in the following form, unnatural sequences such as rUArUArUA (e.g., chimeric sequences), where "rU" represents uridine ribonucleotide (only present in RNA in organisms), and "A" represents adenine deoxynucleotide (only present in DNA in organisms), demonstrate how synthetic biology combines natural components to create novel gene systems. In one way, the unnatural sequence includes (rUA) n RNA or DNA sequences of units. For example, in natural sequences, (rUA) is included n , for example, in DNA sequences, (rUA) is included n units. Where n is any natural number integer. In some ways, natural DNA or RNA sequences include rUA) n nucleic acid sequences, thus becoming unnatural sequences or chimeric sequences.

[0036] Traditional natural DNA has deoxyribonucleotides as its basic building blocks. Deoxyribonucleotides are composed of a base, deoxyribose, and phosphate. The basic structural unit of DNA is deoxynucleotide. According to the different bases it contains, deoxynucleotides have four types: A (adenine), T (thymine), G (guanine), and C (cytosine). The basic building blocks of natural RNA are ribonucleotides, which are composed of one molecule of phosphate, one molecule of ribose, and one molecule of nitrogenous base. Among them, there are four nitrogenous bases, namely adenine (A), uracil (U), guanine (G), and cytosine (C).

[0037] These chimeric sequences are in an intermediate state between natural nucleic acids and engineered substitutes, providing new possibilities for the research and application of the present invention. In synthetic biology, unnatural nucleic acids can be used in editable cells, providing new ideas for more stable gene therapies, biosensors, and synthetic organisms; they also have certain potential in the field of biotechnology. They can be used as probes with a longer shelf life and greater durability in molecular diagnostics or as new components of gene editing systems such as CRISPR; in addition, XNAs can also be used to construct nano-scale scaffolds or as components of programmable biomolecular systems; more importantly, unnatural nucleic acids can achieve the goal of constructing synthetic cells with completely new genetic information, fundamentally expanding human understanding of life and its potential forms.

[0038] In some ways, Cas12 belongs to type II class V RNA-guided endonucleases, which include Cas12a, Cas12b, etc. Among this protein family, the most studied is the Cas12a nuclease. Currently, the most commonly used Cas12a protein comes from the BV3L6 strain of Acidaminococcus and Lachnospiraceae bacterium (LbCas12a). The Cas12a protein has been widely used in multiple species including bacteria, yeast, plants, and human cells.

[0039] Cas12 has both cis-cleavage and trans-cleavage activities. When Cas12 specifically recognizes and cleaves the target nucleic acid (this process is cis-cleavage), this process can activate its trans-cleavage activity, which can fragment any other single-stranded natural DNA in the system into pieces within a short time (this process is trans-cleavage). In the prior art, this property of Cas12 is exactly used to detect various target DNAs. And this scheme discovers that by using this system, it is possible to trans-cleave non-natural sequences or non-natural sequence DNA sequences.

[0040] Cas12 can not only cleave single-stranded DNA, but also cleave single-stranded RNA (cis or trans) through RNase. However, the activity of cleaving RNA is relatively weak. Therefore, in the prior art, it is considered difficult to construct a detection system for target nucleic acids by trans-cleaving natural RNA with Cas12. And this invention proves through a large number of studies that Cas12 can precisely detect target nucleic acids by trans-cleaving non-natural sequences through RNase, thus greatly broadening the application of Cas12 in the field of target nucleic acid detection.

[0041] As used in this invention, "trans-cleavage" means that after being activated by the target nucleic acid, the Cas12 protein can non-specifically cleave any other adjacent nucleic acid sequences, and the nucleic acids include DNA, RNA, or non-natural sequences. The target nucleic acid contains natural DNA or RNA in the sample.

[0042] In some specific embodiments, the non-natural sequence contains both deoxynucleotides and ribonucleotides at the same time.

[0043] It can be understood that natural DNA or RNA only contains deoxynucleotides or ribonucleotides, so the non-natural sequence does not belong to the DNA sequence nor the RNA sequence.

[0044] In some specific embodiments, the deoxynucleotides and ribonucleotides in the non-natural sequence are alternately arranged at intervals of one item, that is, ribonucleotides follow deoxynucleotides, or deoxynucleotides follow ribonucleotides. It can be understood that there is no situation where two deoxynucleotides or two ribonucleotides are connected by a natural backbone in the non-natural sequence. For example: DNA: TT, RNA: UU, etc.

[0045] Furthermore, the Cas12 protein uses its RNase activity to trans-cleave non-natural sequences.

[0046] Previous studies have shown that the Cas12 protein has strong DNase activity for trans-cleavage and weak RNase activity. Therefore, there has been no report on the research or progress of applying this RNase cleavage ability to nucleic acid detection. The present invention explores the RNAase activity of LbCas12a. Compared with traditional systems, there is still much room for improvement in the overall performance of the LbCas12a-ssRNA system. In some ways, in order to improve the efficiency of the Cas12 protein in trans-cleaving natural RNA, we not only performed non-natural modifications on natural RNA sequences but also created non-natural sequences that contain both deoxynucleotides and ribonucleotides; before this, no one had associated the RNase activity of the Cas12 protein, non-natural sequences, and target nucleic acid detection together.

[0047] The present invention designed 1 RNA sequence modified with phosphorothioate bonds (e.g., T*A*rArU*G*C) and 2 non-natural chimeric sequences (rUArUArUA and ArUArUArU), and used conventional LbCas12a protein to perform trans-cleavage on them. It was found that the above 3 non-natural sequences could be trans-cleaved by LbCas12a, and their detection effects were all better than that of ssRNA (rUrUrUrUrUrU or rArArArArArA), and the efficiency of Cas12 in cleaving the 2 non-natural sequences was comparable to that of the positive control ssDNA.

[0048] In summary, the present invention for the first time demonstrates that Cas12a can use its RNase activity to trans-cleave non-natural sequences, and this property can be applied to nucleic acid detection; it also demonstrates that modifying natural RNA can improve the trans-cleavage efficiency of the Cas12a protein, thereby achieving a high signal-to-noise ratio during the detection process, but no one has applied for relevant patents using this feature before.

[0049] On the other hand, the present invention provides the use of a non-natural sequence for preparing a reagent for detecting a target nucleic acid, and the non-natural sequence includes any one or more of the following: (1) containing both deoxynucleotides and ribonucleotides; (2) containing deoxynucleotides and / or ribonucleotides, and the deoxynucleotides and / or ribonucleotides carry artificially created modifications that do not exist under natural conditions; (3) containing deoxynucleotides and / or ribonucleotides, and the backbone composed of the deoxynucleotides and / or ribonucleotides carries artificially created modifications that do not exist under natural conditions; the reagent further includes a Cas12 protein and crRNA.

[0050] In some specific embodiments, the unnatural sequence contains both deoxynucleotides and ribonucleotides.

[0051] In some specific embodiments, the deoxynucleotides and ribonucleotides in the unnatural sequence are arranged alternately every other item, that is, a ribonucleotide follows a deoxynucleotide, or a deoxynucleotide follows a ribonucleotide.

[0052] On the other hand, the present invention provides a probe, which comprises an unnatural sequence, and the unnatural sequence includes any one or more of the following: (1) containing both deoxynucleotides and ribonucleotides; (2) containing deoxynucleotides and / or ribonucleotides, and the deoxynucleotides and / or ribonucleotides carry artificially created modifications that do not exist under natural conditions; (3) containing deoxynucleotides and / or ribonucleotides, and the deoxynucleotides and / or ribonucleotides carry artificially created modifications that do not exist under natural conditions. In some ways, the probe can be trans-cleaved by Cas12 protein. When the Cas12 protein binds to the target nucleic acid, the probe comprising the unnatural sequence can be trans-cleaved by the Cas12 protein.

[0053] The probe refers to introducing a fluorophore at one end of the unnatural sequence and connecting a quencher at the other end; the working principle is as follows: under normal circumstances, the fluorophore emits fluorescence that is quenched by the latter due to the small distance from the quencher; when the Cas protein cleaves the probe, the fluorophore and the quencher are separated, and the fluorophore emits fluorescence normally, which is detected by the instrument, thereby achieving the purpose of detecting the target nucleic acid.

[0054] Further, the unnatural sequence in the probe is composed of nucleotides constituting RNA and nucleotides constituting DNA. The nucleotides constituting RNA are any one or more of uridine ribonucleotide (rU), adenosine ribonucleotide (rA), cytosine ribonucleotide (rC), and guanine ribonucleotide (rG), and the nucleotides constituting DNA are any one or more of thymine deoxynucleotide (T), adenine deoxynucleotide (A), cytosine deoxynucleotide (C), and guanine deoxynucleotide (G).

[0055] Further, the probe is arranged in the order of ribonucleotide - deoxynucleotide or deoxynucleotide - ribonucleotide. The ribonucleotide is any one or more of uridine ribonucleotide, adenosine ribonucleotide, cytosine ribonucleotide, and guanine ribonucleotide, and the deoxynucleotide is any one or more of thymine deoxynucleotide, adenine deoxynucleotide, cytosine deoxynucleotide, and guanine deoxynucleotide.

[0056] In some ways, the length of the probe is 2 - 100bp.

[0057] In some ways, the nucleotides are arranged in the order of rRD or DrR, where rR is any one or more of rU, rA, rC, and rG, and D is any one or more of T, A, C, and G.

