Multiplex detection kit based on crisper-cas system

Abstract

A method for simultaneously detecting two or more target genetic materials present in a biological sample, wherein the method comprises: adding two or more Crispr-Cas systems capable of respectively targeting two or more target genetic materials and two or more probe molecules corresponding to the Crispr-Cas systems into the biological sample; when any of the Crispr-Cas systems encounters the target genetic material it targets, the Cas enzyme in the Crispr-Cas system can be activated by the target genetic material it targets to trans-cleave the probe molecule corresponding to it; detecting the fluorescence signal emitted by the trans-cleaved probe molecule to determine whether the target genetic material is present in the biological sample. Also related are the corresponding kits.

Description

Technical Field

[0001] This application relates to the fields of gene editing and gene diagnostics, and is a nucleic acid detection method developed based on the CRISPR trans-cutting system. It involves the establishment of a technical foundation for the selective cleavage of target DNA by Cas12 family proteins and its variants under RNA mediation, the establishment of RNA multiplex detection methods, and the development of related detection kits. In the future, it can be applied to genetic diagnosis typing, inspection and quarantine, and other fields. Background Technology

[0002] Nucleic acid detection platforms based on CRISPR technology have gained increasing attention due to their high sensitivity, portability, and high specificity. Viruses mutate, and genes exhibit polymorphism; therefore, the need for viral and gene typing necessitates the development of multiplex nucleic acid detection platforms. CRISPR-based multiplex nucleic acid detection, a next-generation molecular diagnostic technology, holds the promise of reshaping the molecular diagnostics field. Cas13, possessing ssRNA trans-cleavage activity, has been developed for RNA detection. The CRISPR-Cas13-based RNA detection platform, called SHERLOCK, combined with isothermal amplification technology, can detect Zika and dengue viruses, identify pathogens, and perform SNP analysis. In 2018, SHERLOCKv2 was established using CRISPR nucleases Cas13a, Cas12a, and Csm6 combined with isothermal amplification technology, enabling multiplex, quantification, and strip-based nucleic acid detection. However, since then, there have been no major breakthroughs in CRISPR-based multiplex detection. However, with the outbreak of various viruses worldwide, and because these viruses produce different symptoms and require different treatments, there is a growing demand for more targeted treatment and recovery, leading to greater societal need for viral strain typing. Therefore, the establishment of dual or multiplex testing platforms has become of even greater practical significance. Summary of the Invention

[0003] This application therefore provides:

[0004] 1. A method for simultaneously detecting two or more target genetic materials present in a biological sample, wherein the method comprises:

[0005] Add two or more CRISPR-Cas systems capable of targeting two or more target genetic materials respectively, and two or more probe molecules corresponding to the CRISPR-Cas systems to the biological sample.

[0006] When any of the Crispr-Cas systems encounters its target genetic material, the Cas enzyme in the Crispr-Cas system can be activated by its target genetic material, thereby trans-cleaving its corresponding probe molecule.

[0007] The presence of the target genetic material in the biological sample is determined by detecting the fluorescent signal emitted by the trans-cleaved probe molecule.

[0008] 2. The method according to item 1, wherein,

[0009] The CRISPR-Cas system includes a Cas enzyme or a nucleic acid capable of expressing a Cas enzyme, and a guide molecule capable of specifically binding to the target genetic material.

[0010] Preferably, the two or more Crispr-Cas systems are selected from two or more of the following group: Crispr-Cas12i system, Crispr-Cas12b system, Crispr-CasX system, and Crispr-Cas12a system; more preferably, the two or more Crispr-Cas systems are Crispr-Cas12i system and Crispr-Cas12b system, and the guide molecule is sgRNA and crRNA.

[0011] 3. The method according to item 2, wherein,

[0012] The two or more probe molecules each have a sequence composed of multiple repeating nucleotides, and the phosphodiester bonds at different positions of the repeating nucleotide sequences of the two or more probe molecules are modified, preferably by thiomodification.

[0013] Furthermore, the two or more probe molecules have fluorescence-quenching groups (FQ) linked to their respective repeating nucleotide sequences; preferably, the two or more fluorescence-quenching groups are selected from two or more of the following: FAM-BHQ1, HEX-BHQ1, Rox-BHQ2, Cy3-Cy5, TET-Dabcyl, FAM-7T-Dabcyl, FAM-BHQ2, Cy3-BHQ2, Cy5-BHQ2, JOE-BHQ1.

[0014] 4. The method according to item 3, wherein the two or more target genetic materials are genetic materials from different biological samples or different genetic materials from the same biological sample.

[0015] 5. The method according to item 4, wherein, among the two or more probe molecules, the structure of the first probe molecule and the second probe molecule is an FQ-poly-7T, FQ-poly-7A or FQ-poly-7C sequence, preferably the first probe molecule and the second probe are both FQ-poly-7C or FQ-poly-7T sequences.

[0016] 6. The method according to item 5, wherein the phosphodiester bond between the first probe molecule and the second T is modified, and the phosphodiester bond between the sixth T and the seventh T is modified, preferably by thiomodification.

[0017] 7. The method according to item 2, wherein, preferably, the Crispr-Cas12b is an engineered Cas12b that has undergone E475R+Q119F+E758R substitution relative to its wild type; preferably, the Crispr-Cas12i is an engineered Cas12i that has undergone N164Y+E176R+K238R+T447R+E563R+E323R+D362R substitution relative to its wild type; wherein the engineered Cas12i is as shown in SEQ ID NO:7, and the engineered Cas12b is as shown in SEQ ID NO:13.