[0058] It can be understood that according to the nucleotide arrangement rule of the non-natural sequence, it can be calculated that there are 4 n (n = the number of nucleotides in the non-natural sequence) types, such as single chimera (poly ArA), double chimera (poly rUArUA), multi-chimera (UrACrGTrA), etc. Any non-natural sequence can be used to prepare a probe to detect the target nucleic acid, and all are within the protection scope of the present invention. Since the types of non-natural sequences cannot be exhausted, the present invention designs 7 representative non-natural sequences and uses these 7 sequences for subsequent research.

[0059] Further, the probe sequence includes any one or more of the following non-natural sequences: rUArUArUA, ArUArUArU, rUrUrArUrUrU, TrUTrUTrU, ArAArAArA, CrCCrCCrC, and GrGGrGGrG, where rU is uridine ribonucleotide, A is deoxyadenosine nucleotide, T is thymidine deoxyribonucleotide, rA is adenosine ribonucleotide, C is cytidine nucleotide, rC is cytidine ribonucleotide, G is deoxyguanosine nucleotide, and rG is guanosine ribonucleotide.

[0060] Further, the probe sequence includes any one or more of rUArUArUA, ArUArUArU, and TrUTrUTrU.

[0061] The present invention selects the commonly used LbCas12a protein and AsCas12a protein to cut the above 7 chimeric sequences respectively, and finds that neither of the two Cas12 proteins can cut the GrGGrGGrG sequence. This may be because the spatial structure of the DNA or RNA sequence composed of guanine is relatively complex, or the sequence itself and the interaction force between them are relatively large, which is not conducive to the cutting of the Cas12 protein; perhaps it is also because the Cas12 family proteins have a preference for not cutting the sequences composed of a large number of Gs. At the same time, the cutting efficiency of the LbCas12a protein and the AsCas12a protein for poly rUA and poly rAU can reach the same level as that for cutting the TTATTT sequence (ssDNA), indicating that poly rUA and poly rAU are expected to become the general cutting sequences of the Cas12a protein family; in addition, the effect of the LbCas12a protein in cutting the TrUTrUTrU sequence is better than its effect in cutting ssDNA.

[0062] In another aspect, the present invention provides a system for detecting a target nucleic acid, which system comprises the above-described probe containing a non-natural sequence, a Cas protein, and a crRNA. The crRNA can bind to the target nucleic acid, thereby activating the Cas protein. After being activated, the Cas protein cleaves the target sequence and also non-specifically trans-cleaves the probe to achieve the effect of detecting the target nucleic acid; the crRNA is designed according to the target nucleic acid sequence and the detection purpose.

[0063] Further, the Cas protein belongs to the Cas12 protein family.

[0064] Further, the Cas protein belongs to the Cas12a and Cas12b subfamilies.

[0065] Further, the Cas protein is any one or more of LbCas12a, AsCas12a, and AapCas12b.

[0066] The present invention explored the effects of chimeric sequences (rUArUArUA and ArUArUArU) on the trans-cleavage activities of Cas12a and Cas12b proteins. The results showed that the enzymatic kinetics of LbCas12a protein and AsCas12a protein when cleaving the chimeric sequences were comparable to or even higher than those when cleaving ssDNA; generally speaking, the trans-cleavage activities of AapCas12b protein on ssDNA, ssRNA, and chimeric sequences were significantly weaker than those of LbCas12a protein and AsCas12a protein, but the effect of AapCas12b on cleaving chimeric sequences was better than that of conventional ssRNA sequences, and it was more inclined to cleave poly ArU sequences.

[0067] In summary, compared with conventional ssRNA, the chimeric sequence can improve the cleavage efficiency of Cas12, and can even achieve or exceed its effect on cleaving ssDNA.

[0068] Further, when the Cas protein is LbCas12a, it is preferable that the probe sequence contains any one or more of rUArUArUA, ArUArUArU, and TrUTrUTrU; when the Cas protein is AsCas12a, it is preferable that the probe sequence contains any one or more of rUArUArUA and ArUArUArU.

[0069] Further, the target is DNA or RNA; when the target is RNA, the system further includes a DNA helper, and the DNA helper is a partial sequence with a PAM sequence and capable of binding to the target RNA or a simple PAM sequence.

[0070] Cas12a was first considered as a DNA-targeting system. If the target sequence is RNA, it needs to be reverse transcribed into cDNA first and then detected. The research of this invention shows that the introduction of a DNA helper with a PAM sequence enables the Cas12a system to effectively target RNA (for the specific principle, see Figure 4a ), thus eliminating the need to pre-reverse transcribe RNA into cDNA.

[0071] On the other hand, this invention provides a method for detecting a target nucleic acid. The method is carried out by using the system for detecting a target nucleic acid as described above, and the type of the target nucleic acid is DNA or RNA.

[0072] In some embodiments, the steps of the method are as follows: Extract DNA or RNA from the sample. If the sample is RNA, reverse transcribe the RNA into cDNA, or add the DNA helper to directly detect the RNA sample. The DNA helper is a pair of DNA sequences that contain a PAM (protospacer adjacent motif) sequence, can complementarily pair with each other and form a stem-loop structure. Prepare a corresponding detection reaction system according to the detection target (DNA or RNA); Take 10 μL - 20 μL of the above reaction solution, quickly transfer it to a 96- or 384-well plate, and place the plate in a real-time fluorescence quantitative PCR instrument. According to the different Cas12 proteins selected, the reaction temperature of Cas12a is 37°C - 42°C, and the heat-resistant Cas12 protein is 60°C - 65°C. Read the data every 30 s - 60 s, and the reaction time is 30 min - 2 h. After the reaction, generate a real-time fluorescence curve or measure the final fluorescence value, and determine whether there is a target nucleic acid in the sample based on the fluorescence curve or fluorescence value; Set 3 replicates and a blank control for each experiment. Pre-amplifying the sample can increase the detection sensitivity of the system.

[0073] On the other hand, this invention provides the use of an unnatural sequence for preparing a reagent for improving the efficiency of detecting a target RNA. The unnatural sequence is rUArUArUA, where rU is uridine ribonucleotide and A is adenine deoxynucleotide. When the detection target is RNA, the detection effect is the best when the Cas12 protein is paired with the rUArUArUA sequence, generating the highest signal-to-noise ratio, and the effect is better than the case when the detection target is ssDNA.

[0074] On the other hand, the present invention provides a use of an unnatural sequence for preparing a reagent for improving the trans-cleavage efficiency of Cas12 protein. The unnatural sequence is any one or more of rUArUArUA and TrUTrUTrU, where rU is uridine ribonucleotide, A is adenine deoxynucleotide, and T is thymine deoxynucleotide. Compared with the conventional ssRNA sequence, Cas12 has higher activity in cleaving the chimeric sequence; and the efficiency of LbCas12a protein in cleaving any one or more chimeric sequences of rUArUArUA and TrUTrUTrU is even higher than that of cleaving the conventional ssDNA (TTATTT).

[0075] The beneficial effects of the present invention include:

[0076] 1. It is found that Cas12 protein can cleave unnatural sequences and be used for nucleic acid (DNA and RNA) detection, and its detection effect is better than that of cleaving ssRNA. In certain cases, it can be comparable to or even better than the conventional ssDNA probe, significantly broadening the uses of Cas12 protein and unnatural sequences;

[0077] 2. The use of unnatural sequences (chimeric sequences) is beneficial to improving the detection effect of the Cas12 system. Especially when the detection target is RNA, it is also proved that modifying the internal structure of ssRNA can enhance the detection sensitivity of ssRNA probes;

[0078] 3. The preference of different Cas12 proteins for trans-cleaving probes is verified, that is, the same Cas12 protein has different efficiencies in cleaving different probes. This property of Cas12 protein can be used to design multi-level detection schemes to achieve rich detection results with the simplest steps;

[0079] 4. LbCas12a and AsCas12a can effectively cleave the chimeric sequences of rUArUArUA and ArUArUArU, providing two general probes that can achieve the detection purpose because they can be effectively cleaved by the Cas12a protein family;

[0080] 5. The Cas12-chimeric sequence detection system is optimized, that is, LbCas12a protein is suitable for use with a variety of probes, and the rUTrUTrU probe is the best, while AsCas12a protein is preferably paired with the chimeric sequences poly rUA and poly rAU;

[0081] 6. A method for detecting RNA without reverse transcription is provided, that is, adding DNA helper;

[0082] 7. Pre-amplification (isothermal amplification or PCR) of the test sample can improve the detection efficiency of the Cas12-chimeric probe system, reaching the single-molecule level;

[0083] 8. The Cas12-chimeric probe system can not only be combined with microfluidic technology for high-throughput detection but also be used in clinical tests;

[0084] 9. It further deepens the understanding of Cas12 and non-natural sequences by researchers, providing a new direction for the optimization of the CRISPR / Cas12 detection system;

[0085] 10. There is no nuclease in nature that degrades non-natural sequences, so the half-life of non-natural sequences is longer than that of DNA and RNA. Therefore, the probes prepared from non-natural sequences can be stored for a longer time; moreover, the detection stability of non-natural sequence probes is relatively high, and the detection results are more reliable. BRIEF DESCRIPTION OF THE DRAWINGS

[0086] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. Obviously, the following drawings are only some embodiments of the present invention. For those of ordinary skill in the art, other drawings can be obtained based on these drawings without creative efforts.

[0087] Figure 1a : Schematic diagram of the working process of the Cas12 protein trans-cleavage system. ssDNA (TTATT), ssRNA (poly rU and poly rA), RNA motif sequences (T*A*rArU*G*C), and chimeric sequences (ArUArUArU and rUArUArUA) are all labeled with fluorescent groups and quenching groups to make probes and applied to the Cas12 protein detection system;

[0088] Figure 1b : Ability of the LbCas12a protein in the activated state to trans-cleave ssDNA probes; NTC: non-template control, as the negative control, the error bars represent the standard deviation, n = 3;

[0089] Figure 1c -e: Ability of the LbCas12a protein in the activated state to trans-cleave ssRNA (rUrUrUrUrUrU, rArArArArArA, and T*A*rArU*G*C) probes; NTC: non-template control, as the negative control, the error bars represent the standard deviation, n = 3;

[0090] Figure 1f-g: The ability of LbCas12a protein in the activated state to trans-cleave chimeric probes (rUArUArUA and ArUArUArU) modified based on ssRNA (poly rU); NTC: non-template control, which is the negative control. Error bars represent standard deviation, n = 3;

[0091] Figure 2a -d: The ability of LbCas12a protein in the activated state to trans-cleave conventional sequences, monomeric ssDNA, monomeric ssRNA, and chimeric sequences; NTC: non-template control, which is the negative control. Error bars represent standard deviation, n = 3;

[0092] Figure 2e -h: The ability of AsCas12a protein in the activated state to trans-cleave conventional sequences, monomeric ssDNA, monomeric ssRNA, and chimeric sequences; NTC: non-template control, which is the negative control. Error bars represent standard deviation, n = 3;

[0093] Figure 3a -b: The effect of chimeric sequences on the trans-cleavage activity of LbCas12a protein; NTC: non-template control, which is the negative control. Error bars represent standard deviation, n = 3; Figure 3a The concentration of the activator of LbCas12a protein in the detection system is 10 nM;

[0094] Figure 3c -d: The effect of chimeric sequences on the trans-cleavage activity of AsCas12a protein; NTC: non-template control, which is the negative control. Error bars represent standard deviation, n = 3; Figure 3c The concentration of the activator of AsCas12a protein in the detection system is 10 nM;

[0095] Figure 3e -f: The effect of chimeric sequences on the trans-cleavage activity of AapCas12b protein; NTC: non-template control, which is the negative control. Error bars represent standard deviation, n = 3; Figure 3e The concentration of the activator of AapCas12b protein in the detection system is 10 nM;

[0096] Figure 4a : Schematic diagram of the principle of RNA detection by the Cas12a system;

[0097] Figure 4b:Real-time reaction curve of RNA detection by AsCas12a system; NTC: non-template control, which is the negative control. Error bars represent standard deviation, n = 3;

[0098] Figure 5a -b: Flow chart of pre-amplification / no-amplification detection samples, where the probe used in Figure a is ssDNA and that in Figure b is the chimeric sequence;

[0099] Figure 5c -d: Detection effect of pre-amplification / no-amplification - LbCas12a - ssDNA / chimeric sequence system; NTC: non-template control, which is the negative control. Error bars represent standard deviation, n = 3;

[0100] Figure 5e -f: Detection effect of pre-amplification / no-amplification - AsCas12a - ssDNA / chimeric sequence system; NTC: non-template control, which is the negative control. Error bars represent standard deviation, n = 3;

[0101] Figure 6a : Schematic diagram of the working process of droplet microfluidics technology;

[0102] Figure 6b : Detection effect of Cas12 - chimeric probe system combined with microfluidic chip;

[0103] Figure 6c : Flow chart of Cas12 - chimeric probe system for detecting COVID-19 clinical saliva samples;

[0104] Figure 6d : Results of Cas12 - chimeric probe system for detecting COVID-19;

[0105] Figure 7a : Flow chart of RAPID detection using different probes and different Cas12 proteins;

[0106] Figure 7b : RAPID performance detection results when using AsCas12a and different probes;

[0107] Figure 7c : RAPID performance detection results when using LbCas12a and different probes. Specific implementation manners

[0108] The present invention will be further elaborated in detail below in conjunction with the accompanying drawings of the specification and specific embodiments. The embodiments are only used to explain the present invention and are not intended to limit the scope of the present invention. LbCas12a and murine RNase inhibitor used in the following embodiments were purchased from New England Biolab (NEB) company, AsCas12a protein was purchased from IDT, and AapCas12b (addgene#153162) was purified according to the previously published article

[15] ; all probes, crRNAs and primers used were synthesized by IDT company. The 5' end of the probe was labeled with a 56-FAM group, and the 3' end was labeled with a 3IABkFQ group; all the activators were from IDT. The remaining materials, reagents, etc. used, unless otherwise specified, are reagents and materials that can be obtained commercially; the test methods used, unless otherwise specified, are conventional methods; when measuring the fluorescence value, 3 replicates were set for each.

[0109] Example 1: Non-DNA sequences can be trans-cleaved by LbCas12a protein

[0110] To study the ability of Cas12 proteins to trans-cleave non-DNA sequences, in this example, LbCas12a protein was used to cleave ssDNA, ssRNA, RNA dinucleotide sequences (sequences containing two ribonucleotides) and unnatural chimeric sequences respectively, and 3 replicates were set for each experimental group. The experimental principle of this example is as Figure 1a shown. Under the guidance of crRNA, the Cas12 protein recognizes the target DNA nucleic acid sequence (activator), and then its trans-cleavage activity is activated, randomly cleaving the adjacent nucleic acid probe. One end of the probe is labeled with a fluorescent group (preferably FAM group in this example), and the other end is labeled with a quenching group (preferably 3IABkFQ group in this example). The cleaved probe can emit fluorescence, which can be recognized by the machine, so as to achieve the purpose of detecting the target nucleic acid. It should be understood that the crRNA sequence in the Cas12 detection system can be replaced according to different detection objects to meet different detection needs; at the same time, the higher the content of the target DNA (activator), the faster and more the activated Cas12 protein, the more and faster the cleaved probes, the stronger the fluorescence emitted, and the more accurate the detection result.

[0111] Specifically, in this embodiment, a partial HPV 18 sequence (SEQ ID NO:1) was used as the activator of Cas12, which was diluted into different gradients (10 nM, 1 nM, 100 pM, 10 pM, and 1 pM) to further explore the effect of different probes on the detection sensitivity of the Cas12 system. Each experiment was set with 3 replicates; the HPV 18 nucleic acid was the same as described in the article

[16] ; the probes were ssDNA (TTATT) (SEQ ID NO:2) with FAM fluorophore and 3IABkFQ quencher, ssRNA (poly rU (SEQ ID NO:3) and poly rA (SEQ ID NO:4)), and unnatural sequences (T*A*rArU*G*C (SEQ ID NO:5, * represents phosphorothioate bond modification), poly ArU (SEQ ID NO:6), and poly rUA (SEQ ID NO:7)), which played the role of reporter elements in the Cas12 detection system; the TTATT (T) was a known ssDNA that could be effectively cleaved by Cas12 protein, and in all embodiments of the present invention, this sequence served as a positive control. The specific experimental operations were as follows: First, prepare 18 μL of the master mix without the Cas12 activator. The components and their final concentrations in the system were as follows: 1×NEB 2.1 buffer (New England Biolab), 50 nM Cas12 (the type of Cas12 protein in this embodiment was LbCas12a), 50 nM crRNA (the specific sequence was as shown in SEQ ID NO:8), 10 U of murine RNase inhibitor, and 125 nM probe. Specifically, 2 μL of NEB 2.1 buffer (10×), 0.5 μL of 2 μM LbCas12a, 0.5 μL of 2 μM crRNA (the specific sequence was as shown in SEQ ID NO:8), 0.25 μL of 40 U / μL murine RNase inhibitor, and 0.5 μL of 5 μM probe; then, add 2 μL of the Cas12 activator (partial synthetic sequence of HPV 18, SEQ ID NO:1) to the master mix. The negative control group was added with 2 μL of ddH2O instead of the activator, and mixed evenly. The Cas12 activator was diluted to the final reaction concentrations of 10 nM, 1 nM, 100 pM, 10 pM, and 1 pM respectively; then, take 10 μL of the above reaction solution and quickly transfer it into a 384-well plate. Use a real-time fluorescence quantitative PCR instrument (Roche LightCycler 480II) to measure the fluorescence generated by each reaction system per minute at 37°C for 2 hours to generate a real-time fluorescence curve. Each reaction system was set with 3 replicates.

[0112] As Figure 1b shown, within a reaction time of 1 hour, when the concentration of HPV 18 was 10 pM, the fluorescence generated by the ssDNA probe could still be detected by the instrument. This might be because the DNase activity of LbCas12a was very strong and the intrinsic detection limit of LbCas12a was at the pM level. As long as a small portion of the LbCas12a protein was activated, the fluorescence intensity emitted by the probe cleaved by it could reach the detectable intensity. On the other hand, this result indicated that when using ssDNA as the probe, the LbCas12a detection system could achieve a sensitivity of 10 pM. In contrast, under the same conditions, when using ssRNA (rUrUrUrUrUrU or rArArArArArA) as the probe, the sensitivity could only reach the nM level ( Figure 1c and d). In addition, these experimental results showed that the activated LbCas12a protein had the ability to trans-cleave ssDNA and ssRNA.