[0018] 8. A kit for multiplex detection of biological samples targeting two or more genetic materials carried by the biological sample, wherein,

[0019] The kit includes two or more CRISPR-Cas systems that target the target genetic material, and probe molecules that can be trans-cleaved by the CRISPR-Cas system and emit fluorescent signals after the CRISPR-Cas system is activated by the target genetic material.

[0020] 9. The kit according to item 8, wherein,

[0021] The CRISPR-Cas system includes a Cas enzyme or a nucleic acid capable of expressing a Cas enzyme, and a guide molecule capable of specifically binding to the target genetic material.

[0022] Preferably, the two or more Crispr-Cas systems are selected from two or more of the following group: Crispr-Cas12i system, Crispr-Cas12b system, Crispr-CasX system, and Crispr-Cas12a system; more preferably, the two or more Crispr-Cas systems are Crispr-Cas12i system and Crispr-Cas12b system, and the guide molecule is sgRNA and crRNA.

[0023] 10. The kit according to item 9, wherein,

[0024] The two or more probe molecules each have a sequence composed of multiple repeating nucleotides, and the phosphodiester bonds at different positions of the repeating nucleotide sequences of the two or more probe molecules are modified, preferably by thiomodification.

[0025] Furthermore, the two or more probe molecules have fluorescence-quenching groups (FQ) linked to their respective repeating nucleotide sequences; preferably, the two or more fluorescence-quenching groups are selected from two or more of the following: FAM-BHQ1, HEX-BHQ1, Rox-BHQ2, Cy3-Cy5, TET-Dabcyl, FAM-7T-Dabcyl, FAM-BHQ2, Cy3-BHQ2, Cy5-BHQ2, JOE-BHQ1.

[0026] 11. The kit according to item 10, wherein the two or more target genetic materials are genetic materials from different biological samples or different genetic materials from the same biological sample.

[0027] 12. The kit according to item 11, wherein, among the two or more probe molecules, the structure of the first probe molecule and the second probe molecule is an FQ-poly-7T, FQ-poly-7A or FQ-poly-7C sequence, preferably the first probe molecule and the second probe are both FQ-poly-7C or FQ-poly-7T sequences.

[0028] 13. The kit according to item 12, wherein the first probe molecule is modified at the phosphodiester bond between the first T and the second T, and the second probe molecule is modified at the phosphodiester bond between the sixth T and the seventh T, preferably by thiomodification.

[0029] 14. The kit according to item 9, wherein the Crispr-Cas12b is an engineered Cas12b with E475R+Q119F+E758R substitution relative to its wild type; and the Crispr-Cas12i is an engineered Cas12i with N164Y+E176R+K238R+T447R+E563R+E323R+D362R substitution relative to its wild type; wherein the engineered Cas12i is as shown in SEQ ID NO:7, and the engineered Cas12b is as shown in SEQ ID NO:13.

[0030] This application also provides:

[0031] 1. A method for simultaneously or sequentially detecting two or more target genetic materials present in a biological sample, wherein the method comprises:

[0032] Add two or more CRISPR-Cas systems capable of targeting two or more of the target genetic materials respectively, and probe molecules corresponding to the CRISPR-Cas systems to the biological sample.

[0033] When any of the Crispr-Cas systems encounters its target genetic material, the Cas enzyme in the Crispr-Cas system can be activated by its target genetic material, thereby trans-cleaving its corresponding probe molecule.

[0034] The presence of the target genetic material in the biological sample is determined by detecting the fluorescence signal or colorimetric result of the trans-cleaved probe molecule.

[0035] 2. The method according to item 1, wherein,

[0036] The CRISPR-Cas system includes a Cas enzyme or a nucleic acid capable of expressing a Cas enzyme, and a guide molecule capable of specifically binding to the target genetic material.

[0037] Preferably, the two or more Crispr-Cas systems are selected from two or more of the following groups: Crispr-Cas12i system, Crispr-Cas12b system, Crispr-CasX system, and Crispr-Cas12a system;

[0038] The guide molecules are sgRNA and / or crRNA.

[0039] 3. The method according to item 2, wherein,

[0040] The probe molecules are of two or more types, each having a sequence composed of multiple repeating nucleotides, and the phosphodiester bonds of the repeating nucleotide sequences in the probe molecules are modified at different positions; the probe molecules have fluorescence-quenching groups (FQ) linked to their respective repeating nucleotide sequences; preferably, the fluorescence-quenching groups are selected from two or more of the following: FAM-BHQ1, HEX-BHQ1, Rox-BHQ2, Cy3-Cy5, TET-Dabcyl, FAM-7T-Dabcyl, FAM-BHQ2, Cy3-BHQ2, Cy5-BHQ2, JOE-BHQ1.

[0041] 4. The method according to item 2, wherein the probe molecule is of only one kind and is unmodified FAM-7T-Biotin and / or FAM-7C-Biotin.

[0042] 5. The method according to item 1, wherein the two or more target genetic materials are genetic materials from different biological samples or different genetic materials from the same biological sample.