[0113] Next, in order to explore whether the activated LbCas12a could non-specifically cleave the RNA motif sequence, in this example, the activated LbCas12a protein was used to cleave the RNA motif sequence (T*A*rArU*G*C, * represents phosphorothioate bond modification), and this experiment was repeated 3 times. The T*A*rArU*G*C sequence can be understood as a sequence formed by adding phosphorothioate bonds and deoxyribonucleotides on the basis of a sequence composed of two ribonucleotides, rA and rU, and the phosphorothioate bond plays a role in preventing the sequence from being cleaved.

[0114] The results were as Figure 1e shown. Compared with the probe composed of only one type of ribonucleotide (rU or rA), the sequence probe composed of two types of ribonucleotides could increase the sensitivity of the Cas12 detection system by at least 10 times. Further, its trans-cleavage ability was affected by the nature of the sequence itself. Therefore, the determination of the trans-cleavage activity of Cas12 should not be limited to special sequence types only, but the scope of the exploration object should be expanded to the bases and other structures inside the nucleic acid.

[0115] Based on the above conclusions, two non-natural sequences, rUArUArUA (poly rUA) and ArUArUArU (poly ArU), were designed in this example. A represents deoxyadenosine monophosphate that constitutes DNA, while rU represents uridine ribonucleotide that constitutes RNA. Each experimental group was set with 3 replicates. It should be noted that these two sequences were synthesized based on RNA sequences and introduced deoxyribonucleotides, which are the building blocks of DNA, so they are also called chimeric sequences or non-natural sequences. Unexpectedly, the limit of detection (LOD) of these chimeric sequences was higher than that of the above RNA motif sequences (T*A*rArU*G*C), and even reached a level similar to that of ssDNA probes ( Figure 1f and g).

[0116] In summary, LbCas12a can non-specifically cleave ssDNA and ssRNA. Under normal circumstances, its activity of cleaving ssDNA is significantly higher than that of ssRNA (rUrUrUrUrUrU, rArArArArArA or T*A*rArU*G*C, Figure 1c and d), but by modifying the ssRNA sequence, such as introducing deoxyribonucleotides (poly rUA or poly ArU), the gap between the activity of LbCas12a in cleaving ssDNA and its activity in cleaving ssRNA can be narrowed ( Figure 1e -g). It should be noted that the modified ssRNA sequences are all non-natural nucleic acid sequences.

[0117] Example 2: Preferences of LbCas12a and AsCas12a for trans-cleaving sequences

[0118] The nuclease activity of LbCas12a (able to cleave both ssDNA and ssRNA) implies that the cleavage mechanisms of other members of the Cas12 protein family are also independent of the sequence type (such as DNA or RNA). Therefore, in this example, two commonly used Cas12 proteins (LbCas12a and AsCas12a) were selected to explore their sequence preferences. In this example, 16 different single-stranded nucleic acid sequences were designed, including ssDNA sequences (TTATTT (SEQ ID NO:9), AAAAAA (SEQ ID NO:10), GGGGGG (SEQ ID NO:11), CCCCCC (SEQ ID NO:12), and TTTTTT (SEQ ID NO:13)), ssRNA sequences (rArArArArArA (SEQ ID NO:4), rGrGrGrGrGrG (SEQ ID NO:14), rCrCrCrCrCrC (SEQ ID NO:15), rUrUrUrUrUrU (SEQ ID NO:3)), and chimeric sequences (poly rUA (SEQ ID NO:7), poly ArU (SEQ ID NO:6), rUrUrArUrUrU (SEQ ID NO:16), TrUTrUTrU (SEQ ID NO:17), ArAArAArA (SEQ ID NO:18), CrCCrCCrC (SEQ ID NO:19), and GrGGrGGrG (SEQ ID NO:20)). The TTATTT is an ssDNA sequence known to be efficiently cleaved by the Cas12 protein. A reaction system with a total volume of 20 μL was constructed. The reaction system and procedure were the same as those described in Example 1. The final reaction concentration of the Cas12 activator was 10 nM, and the reaction was carried out at 37 °C for 2 h. The final fluorescence value was measured using a real-time fluorescence quantitative PCR instrument, QuantStudio 5 real-time PCR system (Thermo Fisher Scientific). The specific experimental results are shown in Figure 2 and Table 1.

[0119] It is worth noting that when cleaving the same probe, the cleavage abilities of the LbCas12a protein and the AsCas12a protein are different, but the sequences that both can cleave are roughly the same, that is, the probes that the LbCas12a protein can cleave, the AsCas12a can also cleave, and vice versa. The cleavage efficiencies of the two Cas12 proteins for the chimeric sequences poly rUA and poly rAU can both reach the level of cleaving the TTATTT sequence ( Figure 2a and e); however, the signal-to-noise ratio generated by cleaving the rUrUrArUrUrU sequence is very low ( Figure 2aand e, Table 1), which means that these two Cas12 proteins have no preference for trans-cleavage of RNA sequences in which a single rU in such poly rU sequences is replaced by rA, and this way of modifying the RNA sequence even results in a signal-to-noise ratio strength lower than that of poly rU and poly rA at the same time( Figure 2c and g).

[0120] When the probe type is monomeric ssDNA( Figure 2b and f, Table 1), the LbCas12a protein produces the highest fluorescence value when cleaving the CCCCCC sequence, followed by the AAAAAA sequence, while the AsCas12a protein has a stronger ability to cleave the TTTTTT sequence and the AAAAAA sequence, but both proteins can hardly cleave the GGGGGG sequence. The same phenomenon also occurs when cleaving the rGrGrGrGrGrG sequence and the chimeric sequence GrGGrGGrG belonging to the ssRNA type, indicating that the two Cas12 proteins cannot cleave nucleotide sequences composed of guanine, whether the guanine is linked to deoxyribose or ribose. It may be because the above three poly(r)G sequences can form relatively complex and stable spatial structures by themselves or among them, which are not easy to be cleaved by Cas12 proteins; or these sequences tend to bind to repetitive sequences on chromosomes (chromatin), and these repetitive sequences are far from the Cas12 proteins that recognize and bind to the target sequences, and Cas12 can only non-specifically cleave adjacent sequences, so the poly(r)G sequences cannot be cleaved by Cas12 proteins. In addition, when cleaving ssDNA sequences, the background fluorescence generated by the AsCas12a system is higher than that of LbCas12a, which is consistent with previous reports

[17] . This may be because AsCas12a has a certain trans-cleavage activity when not guided by crRNA to recognize the target sequence, and this activity is stronger than that of LbCas12a. When the probe type is monomeric ssRNA( Figure 2c and g, Table 1), the LbCas12a protein is most suitable for cleaving the rUrUrUrUrUrU sequence, while AsCas12a is suitable for cleaving the rCrCrCrCrCrC sequence. Introducing ribonucleotides based on ssRNA can improve the cleavage efficiency of Cas12 proteins to a certain extent( Figure 2dand h, Table 1), for example, on the basis of the rUrUrUrUrUrU sequence, thymidine deoxyribonucleotide is introduced to prepare the TrUTrUTrU sequence, and the cleavage effect of LbCas12a on the latter is far better than that on the former, and even better than that on the TTATTT sequence; at the same time, transforming the rUrUrUrUrUrU sequence into the TrUTrUTrU sequence or transforming the rArArArArArA sequence into ArAArAArA can also improve the cleavage effect of AsCas12a.

[0121] Table 1 Effects of different Cas12 proteins on trans-cleavage of different nucleic acid sequences

[0122]

[0123] In summary, both LbCas12a protein and AsCas12a protein have the ability to cleave chimeric sequences (except GrGGrGGrG), and even the trans-cleavage activity when cleaving some chimeric sequences is equivalent to or better than that of cleaving TTATTT; further, when applying the two proteins of LbCas12a and AsCas12a to the field of nucleic acid detection, the sequence of the chimeric probe can be selected according to their preference for trans-cleaving sequences

[18] , conversely, the suitable Cas12 protein can also be selected through the sequence of the chimeric probe. In this design concept, different variants of Cas12 protein can show limited trans-cleavage activity on certain sequences, but exhibit high enzyme kinetics on specific sequences; then, by combining different Cas12 variants with specific probe materials, each combination is used to detect different target nucleic acid sequences. In the reaction, the recognition of a specific probe corresponds to the activation of a specific Cas protein, which further indicates that a specific crRNA has recognized a certain sequence, allowing the recognition of different targets. See Table 2 specifically, √ represents a better choice, and × represents inapplicable. As can be seen from Table 2, compared with AsCas12a protein, the applicable range of LbCas12a protein is wider; on the other hand, both chimeric sequences polyrUA and polyrAU can be effectively cleaved by the two proteins, or can be used as universal chimeric nucleic acid probes. Further, when the cleavage object is a chimeric sequence, the LbCas12a protein is most suitable to be used in combination with the TrUTrUTrU probe, followed by the poly rUA probe and the poly rAU probe, while the AsCas12a protein is preferably combined with the chimeric sequences poly rUA and poly rAU. Generally speaking, the research results of this example emphasize the sequence preference of LbCas12a and AsCas12a in the trans-cleavage reaction, which is reflected in the type of genetic material or specific sequences. Understanding these preferences is crucial for the design and development of multi-level one-to-one diagnostics using Cas12 family proteins.

[0124] Table 2 Preferences of LbCas12a and AsCas12a for Trans-Cleaving Nucleic Acid Sequences Example 3: Effect of Chimeric Sequences on the Trans-Cleavage Activity of Cas12a and Cas12b Proteins

[0125] To explore the effect of pairing with non-ssDNA probes on the detection limits (LOD) of different Cas12 proteins, in this example, LbCas12a, AsCas12a, and AapCas12b were used as the research objects, and 5 different types of probes were selected, including one ssDNA probe (TTATT, SEQ ID NO:2), two ssRNA probes (rUrUrUrUrUrU (SEQ ID NO:3) and rArArArArArA (SEQ ID NO:4)), and two chimeric probes (rUArUArUA (SEQ ID NO:7) and ArUArUArU (SEQ ID NO:6)), and the Cas proteins were activated with activators at different concentrations (1 nM, 100 pM, and 10 pM). The 3 proteins represent two subfamilies of Cas12 proteins (Cas12a and Cas12b protein families) and are commonly used in the fields of diagnosis and gene editing [15, 19]. The Cas12a protein can carry out enzymatic reactions at room temperature (37°C), while AapCas12b is a thermostable protein that functions at 60°C. Therefore, the type of Cas12 can be selected by temperature conditions, and then the preferred chimeric probe can be paired with it.

[0126] In this example, the Cas12 protein activator used is the post-transcriptional DNA sequence of the N-terminal part of the COVID-19 gene (SEQ ID NO:21), and the crRNA sequence is the same as that described in SEQ ID NO:22; the remaining components and detection steps of the LbCas12a and AsCas12a detection systems are the same as those described in Examples 1-2. The AapCas12b cleavage experimental steps are as follows: Prepare a reaction system with a total volume of 20 μL, and the components and their final concentrations in the system are as follows: 1×isothermal amplification buffer (LAMP, NEB), 100 nM AapCas12b protein, 100 nM crRNA (specific sequence as shown in SEQ ID NO:22), 125 nM probe, and 2 μL DNA activator (SEQ ID NO:21). React at 60°C for 2 h, and the remaining steps are the same as those described in Examples 1-2.

[0127] Existing studies have shown that the trans-cleavage of ssDNA probes by Cas12a protein can be used to detect target nucleic acids at the picomolar level [6, 20]. Therefore, in this example, the ssDNA (TTATT) probe was used as a positive control.

[0128] Consistent with the experimental results of Example 2, the enzyme kinetics of the LbCas12a protein and the AsCas12a protein when cleaving the chimeric sequence are comparable to, or even higher than, those when cleaving ssDNA ( Figure 3a-d); In addition, the use of chimeric probes can also achieve a detection limit of picomolar level for the above two proteins. It should be noted that AsCas12a exhibits stronger enzymatic kinetics than LbCas12a, that is, the time to reach the signal peak is shorter ( Figure 3a and c). Conversely, the trans-cleavage activity of the AapCas12b protein is significantly weaker than that of the above two Cas12a proteins ( Figure 3e -f). First, the AapCas12b protein shows a slower reaction rate, that is, it takes at least 60 minutes to reach the signal peak; second, the RNase activity of the AapCas12b protein is weaker; third, the background fluorescence generated during the cleavage of AapCas12b is stronger. It should be noted that the AapCas12b protein has a better cleavage effect on the chimeric sequence than the conventional ssRNA sequence. Further, AapCas12b is more inclined to cleave the poly ArU sequence rather than the poly rUA sequence, which is contrary to the tendency of the Cas12a protein.

[0129] In summary, compared with the conventional ssRNA, the chimeric sequence can improve the cleavage efficiency of Cas12, and even achieve or exceed its cleavage effect on ssDNA.

[0130] Example 4: The effect of using AsCas12a and unnatural sequences to detect RNA

[0131] Cas12a was first considered a DNA-targeting system. If the target sequence is RNA, it needs to be reverse transcribed into cDNA first and then detected. However, our latest research shows that the introduction of a DNA helper with a PAM sequence can enable the Cas12a system to effectively target RNA (for the specific principle, see Figure 4a), so in this embodiment, the recognition ability of the above system for target RNA is optimized by screening the types of reverse cleavage sequences. The types of the reverse cleavage sequences are the same as those described in Embodiment 3 (). The concentration of the target RNA is 50 nM, and the RNA sequence is as shown in SEQ ID NO: 23. Each experimental group is set with 3 replicates. The specific experimental results are as follows: Prepare a 50 μL reaction system, and the components and their final concentrations in the system are as follows: 1×NEB 2.1 buffer, 90 nM AsCas12a, 50 U RNase inhibitor, 90 nM crRNA (the specific sequence is the same as that shown in SEQ ID NO: 24), 20 nM DNA helper (the helper sequences are as shown in SEQ ID NO: 25 and SEQ ID NO: 26), 31.25 mM MgCl2, 500 nM probe, and 50 nM RNA activator (the specific sequence is the same as that shown in SEQ ID NO: 23); Take 15 μL of the above reaction solution into a 384-well plate and set 3 replicates; Using a real-time fluorescence quantitative instrument (QuantStudio 5 real-time PCR system ThermoFisher Scientific), measure the fluorescence intensity once per minute at 37 °C for 2 h, and then generate a real-time fluorescence curve and detect the final fluorescence value.

[0132] Overall, all probes can be cleaved and then fluoresce, confirming that DNA helper can indeed assist the Cas12a system to directly detect RNA without first reverse transcribing RNA into cDNA and then performing detection; however, among the 5 types of probes, the chimeric sequence poly rUA shows the best detection effect, that is, it produces the highest signal-to-noise ratio, which is better than ssDNA ( Figure 4b ). Surprisingly, compared with the effect of detecting DNA, the monomeric ssRNAs (poly rU and poly rA) show very weak RNA detection ability, indicating that although the auxiliary method can enable the Cas12 protein to directly recognize RNA and activate its trans-cleavage activity, the trans-cleavage activity of the RNA-activated Cas12 protein is weaker than that activated by DNA. In summary, combining the results of Embodiments 2 and 3, this embodiment further proves the role of the chimeric sequence in improving the detection effect of the Cas12 system, and further expands the application field of the Cas12 protein detection system, that is, the detection object can be either DNA or RNA; and no reverse transcription process is required.

[0133] Embodiment 5: Nucleic acid amplification can effectively improve the sensitivity of the Cas12-chimeric probe system

[0134] Examples 2 to 4 demonstrated the role of the chimeric sequence in the Cas12 detection system, provided a new solution for the trans-cleavage of XNA, and provided an optimized solution for selecting different chimeric sequences according to different detection purposes. On the other hand, there was no amplification-free direct detection of DNA or RNA in the above Examples (1-4). In this example, a pre-amplification step was introduced to optimize the Cas12a-chimeric sequence detection system. The pre-amplification in this example was recombinase polymerase amplification (RPA), but it can be understood that any other method for amplifying target nucleic acids is still applicable. Figure 5a Figures a and b are flowcharts for detecting target nucleic acid fragments by pre-amplification or amplification-free methods. The target nucleic acid fragments include RNA and DNA, where Figure 5a the probes used are conventional ssDNAs, such as TTATT; Figure 5bIt is a chimeric probe with an RNA backbone and DNA modification, such as rUArUArUA. It can be understood that when the detection target is RNA, reverse transcription reaction needs to be carried out first, that is, cDNA is synthesized using RNA as a template. The specific transcription process can refer to the RPA reaction of related amplification method suppliers such as TwistAmp; then isothermal amplification method or polymerase chain reaction (PCR) is used to amplify the target fragment. The specific experimental steps are as follows: Use the TwistAmp Basic kit to complete RPA isothermal amplification. The amplification primers are the same as those shown in SEQ ID NO:27 and SEQ ID NO:28. The total volume of the amplification reaction system is 20 μL, including 19 μL of reaction solution (1 μL of each of the forward and reverse primers at 10 μM, 12 μL of primer-free rehydration buffer, 1 μL of amplification template, and 4 μL of ddH2O) and 1 μL of 280 mM MgOAc initiator (final concentration 14 mM). The amplification template is the HPV18 sequence (SEQ ID NO:1). The reaction temperature is 37 °C and the reaction duration is 20 min. For the detailed reaction system and process, refer to the TwistAmp product manual; Take 2 μL of the above amplified product and add it to 18 μL of the Cas12a detection reaction solution to form a detection system with a total volume of 20 μL. The components and total concentrations of the detection reaction solution are as follows: 1×NEB 2.1 buffer, 50 nM Cas12a (LaCas12a / AsCas12a), 1 U / μL RNase inhibitor, 50 nM crRNA (SEQ ID NO:8), and 125 nM probe (ssDNA (TTATT, SEQ ID NO:2) or rUArUArUA (SEQ ID NO:7)). Then use the Roche Lightcycler 480II instrument to measure the fluorescence value. Set the temperature at 37 °C, measure once every 30 s, and the measurement duration is 30 min.

[0135] As can be seen from Figure 5c -f, under the condition of no amplification, whether it is the LbCas12a-rUArUArUA system ( Figure 5d ) or the AsCas12a-rUArUArUA system ( Figure 5f ), the detection limit is the same as that of LaCas12a / AsCas12a-TTATT ( Figure 5c and e), reaching 1×10 6cp / μL. This result is consistent with that of Example 2 above, indicating that the result of detecting the target nucleic acid using the chimeric sequence has high stability; after 20 minutes of RPA pretreatment of the test sample, the detection sensitivity of the above 4 systems is increased by 1,000,000 times, reaching the single-molecule level. In summary, RPA can improve the detection level of the chimeric probe provided by the present invention, and on the other hand, it also indicates that the chimeric probe is also applicable to the conventional detection process.