[0043] 6. The method according to item 3, wherein the Crispr-Cas system is of two types, namely Crispr-Cas12i and Crispr-Cas12b, and the probe molecule is of two types, namely a first probe molecule and a second probe molecule, and the structure of the first probe molecule and the second probe molecule is an FQ-poly-7T, FQ-poly-7A or FQ-poly-7C sequence, preferably the first probe molecule and the second probe are both FQ-poly-7C or FQ-poly-7T sequences.

[0044] 7. The method according to item 6, wherein the first probe molecule has only one unmodified phosphodiester bond located between the m-th nucleotide and the (m+1)-th nucleotide, and the second probe molecule has only one unmodified phosphodiester bond located between the n-th nucleotide and the (n+1)-th nucleotide, wherein m is less than n.

[0045] 8. The method as described in item 7, wherein m is an integer selected from 1 to 6, and n is an integer selected from 1 to 6, wherein the modification is a thiomodification, preferably m is 1, 2, or 3, and n is 4, 5, or 6.

[0046] 9. The method according to item 6, wherein the Crispr-Cas12b is an engineered Cas12b that has undergone E475R+Q119F+E758R substitution relative to its wild type; and the Crispr-Cas12i is an engineered Cas12i that has undergone N164Y+E176R+K238R+T447R+E563R+E323R+D362R substitution relative to its wild type; wherein the engineered Cas12i is as shown in SEQ ID NO:7, and the engineered Cas12b is as shown in SEQ ID NO:13.

[0047] 10. A kit for simultaneously or sequentially detecting two or more target genetic materials carried by a biological sample, wherein,

[0048] The kit includes two or more CRISPR-Cas systems that target the target genetic material, and probe molecules that can be trans-cleaved by the CRISPR-Cas system and emit fluorescent signals after the CRISPR-Cas system is activated by the target genetic material.

[0049] 11. The kit according to item 10, wherein,

[0050] The CRISPR-Cas system includes a Cas enzyme or a nucleic acid capable of expressing a Cas enzyme, and a guide molecule capable of specifically binding to the target genetic material.

[0051] Preferably, the two or more Crispr-Cas systems are selected from two or more of the following groups: Crispr-Cas12i system, Crispr-Cas12b system, Crispr-CasX system, and Crispr-Cas12a system;

[0052] The guide molecules are sgRNA and / or crRNA.

[0053] 12. The kit according to item 11, wherein,

[0054] The probe molecules are of two or more types, each having a sequence composed of multiple repeating nucleotides, and the phosphodiester bonds of the repeating nucleotide sequences in the probe molecules are modified at different positions; the probe molecules have fluorescence-quenching groups (FQ) linked to their respective repeating nucleotide sequences; preferably, the fluorescence-quenching groups are selected from two or more of the following: FAM-BHQ1, HEX-BHQ1, Rox-BHQ2, Cy3-Cy5, TET-Dabcyl, FAM-7T-Dabcyl, FAM-BHQ2, Cy3-BHQ2, Cy5-BHQ2, JOE-BHQ1.

[0055] 13. The kit according to item 11, wherein the probe molecule is of only one kind and is unmodified FAM-7T-Biotin and / or FAM-7C-Biotin.

[0056] 14. The kit according to item 10, wherein the two or more target genetic materials are genetic materials from different biological samples or different genetic materials from the same biological sample.

[0057] 15. The kit according to claim 12, wherein the Crispr-Cas system is of two types, namely Crispr-Cas12i and Crispr-Cas12b, and the probe molecules are of two types, namely a first probe molecule and a second probe molecule, and the structure of the first probe molecule and the second probe molecule is an FQ-poly-7T, FQ-poly-7A or FQ-poly-7C sequence, preferably the first probe molecule and the second probe are both FQ-poly-7C or FQ-poly-7T sequences.

[0058] 16. The kit according to item 15, wherein the first probe molecule has only one unmodified phosphodiester bond located between the m-th nucleotide and the (m+1)-th nucleotide, and the second probe molecule has only one unmodified phosphodiester bond located between the n-th nucleotide and the (n+1)-th nucleotide, wherein m is less than n.

[0059] 17. The kit of item 16, wherein m is an integer selected from 1 to 6, and n is an integer selected from 1 to 6, wherein the modification is a thiomodification, preferably m is 1, 2, or 3, and n is 4, 5, or 6.

[0060] 18. The kit according to claim 15, wherein the Crispr-Cas12b is an engineered Cas12b with E475R+Q119F+E758R substitution relative to its wild type; and the Crispr-Cas12i is an engineered Cas12i with N164Y+E176R+K238R+T447R+E563R+E323R+D362R substitution relative to its wild type; wherein the engineered Cas12i is as shown in SEQ ID NO:7, and the engineered Cas12b is as shown in SEQ ID NO:13.

[0061] The beneficial technical effects achieved by the technical solution of this application

[0062] This invention provides a multiplex nucleic acid detection method based on different Cas12 family proteins, capable of specifically detecting DNA and RNA. It utilizes a multiplex target detection platform technology built upon the Cas12 nuclease's preference for cleavage by different modified probes. This technology offers a rapid and convenient method for DNA or RNA detection and typing, applicable to various fields such as infectious disease screening, genotyping and detection, genetic diagnosis, medicine and healthcare, agriculture, and the chemical industry. It is also suitable for various application scenarios, especially the single-tube dual-detection system, which can be used in hospitals with limited equipment for pathogen typing, providing sensitive and specific sequence analysis of viral strains. The test strip and visual detection system are more suitable for home and customs settings, also exhibiting good sensitivity. While it cannot detect multiple targets in a single tube, separate tube detection offers better accuracy and greater portability. Attached Figure Description

[0063] Figure 1 SDS-PAGE image of purified Cas12 family protein a

[0064] Figure 2. (a) Comparison of trans-cleavage activities of different Cas12b variants. Cas12b trans-cleavage activity was activated at 37°C using a 40 nM activator. A 200 nM 5'-FAM-TTTTTTT-BHQ1-3' probe was cleaved using 100 nM sgRNA and 30 nM of different Cas protein variants. The results are shown in the figure. Cas12b-E475R+Q119F+E758R exhibited better trans-cleavage activity. (b) Comparison of trans-cleavage activities of different Cas12i variants. The trans-cleavage activity of Cas12b was activated at 37°C using a 40 nM activator. The 200 nM 5'-FAM-TTTTTTT-BHQ1-3' probe was cleaved with 200 nM crRNA and 100 nM of different Cas protein variants. The results are shown in the figure. Cas12b-N164Y+E176R+K238R+T447R+E563R+E323R+D362R exhibited good trans-cleavage activity. (c) Comparison of trans-cleavage activities of different Cas12X variants. The trans-cleavage activity of CasX was activated at 37°C using a 40 nM activator. The 200 nM 5'-FAM-TTTTTTT-BHQ1-3' probe was cleaved with 1 µM sgRNA and 100 nM of different Cas protein variants. The results are shown in the figure. CasX-T26R+K610R+K808R exhibited good trans-cleavage activity.

[0065] Figure 3 Analysis of the cleavage preference of Cas12i variants for different sequence probes. Cas12i was used for cleavage experiments with poly_T-FQ, poly_A-FQ, poly_G-FQ, and poly_C-FQ probes under crRNA guidance and target DNA activation, and the results are shown in the figure.

[0066] Figure 4 Probe preference analysis for cleavage of two Cas12 family enzymes with different modification groups. The trans-cleavage activities of Cas12i and Cas12b were activated at 37°C using a 40 nM activator. 200 nM 5'-FAM-TTTTTTT-BHQ1-3' and 5'-BHQ1-TTTTTTT-FAM-3' probes were cleaved with 200 nM crRNA, 100 nM Cas12i, or 30 nM Cas12b protein variants. The results are shown in the figure. The Cas12b and Cas12i variants exhibited opposite preferences for the two probes.

[0067] Figure 5Preferred cleavage patterns of poly-T probes modified with thiophosphate bonds at different phosphodiester bond positions by two Cas12 family enzymes. The trans-cleavage activities of Cas12i and Cas12b were activated at 37°C using a 40 nM activator. 1 µM probes with thiophosphate bonds modified at different phosphodiester bond positions were cleaved using 200 nM crRNA, 100 nM Cas12i, or 30 nM Cas12b protein variants. The results are shown in the figure, indicating a preference trend of increasing trans-cleavage activity from the 5' to 3' end for Cas12b. The preference trend of increasing trans-cleavage activity from the 5' to 3' end for Cas12i is also shown.

[0068] Figure 6 This study analyzed the cleavage preference of two Cas12 family enzymes for poly-T probes modified with different fluorescent groups (HEX modification) at different phosphodiester bond thioversion positions. The trans-cleavage activities of Cas12i and Cas12b were activated at 37°C using a 40 nM activator. Probes modified with 1 µM of phosphodiester bond thioversion at different positions were cleaved using 200 nM crRNA, 100 nM Cas12i, or 30 nM Cas12b protein variants. The results, as shown in the figure, indicate a preference trend of increasing trans-cleavage activity from the 5' to 3' end for Cas12b. The preference trend of increasing trans-cleavage activity from the 5' to 3' end for Cas12i was also observed, and changing the fluorescent modification group did not alter this preference trend.

[0069] Figure 7 This study analyzed the cleavage preference of two Cas12 family enzymes for poly-C probes modified with different phosphodiester bond thiophosphate sequences. The trans-cleavage activities of Cas12i and Cas12b were activated at 37°C using a 40 nM activator. Probes modified with 1 µM of phosphodiester bond thiophosphate at different positions were cleaved using 200 nM crRNA, 100 nM Cas12i, or 30 nM Cas12b protein variants. The results, as shown in the figure, indicate a preference trend of increased trans-cleavage activity from the 5' to 3' end for Cas12b. The preference trend of increased trans-cleavage activity from the 5' to 3' end for Cas12i was also observed. Replacing the base sequence with poly-C did not alter this preference trend.

[0070] Figure 8 Analysis of the thymine homopolymer cleavage preference of various Cas12a homologs for the modified phosphodiester bonds of the FAM-BHQ1 combination.

[0071] Figure 9The screening of HPV16 and HPV18 E6 and E7 gene sequences was based on the efficient sgRNA sites of Cas12b. The corresponding fragments were obtained by PCR and purification, and the concentration was determined using Nanodrop as the activator fragment. At 37°C, with 40 nM activator and 20 nM sgRNA, a 200 nM 5'-FAM-TTTTTTT-BHQ1-3' probe was cleaved to maximize the separation of the two viral sequences into the selected sgRNA.