[0136] Example 6: Application of the Cas12a-chimeric sequence detection system

[0137] To further expand the application of the Cas12a-chimeric sequence detection system, in this example, the above system is combined with microfluidic technology to explore whether the Cas12a-chimeric sequence detection system is applicable to microarray reactions. The experimental principle is as Figure 6a shown. The detection system flows through the pipeline of the microfluidic chip as the aqueous phase, and at the same time, the oil phase flows into the pipeline from the other end, and then the aqueous phase and the oil phase are mixed. Since the aqueous phase and the oil phase are immiscible, individual droplets are formed. The experimental steps are as follows: Prepare a master mixture with a total volume of 20 μL containing 1×NEB 2.1 buffer, 100 nM AsCas12a, 1 U / μL RNase inhibitor, 100 nM crRNA (SEQ ID NO: 8), 1 nM DNA (SEQ ID NO: 1) for activating Cas12 protein (not added in the negative control), and 500 nM chimeric reporter (rUArUArUA, SEQ ID NO: 7) on ice, and load it onto the microfluidic chip for droplet dispensing. The microfluidic chip is prepared according to the standard of SU-8 (MicroChem, Westborough, MA, USA), and the specific steps are the same as described in reference

[23] . The oil phase (Evagreen Digital PCR oil (#1864034)) used in this experiment is purchased from Bio-rad; Use 20 μL of the above main reaction solution as the aqueous phase to form a droplet with a diameter of 35 μm within 2 minutes, incubate the droplet at 37°C for 1 h, and then observe the luminescence of the droplet under a fluorescence microscope (Nikon).

[0138] The results are as Figure 6b shown. Since the negative control (NTC) does not contain a DNA activator, no fluorescence can be detected; on the other hand, the droplets with green fluorescence observed in the experimental group indicate that the chimeric sequence can still be effectively cleaved by the Cas12a protein under microscale reaction conditions, indicating that the Cas12a-chimeric sequence detection system is applicable to microscale reaction conditions, which is beneficial for high-throughput detection.

[0139] In addition, in this embodiment, the Cas12a-chimeric sequence system is applied to the detection of COVID-19, and the specific steps are as follows( Figure 6c ): Collect the saliva samples of the subjects to be tested, and use the RNA extraction kit QIAamp Viral RNA MiniKit (Qiagen, 52906) to extract the RNA in the samples, and perform reverse transcription loop-mediated isothermal amplification (RT-LAMP) on the samples for 20 min

[24] . The RT-LAMP experiment uses WarmStart LAMP 2×Master Mix (E1700S) from NEB. The total volume of each reaction system is 30 μL. The system contains 0.2 μM LAMP-F3 (SEQ ID NO: 29) and 0.2 μM LAMP-B3 (SEQ ID NO: 30), 1.6 μM LAMP-FIP (SEQ ID NO: 31) and 1.6 μM LAMP-BIP (SEQ ID NO: 32) primers, 0.4 μM LAMP-LF (SEQ ID NO: 33) and 0.4 μM LAMP-LB (SEQ ID NO: 34) primers and 1.0 μL of the RNA of the subject to be tested, and the system is made up to 30 μL with ddH2O; react at 65 °C for 30 min. The amplification product is mixed with the master mixture containing 1×NEB buffer 2.1, 50 nM Cas12a (LbCas12a), 1 U / μL RNase inhibitor, 50 nM crRNA (the specific sequence is the same as that described in SEQ ID NO: 35), and 500 nM chimeric probe (rUArUArUA, SEQ ID NO: 7). Then, 15 μL of the mixed solution is aspirated and transferred to a 384-well plate, and incubated at 37 °C for 1 h, and the final fluorescence value of the reaction system is measured. This experiment has a negative control and 3 replicates.

[0140] As Figure 6d The results show that all 5 pre-determined COVID-19 positive samples are accurately detected, which is consistent with the results of RT-PCR detection, that is, the detection accuracy of the Cas12a-chimeric sequence system is as high as 100%. The QuantiNova Probe RT-PCR Kit (Qiagen, 208354) was used in the RT-qPCR experiment. This clinical verification further proves the practical application of the chimeric sequence in the field of diagnosis.

[0141] Example 7: A nucleic acid detection system

[0142] Combined with the experimental results of Examples 1 to 6, this example provides a nucleic acid detection system. When the detection target is DNA, the reaction volume of the detection system is 10 to 100 μL, and the components and their final concentrations of the reaction system are as follows: 1× NEB 2.1 buffer, 50 nM Cas12, 50 nM crRNA, 10 U rat RNase inhibitor, 500 nM probe, and 1× DNA sample to be detected (the DNA sample concentration is preferably greater than 1 pM). Among them, when the Cas12 protein is LbCas12a protein, the probes used are any one or more of TrUTrUTrU (SEQ ID NO: 17), rUArUArUA (SEQ ID NO: 7), and ArUArUArU (SEQ ID NO: 6); when the Cas12 protein is AsCas12a protein, the probes used are any one or more of rUArUArUA (SEQ ID NO: 7) and ArUArUArU (SEQ ID NO: 6); the crRNA sequence is designed according to the specific sequence of the detection target.

[0143] When the detection target is RNA, cDNA needs to be generated through reverse transcription reaction, and the cDNA detection system is the same as the above-mentioned DNA detection system (amplify in advance before detection); or a DNA helper sequence is introduced into the detection system to directly detect RNA (it can be without prior amplification). Specifically, the reaction volume of the detection system is 10 to 100 μL, and the components and their final concentrations of the reaction system are as follows: 1× NEB 2.1 buffer, 90 nM AsCas12a, 50 U RNase inhibitor, 20 nM DNA helper, 90 nM gRNA, 31.25 mM MgCl2, 500 nM probe (rUArUArUA), and 1× RNA sample to be detected (the RNA sample concentration is preferably greater than 1 pM). The DNA helper contains a PAM sequence and a partial spacer sequence. In some specific embodiments, the DNA helper is shown in SEQ ID NO: 25 and SEQ ID NO: 26, and the crRNA sequence is designed according to the specific sequence of the detection target.

[0144] The above reaction system for detecting DNA or RNA can be used in combination with experiments such as isothermal amplification, PCR, or RPA. Amplification can improve the detection sensitivity of the system.

[0145] Combined with Examples 1 to 6, this example provides a method for detecting nucleic acids using the system described in Example 7. The specific steps are as follows: Extract DNA or RNA from the sample. If the sample is RNA, reverse transcribe the RNA into cDNA or directly detect the RNA using DNA helper and Cas12a protein (preferably AsCas12a in this example). Prepare a detection reaction system according to the detection target (DNA or RNA), and the specific system is the same as described above; Take 10 μL to 20 μL of the above reaction solution, quickly transfer it to a 96- or 384-well plate, and place the plate in a real-time fluorescence quantitative PCR instrument. The reaction temperature is determined by the type of Cas12 protein (the reaction temperature of LbCas12a and AsCas12a is 37 °C, and that of AapCas12b is 60 °C). Read the data once every 30 s to 60 s, and the reaction time is 30 min to 2 h. After the reaction, generate a real-time fluorescence curve or directly read the final fluorescence value, and judge whether the detection result is positive according to the fluorescence value. Set 3 replicates and a blank control for each experiment.

[0146] Example 8: Application of Chimeric Sequences in the RAPID System

[0147] The RAPID (RNA / DNA Affinity Precision Innovative Diagnostics) system is a CRISPR / Cas12a nucleic acid diagnostic system independently developed by this research team. By introducing nicks or incisions in the target sequence, the activity of Cas12a to cleave the target sequence can be activated. Cas12a can directly target the sequence downstream of the nick site and cleave it, while also cleaving the probe (natural or unnatural probe) in the reverse direction, thus enabling the detection of the target sequence without relying on the PAM sequence. Under the introduction of such nicks or incisions, the activity of Cas12a is activated, thereby enhancing its activity to trans-cleave sequences.

[0148] Here, the "nick" or "incision" can be a nick in the target nucleic acid sequence itself, or a nick formed by the binding of other sequences to the target sequence (here, there is no nick in the target sequence, but an unpaired base similar to an incision formed by binding to other sequences). As shown in the figure, when the protein binds to the target nucleic acids 101, 102, or 104, and the guide sequence 100 binds to the target sequence, one or more bases do not pair, forming a nick. The formation of such a nick can be directly formed by the guide sequence binding to the target sequence (base non-pairing), or together with other sequences to form one or more nicks. As Figure 7a shown, the leader or guide sequence 100 forms a nick 105 together with the sequences 101 and 104. Here, the target sequence can be single-stranded or double-stranded nucleic acid sequences, such as DNA or RNA.

[0149] To improve the performance of RAPID and expand its applications, this example investigated the impact of using non-ssDNA probes for RAPID detection of different Cas12 proteins. The schematic diagram of the process is shown in Figure 7a . Taking AsCas12a and LbCas12a as the research objects respectively, three different types of probes were selected, namely ssDNA probe, ssRNA probe, and chimeric sequence probe (one of the non-natural sequences), to investigate whether different probes can all be used for the trans-cleavage of the RAPID system to achieve detection.

[0150] Specifically, the target sequence with a nick site is the double-stranded binding product of DNA helper sense and DNA helper anti-sense (SEQ ID NO:36, SEQ ID NO:37). The probe is labeled with a FAM fluorophore and a 3IABkFQ quencher. The ssDNA probes include: R1 (TTATT) (SEQ ID NO:2), R5 ((T)6) (SEQ ID NO:13), R6 ((A)6) (SEQ ID NO:10), R7 ((C)6) (SEQ ID NO:12), R8 ((G)6) (SEQ ID NO:11); the ssRNA probes include: R2 (UUAUU) (SEQ ID NO:38), R9 ((rA)6) (SEQ ID NO:4), R10 ((rU)6) (SEQ ID NO:3), R11 ((rC)6) (SEQ ID NO:15), R12 ((rG)6) (SEQ ID NO:14); the chimeric sequence probes include: R3 ((rUA)3) (SEQ ID NO:7), R4 ((ArU)3) (SEQ ID NO:6), R13 ((ArA)3) (SEQ ID NO:18), R14 ((TrU)3) (SEQ ID NO:17), R15 ((GrG)3) (SEQ ID NO:20), R16 ((CrC)3) (SEQ ID NO:19). The specific experimental procedures are as follows: Prepare a 50 μL reaction system: 1×NEB 2.1 buffer (New England Biolab), 90 nM Cas12 (the types of Cas12 proteins in this example are AsCas12a or LbCas12a), 90 nM gRNA of LbCas12a or AsCas12a (the specific sequences are shown in SEQ ID NO:39 and SEQ ID NO:40), 50 U murine RNase inhibitor, 31.25 mM MgCl2, and 500 nM probe. Then add 50 nM target sequence with a nick site (SEQ ID NO:36, SEQ ID NO:37). The activated single-stranded sequence is (SEQ ID NO:41). In the negative control group, 2 μL ddH2O is added instead of the target sequence. Mix well; Pipette 15 μL of the mixture and transfer it to a 384-well plate. Incubate at 37 °C for 1 h, and measure the final fluorescence value of the reaction system. Each reaction system is set with 3 replicates, and the results are expressed as the mean ± standard deviation (n = 3) in relative fluorescence units (RFU). The bar graph represents the arithmetic mean ± SD. The detection results are as Figure 7b and Figure 7cAs shown, the performance detection results of RAPID when using AsCas12a and different probes are shown in Figure 7b , and the performance detection results of RAPID when using LbCas12a and different probes are shown in Figure 7c .

[0151] According to Figure 7b and Figure 7c , it can be seen that when the probe is the ssDNA sequence (R1: TTATT), the trans-cleavage performance of RAPID using AsCas12a and LbCas12a is consistent with that of the traditional Cas12a system. Compared with LbCas12a, AsCas12a shows a high non-specific signal in the absence of the target sequence (negative control group) ( Figure 7b (i) in Figure 7c (i)). When the probe is the ssRNA sequence for trans-cleavage (R2: rUrUrArUrU), the RNase activity of AsCas12a is stronger than that of LbCas12a, but the relative signals of both are weak. However, when DNA bases are introduced on the basis of ssRNA to construct chimeric sequences (R3: (rUA)3 and R4: (ArU)3), surprisingly, the trans-cleavage signals of these chimeric sequences are significantly improved compared with the RNA-based sequences of AsCas12a and LbCas12a ( Figure 7b (i) in Figure 7c (i)), and it can also significantly reduce the background signal of AsCas12, especially in the case of the chimeric sequence R3 as the probe.

[0152] When using ssDNA homopolymers (R5: (T)6, R6: (A)6, R7: (C)6 and R8: (G)6) as probes, the DNase activity was investigated in the presence and absence of the target sequence. It was found that compared with the high signal and low background of LbCas12a, ssDNA homopolymers produced very high background signals for R5, R6 and R7 of AsCas12a ( Figure 7b (ii)), and no trans-cleavage signal was observed for the poly-G (R8) probe in both AsCas12a and LbCas12a, which is similar to the results of Example 2.

[0153] When using ssRNA homopolymers (R9: (rA)6, R10: (rU)6, R11: (rC)6 and R12: (rG)6) as probes, RAPID was tested to evaluate its RNase activity. The RNase activities of R10 and R11 in AsCas12a and R9, R10 and R11 in LbCas12a are weak, and the activities of R9 and R12 with AsCas12a and R12 with LbCas12a are extremely low ( Figure 7bin (iii) and Figure 7b in (iii)).

[0154] When RAPID was applied to trans-cleaving chimeric homopolymers (R13: (ArA)3, R14: (TrU)3, R15: (GrG)3, and R16: (CrC)3), R13, R14, and R16 showed good trans-cleavage effects with AsCas12a and LbCas12a, while R15 was hardly cleaved ( Figure 7b in (iv) and Figure 7b in (iv)).

[0155] The above results indicate that both chimeric sequence probes and RNA probes can also be used for the detection of RAPID. Among them, the performance of chimeric sequence probes is comparable to that of some ssDNA sequences. For example, when using the chimeric sequence probe R3, the performance is enhanced, and the non-specific background signal of AsCas12a is significantly reduced, indicating that AsCas12 has a wider tolerance for chimeric sequence probes than LbCas12a. In addition, the background signal from the ssDNA probe R1 (the traditional reporter substrate of Cas12a) is significantly higher than that of the chimeric sequence R3, indicating that chimeric sequence probes can not only achieve the cleavage efficiency of ssDNA probes but also minimize background interference, and the comprehensive effect is significantly better than that of traditional ssDNA probes.

[0156] When using LbCas12a, the ssDNA sequence also showed superior trans-cleavage activity compared to RNA probes, highlighting the inherent preference of LbCas12a for ssDNA probes in the RAPID system. The ability of LbCas12a to cleave chimeric sequence probes R3 and R4 was slightly lower than that of ssDNA R1, but the ability to cleave chimeric sequence probes R14 and R16 was excellent, which also indicates that the RAPID system has flexible trans-cleavage substrate specificity.

[0157] In summary, when using traditional ssDNA probes, AsCas12a is usually affected by high background signals, while the chimeric sequence probe (R3) overcomes this limitation. For LbCas12a, chimeric sequence probes provide an effective solution to improve the signal-to-noise ratio. Based on the nucleic acid detection of LbCas12a and AsCas12a in the RAPID system, combined with chimeric sequence probes (such as R3: (rUA)3, etc.), the detection sensitivity of target sequences can be further improved.

[0158] All patents and publications mentioned in the specification of this invention indicate that these are publicly known technologies in the field and can be used in this invention. All patents and publications cited herein are equally listed in the references, as if each publication were specifically individually referenced. The invention described herein can be implemented in the absence of any one or more elements, one or more limitations, where such limitations are not specifically stated. For example, in each instance herein, the terms "comprising", "consisting essentially of", and "consisting of" can be replaced by either of the remaining two terms. The so-called "a" herein merely means "one", and does not exclude including only one, nor does it exclude including more than two. The terms and expressions used herein are for descriptive purposes and are not limiting, and there is no intention to indicate that the terms and interpretations described in this book exclude any equivalent features, but it can be understood that any suitable changes or modifications can be made within the scope of this invention and the claims. It is understood that the embodiments described in this invention are all preferred embodiments and features, and any person of ordinary skill in the art can make some changes and variations based on the essence described in this invention, and such changes and variations are also considered to be within the scope of this invention and the scope limited by the independent claims and the dependent claims.

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[0182]

[0183]

Claims

1. Use of a Cas12 protein for preparing a reagent for trans-cleaving an unnatural sequence to detect a target nucleic acid.

2. The use according to claim 1, wherein The unnatural sequence includes any one or more of the following: (1) Containing both deoxynucleotides and ribonucleotides; (2) Containing deoxynucleotides and / or ribonucleotides, and the deoxynucleotides and / or ribonucleotides carry artificial modifications that do not exist under natural conditions; (3) Containing deoxynucleotides and / or ribonucleotides, and the backbone composed of the deoxynucleotides and / or ribonucleotides carries artificial modifications that do not exist under natural conditions.

3. The use according to claim 1 or 2, characterized in that, The unnatural sequence contains both deoxynucleotides and ribonucleotides.

4. The use according to any one of claims 1 to 3, characterized in that, The deoxynucleotides and ribonucleotides of the unnatural sequence are alternately arranged at an interval of one item, that is, a ribonucleotide follows a deoxynucleotide, or a deoxynucleotide follows a ribonucleotide.

5. Use according to any one of claims 1 - 4, characterized in that, The Cas12 protein uses its RNase activity to trans-cleave the unnatural sequence.

6. The use according to any one of claims 1-5, wherein the Cas12 protein includes a Cas12a protein or a Cas12b protein.

7. The use according to any one of claims 1 to 6, characterized in that, The unnatural sequence is composed of nucleotides constituting RNA and nucleotides constituting DNA. The nucleotides constituting RNA are any one or more of uridine ribonucleotide, adenine ribonucleotide, cytosine ribonucleotide, and guanine ribonucleotide. The nucleotides constituting DNA are any one or more of thymine deoxynucleotide, adenine deoxynucleotide, cytosine deoxynucleotide, and guanine deoxynucleotide.

8. The use according to any one of claims 1 to 7, characterized in that, The unnatural sequence is arranged in the order of ribonucleotide - deoxynucleotide or deoxynucleotide - ribonucleotide. The ribonucleotide is any one or more of uridine ribonucleotide, adenine ribonucleotide, cytosine ribonucleotide, and guanine ribonucleotide. The deoxynucleotide is any one or more of thymine deoxynucleotide, adenine deoxynucleotide, cytosine deoxynucleotide, and guanine deoxynucleotide.