[0072] Figure 10 Feasibility analysis of multiplex detection in a single tube. Based on probes that specifically cleave two Cas12 proteins, the cleavage systems of the two proteins were used to test the distinguishing ability of the two target detection systems for the two targets. Simultaneously, the two systems were mixed together, and detection was performed in a single mixture to analyze its feasibility.

[0073] Figure 11 Sensitivity analysis of single-tube multiplex genomic analysis. The genomic genome, quantified by qPCR, was first amplified isothermally, followed by multiplex analysis to determine the limit of detection.

[0074] Figure 12 Sensitivity and specificity analysis of SARS-CoV-2 wild-type and Omicron variant typing. Using a real-world example, specific sgRNAs were screened using DNA from the synthesized SARS-CoV-2 wild-type strain genome and the RNA nucleic acid of the Omicron variant.

[0075] Figure 13 A single-tube method was used to analyze the sensitivity of dual-detection genotyping for SARS-CoV-2. The CRISPR / Cas12b and CRISPR / Cas12i systems were mixed in the same system to perform genotyping detection of RNA from the original SARS-CoV-2 strain and the XBB1.5 variant.

[0076] Figure 14 Lateral flow chromatography was used for dual-detection genotyping of SARS-CoV-2. The CRISPR / Cas12b and CRISPR / Cas12i systems were mixed in the same system to perform genotyping of RNA from the original SARS-CoV-2 strain and the XBB1.5 variant.

[0077] Figure 15 Lateral flow chromatography was used for sensitivity analysis of dual-detection SARS-CoV-2 genotyping. The CRISPR / Cas12b and CRISPR / Cas12i systems were mixed in the same system to perform genotyping and sensitivity analysis of RNA from the original SARS-CoV-2 strain and the XBB1.5 variant.

[0078] Figure 16 Visual observation of dual-detection SARS-CoV-2 genotyping. The CRISPR / Cas12b and CRISPR / Cas12i systems were mixed in the same system to perform genotyping detection on the RNA of the original SARS-CoV-2 strain and the XBB1.5 variant.

[0079] Figure 17 Visual observation of SARS-CoV-2 dual detection genotyping sensitivity analysis. The CRISPR / Cas12b and CRISPR / Cas12i systems were mixed in the same system to perform genotyping detection and sensitivity analysis of the RNA of the original SARS-CoV-2 strain and the XBB1.5 variant. Detailed Implementation

[0080] Specific embodiments of the invention will now be described in more detail with reference to the accompanying drawings. While specific embodiments of the invention are shown in the drawings, it should be understood that the invention can be implemented in various forms and should not be limited to the embodiments set forth herein. Rather, these embodiments are provided to enable a more thorough understanding of the invention and to fully convey the scope of the invention to those skilled in the art.

[0081] It should be noted that certain terms are used in the specification and claims to refer to specific components. Those skilled in the art will understand that different terms may be used to refer to the same component. This specification and claims do not distinguish components based on differences in terminology, but rather on differences in function. The terms "comprising" or "including" used throughout the specification and claims are open-ended and should be interpreted as "comprising but not limited to." The following descriptions are preferred embodiments for carrying out the invention; however, these descriptions are for the purpose of understanding the general principles of the specification and are not intended to limit the scope of the invention. The scope of protection of this invention is determined by the appended claims.

[0082] As used herein, "substantially free of" with respect to a particular component is used to indicate that the particular component was not intentionally formulated into the composition and / or is present only as a contaminant or in trace amounts. Therefore, the total amount of the particular component resulting from any accidental contamination of the composition is less than 0.05%, preferably less than 0.01%. Most preferably, the composition is in which the amount of the particular component is undetectable by standard analytical methods.

[0083] As used in this specification, "a" or "an" may mean one or more. As used in the claims, when used with the word "comprising," the word "a" or "an" may mean one or more.

[0084] The term “or” is used in the claims to mean “and / or” unless it is explicitly stated that it refers only to an alternative or that the alternatives are mutually exclusive, although this disclosure supports the definition of referring only to an alternative and “and / or”. As used herein, “another” can mean at least a second or more.

[0085] Throughout this application, the term “about” is used to indicate a value that includes the inherent variation of the error of the apparatus, the method used to determine that value, or the variation that exists between subjects under study.

[0086] The methods for obtaining various biological materials described in the embodiments are merely to provide experimental methods for specific disclosure purposes and should not be construed as limiting the sources of biological materials used in this invention. In fact, the sources of biological materials used are wide-ranging, and any biological material that can be obtained without violating laws and ethical standards can be substituted and used according to the suggestions in the embodiments.

[0087] This application relates in its first aspect to a method for detecting genetic material.

[0088] One specific embodiment provides a method for simultaneously or sequentially detecting two or more target genetic materials present in a biological sample, wherein the method includes:

[0089] Add two or more CRISPR-Cas systems capable of targeting two or more of the target genetic material respectively, and probe molecules corresponding to the CRISPR-Cas systems to the biological sample.

[0090] When any of the Crispr-Cas systems encounters its target genetic material, the Cas enzyme in the Crispr-Cas system can be activated by its target genetic material, thereby trans-cleaving its corresponding probe molecule.

[0091] The presence of the target genetic material in the biological sample is determined by detecting the fluorescence signal or colorimetric result of the trans-cleaved probe molecule.

[0092] In the context of this instruction manual, the results obtained by "simultaneous" (referred to as the "one-tube method") and "sequential" (referred to as the "test strip method") detection correspond to "fluorescence signal" (usually measured in an EP tube or cuvette) and "color development result" (usually developed on commercially available test strips), respectively. In the "simultaneous" detection protocol, two or more Cas enzymes, two or more sgRNA / crRNAs, and two or more probes are added to the sample together, and the color development result is observed in an EP tube or cuvette. In the "sequential" detection protocol, two or more Cas enzymes, two or more sgRNA / crRNAs, and probes that can be trans-cleaved by them are added to the sample sequentially (and the reacted sample is then dropped onto test strips), and the color development result corresponding to the two or more genetic materials is read twice on the test strips.

[0093] In the context of this specification, "trans-cleavage" specifically refers to the cleavage of non-target nucleic acids by Cas enzymes induced by cis-cleavage (cleavage of the target nucleic acid).

[0094] In yet another specific embodiment, the above method is provided, wherein,

[0095] The CRISPR-Cas system includes a Cas enzyme or a nucleic acid capable of expressing a Cas enzyme, and a guide molecule capable of specifically binding to the target genetic material.

[0096] Preferably, the two or more Crispr-Cas systems are selected from two or more of the following groups: Crispr-Cas12i system, Crispr-Cas12b system, Crispr-CasX system, and Crispr-Cas12a system;

[0097] The guide molecules are sgRNA and / or crRNA.

[0098] In the context of this specification, when using the CRISPR-Cas12b system, the corresponding guide molecule is sgRNA, and when using the CRISPR-Cas12i system, the corresponding guide molecule is crRNA.

[0099] In yet another embodiment, the above method is provided, wherein,

[0100] The probe molecules are of two or more types, each having a sequence composed of multiple repeating nucleotides, and the phosphodiester bonds of the repeating nucleotide sequences in the probe molecules are modified at different positions; the probe molecules have fluorescence-quenching groups (FQ) linked to their respective repeating nucleotide sequences; preferably, the fluorescence-quenching groups are selected from two or more of the following: FAM-BHQ1, HEX-BHQ1, Rox-BHQ2, Cy3-Cy5, TET-Dabcyl, FAM-7T-Dabcyl, FAM-BHQ2, Cy3-BHQ2, Cy5-BHQ2, JOE-BHQ1.

[0101] In the context of this specification, the principle of a fluorescence quenching group refers to the phenomenon in fluorescence analysis where the fluorescence intensity decreases or disappears after a fluorescent substance interacts with certain substances; this phenomenon is called fluorescence quenching, and the substance that causes fluorescence quenching is called a fluorescence quencher; specifically, when the fluorescence-quenching group (FQ) of this application is trans-cleaved by Cas enzyme, the F- and Q- groups move away from each other, the quenching effect disappears, and the fluorescence emitted by the original probe molecule can be observed.

[0102] In another embodiment, the above method is provided, wherein the probe molecule is unmodified FAM-7T-biotin and / or FAM-7C-biotin. Typically, when a CRISPR-Cas system is selected for sequential color development using a "test strip method," the CRISPR-Cas12i and / or CRISPR-Cas12b system is selected; two or more target genetic materials are distinguished by the specificity of specific sgRNAs and / or crRNAs against the target genetic material. Typically, when selecting probes for sequential color development using a "test strip method," FAM-7T-biotin can be used in both the first and second tests; FAM-7C-biotin can be used in both the first and second tests; FAM-7T-biotin can be used in the first test and FAM-7C-biotin in the second test; or FAM-7C-biotin can be used in the first test and FAM-7T-biotin in the second test.

[0103] In the context of this specification, FAM-7T-Biotin also functions as a probe that, upon trans-cleavage, induces a colorimetric reaction on biotin-avidin test strips, which are commercially available, such as Milenia Genline Dipsticks (Germany, MGDS) from Milenia Biotec.

[0104] In another embodiment, the method described above is provided, wherein the two or more target genetic materials are genetic materials from different biological samples or different genetic materials from the same biological sample.

[0105] One embodiment provides the above method, typically using a CRISPR-Cas system for simultaneous colorimetric development via a single-tube method. The CRISPR-Cas system comprises two types: CRISPR-Cas12i and CRISPR-Cas12b, and the probe molecules comprise two types: a first probe molecule and a second probe molecule. The first and second probe molecules have the structure of an FQ-poly-7T, FQ-poly-7A, or FQ-poly-7C sequence, preferably both being FQ-poly-7T sequences. Specifically, when both the first and second probe molecules are FQ-poly-7T sequences, they can be selected from FQ-S-1-7T, FQ-S-2-7T, FQ-S-3-7T, FQ-S-4-7T, FQ-S-5-7T, and FQ-S-6-7T. For example, FQ-S-1-7T means that in the FQ-poly-7T sequence, only the phosphodiester bond between the first T and the second T is not modified, while FQ-S-6-7T means that in the FQ-poly-7T sequence, only the phosphodiester bond between the sixth T and the seventh T is not modified.

[0106] In one embodiment, the above method is provided, wherein the first probe molecule has only one unmodified phosphodiester bond located between the m-th nucleotide and the (m+1)-th nucleotide, and the second probe molecule has only one unmodified phosphodiester bond located between the n-th nucleotide and the (n+1)-th nucleotide, wherein m is less than n; more specifically, m is an integer selected from 1 to 6, and n is an integer selected from 1 to 6, wherein the modification is a thiomodification, preferably m is 1, 2, or 3, and n is 4, 5, or 6.