9. The use according to any one of claims 1-8, characterized in that, The unnatural sequence includes any one or more of rUArUArUA, ArUArUArU, rUrUrArUrUrU, TrUTrUTrU, ArAArAArA, CrCCrCCrC, and GrGGrGGrG. The rU is uridine ribonucleotide, the A is adenine deoxynucleotide, the T is thymine deoxynucleotide, the rA is adenine ribonucleotide, the C is cytosine deoxynucleotide, the rC is cytosine ribonucleotide, the G is guanine deoxynucleotide, and the rG is guanine ribonucleotide.

10. The use according to any one of claims 1-9, characterized in that, The unnatural sequence includes any one or more of rUArUArUA, ArUArUArU, and TrUTrUTrU.

11. Use according to any one of claims 1 to 10, wherein, The reagent includes the necessary reagents for amplifying the target nucleic acid.

12. Use according to any one of claims 1 to 10, wherein, The reagent includes a Cas12 protein and an unnatural sequence.

13. The use according to claim 1, wherein The unnatural sequence includes nucleic acid sequences that cannot be generated during the long evolutionary process in nature or that cannot be stably inherited once generated.

14. The use according to claim 1, wherein the target nucleic acid is present in a sample.

15. The use according to claim 1, wherein the sample is saliva, blood, urine, or nasal secretion.

16. The use according to claim 1, wherein the target nucleic acid comprises DNA or RNA.

17. Use of a non-natural sequence for preparing a reagent for detecting a target nucleic acid, characterized in that, The unnatural sequence comprises any one or more of the following: (1) containing both deoxynucleotides and ribonucleotides; (2) containing deoxynucleotides and / or ribonucleotides, and the deoxynucleotides and / or ribonucleotides carry artificially created modifications that do not exist under natural conditions; (3) containing deoxynucleotides and / or ribonucleotides, and the backbone composed of the deoxynucleotides and / or ribonucleotides carries artificially created modifications that do not exist under natural conditions.

18. The use according to claim 17, wherein, The reagent further comprises Cas12 protein.

19. The use according to claim 17, wherein The unnatural sequence contains both deoxynucleotides and ribonucleotides.

20. A probe, characterized in that, The probe comprises an unnatural sequence, and the unnatural sequence comprises any one or more of the following: (1) containing both deoxynucleotides and ribonucleotides; (2) containing deoxynucleotides and / or ribonucleotides, and the deoxynucleotides and / or ribonucleotides carry artificially created modifications that do not exist under natural conditions; (3) containing deoxynucleotides and / or ribonucleotides, and the backbone composed of the deoxynucleotides and / or ribonucleotides carries artificially created modifications that do not exist under natural conditions.

21. The probe according to claim 20, wherein The probe is composed of nucleotides that make up RNA and nucleotides that make up DNA. The nucleotides that make up RNA are any one or more of uridine ribonucleotide, adenine ribonucleotide, cytosine ribonucleotide, and guanine ribonucleotide. The nucleotides that make up DNA are any one or more of thymine deoxynucleotide, adenine deoxynucleotide, cytosine deoxynucleotide, and guanine deoxynucleotide.

22. The probe according to claim 20, wherein, The probe is arranged in the order of ribonucleotide - deoxynucleotide or deoxynucleotide - ribonucleotide. The ribonucleotide is any one or more of uridine ribonucleotide, adenine ribonucleotide, cytosine ribonucleotide, and guanine ribonucleotide. The deoxynucleotide is any one or more of thymine deoxynucleotide, adenine deoxynucleotide, cytosine deoxynucleotide, and guanine deoxynucleotide.

23. The probe according to claim 20, wherein The probe sequence comprises any one or more of rUArUArUA, ArUArUArU, rUrUrArUrUrU, TrUTrUTrU, ArAArAArA, CrCCrCCrC, and GrGGrGGrG. The rU is uridine ribonucleotide, the A is adenine deoxynucleotide, the T is thymine deoxynucleotide, the rA is adenine ribonucleotide, the C is cytosine deoxynucleotide, the rC is cytosine ribonucleotide, the G is guanine deoxynucleotide, and the rG is guanine ribonucleotide.

24. The probe according to claim 20, wherein, The probe sequence comprises any one or more of rUArUArUA, ArUArUArU, and TrUTrUTrU.

25. The probe according to claim 20, wherein the probe can be trans-cleaved by Cas12 protein.

26. A kit for detecting target nucleic acid in a sample, characterized in that, The kit includes: Cas12 protein and a non-natural sequence that can be trans-cleaved by Cas12 protein.

27. The kit according to claim 26, wherein, The Cas12 protein includes Cas12a and Cas12b subfamilies.

28. The kit according to claim 26, wherein, The Cas12 protein is any one or more of LbCas12a, AsCas12a, and AapCas12b.

29. The kit according to any one of claims 26-28, characterized in that, The non-natural sequence includes any one or more of the following: (4) Containing both deoxynucleotides and ribonucleotides; (5) Containing deoxynucleotides and / or ribonucleotides, and the deoxynucleotides and / or ribonucleotides carry artificially created modifications that do not exist under natural conditions; (6) Containing deoxynucleotides and / or ribonucleotides, and the backbone composed of the deoxynucleotides and / or ribonucleotides carries artificially created modifications that do not exist under natural conditions.

30. The kit according to any one of claims 26 - 29, characterized in that, The non-natural sequence contains both deoxynucleotides and ribonucleotides.

31. The kit according to any one of claims 26-30, characterized in that, The deoxynucleotides and ribonucleotides of the non-natural sequence are alternately arranged every other item, that is, ribonucleotides follow deoxynucleotides, or deoxynucleotides follow ribonucleotides.

32. The kit according to any one of claims 26 - 31, characterized in that, The non-natural sequence includes a sequence composed of nucleotides that make up RNA and nucleotides that make up DNA. The nucleotides that make up RNA are any one or more of uridine ribonucleotide, adenine ribonucleotide, cytosine ribonucleotide, and guanine ribonucleotide. The nucleotides that make up DNA are any one or more of thymine deoxynucleotide, adenine deoxynucleotide, cytosine deoxynucleotide, and guanine deoxynucleotide.

33. The kit according to any one of claims 26 - 31, characterized in that, The non-natural sequence is arranged in the order of ribonucleotide - deoxynucleotide or deoxynucleotide - ribonucleotide in sequence. The ribonucleotide is any one or more of uridine ribonucleotide, adenine ribonucleotide, cytosine ribonucleotide, and guanine ribonucleotide. The deoxynucleotide is any one or more of thymine deoxynucleotide, adenine deoxynucleotide, cytosine deoxynucleotide, and guanine deoxynucleotide.

34. The kit according to any one of claims 26-33, wherein, The non-natural nucleic acid sequence described includes (rUA) n nucleic acid sequence, where n is any natural number integer.

35. The kit according to claim 34, wherein, n＝1-100。 36. The kit according to any one of claims 26-35, characterized in that The non-natural sequence includes any one or more of rUArUArUA, ArUArUArU, rUrUrArUrUrU, TrUTrUTrU, ArAArAArA, CrCCrCCrC, and GrGGrGGrG. The rU is uridine ribonucleotide, the A is adenine deoxynucleotide, the T is thymine deoxynucleotide, the rA is adenine ribonucleotide, the C is cytosine deoxynucleotide, the rC is cytosine ribonucleotide, the G is guanine deoxynucleotide, and the rG is guanine ribonucleotide.

37. The kit according to claim 36, characterized in that, The non-natural sequence includes any one or more of rUArUArUA, ArUArUArU, and TrUTrUTrU.

38. The kit according to claim 26, wherein, The kit further includes necessary reagents for amplifying the target nucleic acid.

39. The kit according to claim 38, wherein, The necessary reagents include enzymes, primers, and inorganic salt reagents.

40. The kit according to claim 26, wherein, The target nucleic acid includes natural DNA or natural RNA.

41. The kit according to claim 26, wherein, The DNA or RNA in the target nucleic acid includes double-stranded or single-stranded forms.

42. The kit according to claim 26, wherein, The DNA in the target nucleic acid is double-stranded and the RNA is single-stranded.

43. The kit according to claim 26, wherein The target nucleic acid includes a nicked target nucleic acid.

44. The kit according to claim 26, wherein, The unnatural sequence includes a labeling substance, and the labeling substance is a fluorescent labeling substance.

45. The kit according to claim 26, wherein, The unnatural sequence includes a chimeric sequence.

46. The kit according to claim 26, wherein, The kit further includes a reagent for reverse-transcribing RNA into cDNA or a DNA helper.

47. The kit according to claim 45, wherein, The chimeric sequence includes: single chimeric (poly ArA), double chimeric (poly rUArUA), and multiple chimeric (UrACrGTrA).

48. The kit according to claim 26, wherein, The unnatural sequence includes a nucleic acid sequence that cannot be generated during the long evolutionary process in nature or a nucleic acid sequence that, once generated, cannot be stably inherited.

49. The kit according to claim 1, wherein the target nucleic acid is present in a sample.

50. The kit according to claim 26, wherein, When the Cas12 protein is LbCas12a, the probe sequence comprises any one or more of rUArUArUA, ArUArUArU, and TrUTrUTrU; when the Cas protein is AsCas12a, the probe sequence comprises any one or more of rUArUArUA and ArUArUArU.

51. The kit according to claim 46, characterized in that, The DNA helper is a partial sequence with a PAM sequence and capable of binding to the target RNA or a simple PAM sequence.

52. The kit according to claim 26, wherein The target sequence has a nick or a cut.

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