[0107] Typically, the first probe molecule is FQ-S-1-7T, while the second probe molecule is FQ-S-6-7T; Cas12i has a very strong cleavage effect on the first probe molecule but a very weak cleavage effect on the second probe molecule; correspondingly, Cas12b has a very strong cleavage effect on the second probe molecule but a very weak cleavage effect on the first probe molecule.

[0108] In another specific embodiment, the above method is provided, wherein the Crispr-Cas12b is an engineered Cas12b that has undergone E475R+Q119F+E758R substitution relative to its wild type; and the Crispr-Cas12i is an engineered Cas12i that has undergone N164Y+E176R+K238R+T447R+E563R+E323R+D362R substitution relative to its wild type; wherein the engineered Cas12i is shown as SEQ ID NO:7, and the engineered Cas12b is shown as SEQ ID NO:13.

[0109] This application provides a reagent kit in a second aspect.

[0110] One embodiment provides a kit for simultaneously or sequentially detecting two or more target genetic materials carried by a biological sample, wherein...

[0111] The kit includes two or more CRISPR-Cas systems that target the target genetic material, and probe molecules that can be trans-cleaved by the CRISPR-Cas system and emit fluorescent signals after the CRISPR-Cas system is activated by the target genetic material.

[0112] In yet another embodiment, the above-described kit is provided, wherein,

[0113] The CRISPR-Cas system includes a Cas enzyme or a nucleic acid capable of expressing a Cas enzyme, and a guide molecule capable of specifically binding to the target genetic material.

[0114] Preferably, the two or more Crispr-Cas systems are selected from two or more of the following groups: Crispr-Cas12i system, Crispr-Cas12b system, Crispr-CasX system, and Crispr-Cas12a system;

[0115] The guide molecules are sgRNA and / or crRNA.

[0116] In yet another embodiment, the above-described kit is provided, wherein,

[0117] The probe molecules are of two or more types, each having a sequence composed of multiple repeating nucleotides, and the phosphodiester bonds of the repeating nucleotide sequences in the probe molecules are modified at different positions; the probe molecules have fluorescence-quenching groups (FQ) linked to their respective repeating nucleotide sequences; preferably, the fluorescence-quenching groups are selected from two or more of the following: FAM-BHQ1, HEX-BHQ1, Rox-BHQ2, Cy3-Cy5, TET-Dabcyl, FAM-7T-Dabcyl, FAM-BHQ2, Cy3-BHQ2, Cy5-BHQ2, JOE-BHQ1.

[0118] In one embodiment, the kit described above is provided, wherein the probe molecule is of only one type and is unmodified FAM-7T-Biotin and / or FAM-7C-Biotin.

[0119] In another embodiment, the above-described kit is provided, wherein the two or more target genetic materials are genetic materials from different biological samples or different genetic materials from the same biological sample.

[0120] In another embodiment, the above-described kit is provided, wherein the Crispr-Cas system is of two types, namely Crispr-Cas12i and Crispr-Cas12b, and the probe molecules are of two types, namely a first probe molecule and a second probe molecule, and the structure of the first probe molecule and the second probe molecule is an FQ-poly-7T, FQ-poly-7A or FQ-poly-7C sequence, preferably the first probe molecule and the second probe are both FQ-poly-7T sequences.

[0121] In one embodiment, the kit described above is provided, wherein the first probe molecule has only one unmodified phosphodiester bond located between the m-th nucleotide and the (m+1)-th nucleotide, and the second probe molecule has only one unmodified phosphodiester bond located between the n-th nucleotide and the (n+1)-th nucleotide, wherein m is less than n.

[0122] In another embodiment, the above-described kit is provided, wherein m is an integer selected from 1 to 6, and n is an integer selected from 1 to 6, wherein the modification is a thiomodification, preferably m is 1, 2, or 3, and n is 4, 5, or 6.

[0123] In another embodiment, the above-described kit is provided, wherein the Crispr-Cas12b is an engineered Cas12b that has undergone E475R+Q119F+E758R substitution relative to its wild type; and the Crispr-Cas12i is an engineered Cas12i that has undergone N164Y+E176R+K238R+T447R+E563R+E323R+D362R substitution relative to its wild type; wherein the engineered Cas12i is shown as SEQ ID NO:7, and the engineered Cas12b is shown as SEQ ID NO:13.

[0124] Example Section

[0125] Experimental Materials and Methods

[0126] Protein expression and purification

[0127] Based on previous reports, Cas12i and Cas12b proteins were purified. In short, BPK2014-Cas12-His10 protein was expressed in *E. coli* strain BL21(λDE3) and induced with 0.5 mM IPTG at 16 °C for 16 h. The cell pellet was harvested and lysed, and purified using His60 Ni Superflow Resin (Thermo Fisher) followed by imidazole gradient elution. The purified Cas12 protein was dialyzed, concentrated, and quantified using a protein BCA quantification kit (Thermo Fisher).

[0128] Nucleic acid preparation and RNA transcription

[0129] DNA oligonucleotides were synthesized from Tianyi Huiyuan, and chemically modified probes were commercially available (GenScript). Double-stranded DNA activators were obtained by PCR amplification and purified using the Oligo Clean & Concentrator Kit (ZYMO Research). crRNA and sgRNA transcription templates were obtained by PCR gel recovery or annealing, followed by in vitro transcription using the HiScribe™ T7 High YieldRNA Synthesis Kit (NEB) and purification using the Oligo Clean & Concentrator Kit (NEB). Qubit quantification was performed.

[0130] Reporting assay of fluoroquinolone quencher (FQ) labeled

[0131] Using a specific concentration of Cas12i or Cas12b, guide RNA, activator, custom-synthesized modified ssDNA / ssRNA FQ reporter gene, and reaction buffer (unless otherwise specified), a 15 μl reaction was performed in a Corning 384-well polystyrene NBS microplate / Axygene 96-well plate for assay. The reaction mixture was incubated at 37 °C to indicate the time on a fluorescence plate reader (BioTek Synergy 4), with fluorescence kinetics measured every 5 minutes (λex = 485 nm; λem = 528 nm, transmission gain = 61). Alternatively, incubation was performed for 30–90 minutes at a specific temperature in an Applied Biosystems real-time PCR system (Thermo Fisher), with fluorescence kinetics measured every minute. Fluorescence / ΔRn results were analyzed using GraphPad software.

[0132] Recombinase polymerase amplification (RPA) reaction

[0133] According to the manufacturer's instructions, recombinase polymerase amplification (RPA) was performed using TwistAmp Basic (TwistDx). 50 μl RPA reaction systems containing different amounts of DNA input were incubated at 39°C for 20 minutes. As described above, 5 μl of the RPA product was directly transferred to a 55 μl reporter system assay to detect the fluorophore quencher (FQ)-labeled product.

[0134] Reverse transcriptase-loop-mediated isothermal amplification (RT-LAMP) experiment

[0135] Following the manufacturer's instructions, use Bst2.0 DNA polymerase, Bst3.0 DNA polymerase, and hot-start RTx reverse transcriptase, and mix the corresponding amplification primers. Incubate 50 μl LAMP reaction systems containing different amounts of DNA input at 65°C for 40–60 minutes. As described above, directly transfer 5 μl of LAMP product to 55 μl of a reporter system assay to detect fluorophore quencher (FQ)-labeled samples.

[0136] Cell culture

[0137] SiHa cell lines (containing the HPV16 fragment to be tested) were cultured at 37°C in 12-well plates using commercially available complete culture medium (Procell). HeLa cell lines (containing the HPV18 fragment to be tested) were cultured at 37°C in 12-well plates using self-prepared complete culture medium (DMEM + 10% FBS + 2% penicillin antibody). Farage cell lines (excluding HPV16 and HPV18 fragments to be tested) were cultured at 37°C in 12-well plates using commercially available complete culture medium (Procell). After each well of the three cell lines reached confluence, nucleic acids were extracted by genomic DNA extraction or by lysing the genome using lysis buffer, followed by qPCR quantification. Samples containing nucleic acids were then used for subsequent detection experiments.

[0138] qPCR analysis of sample CT values

[0139] The nucleic acids were quantified using commercially available qPCR reagents (Novizan), following the procedures and dosages recommended on the official website. The CT values ​​of the samples were measured using an ABI Q6 real-time PCR instrument.

[0140] Multi-detection platform system

[0141] The detection template was first amplified using RT-LAMP / LAMP, and then 2 μL was added to an 18 μL detection system. The detection system contained specific concentrations of Cas12i and Cas12b guide RNAs, activators, a custom-synthesized modified ssDNA FQ reporter gene, and a reaction buffer (unless otherwise specified). 20 μL of the reaction was performed in an Axygene 96-well plate for detection. The reaction mixture was incubated at 47 °C for 30–90 minutes using an Applied Biosystems real-time PCR system (Thermo Fisher), with fluorescence kinetics of FAM and HEX measured every minute. The fluorescence / ΔRn results were analyzed using GraphPad software.

[0142] Test strips and visual fluorescence analysis

[0143] The amplified product is incubated in a detection system containing Cas12i or Cas12b variant protein, crRNA / sgRNA, activator, custom-synthesized modified homopolymer ssDNA / ssRNA, FB reporter gene, and reaction buffer (unless otherwise specified). The product is diluted with diluent according to the instructions and then inserted into a test strip (Milenia HybriDetect Dipstick) to interpret the results. The acquired images are converted to 8-bit grayscale using Photoshop and imported into ImageLab software (BioRad ImageLab software). The image is inverted. In the same image, the band widths and background adjustments remain unchanged between bands. The calculation method is to divide the intensity of the top (test) band by the intensity of the bottom (control) band.

[0144] Fluorescence analysis involves trans-cleavage of Cas12i or Cas12b variant proteins, crRNA / sgRNA, activators, custom-synthesized modified homopolymers ssDNA / ssRNA, and FB reporter genes, followed by UV irradiation. Fluorescence and visible light conditions are then captured directly using a mobile phone or instrument.

[0145] Example 1. Trans-cleavage activity analysis of Cas12 family variants

[0146] To establish a Cas12 multiplex detection-based trans-cleavage activity assay, we first selected Cas12i and Cas12b variants (SED ID NO. 1-13) from our previously published work.

[0147] Protein expression and purification ( Figure 1 Following this, the trans-cleavage activity was analyzed and compared. In Example 1, we used a 100 bp DNA fragment purified from the PCR product as the activator, and measured the trans-cleavage activity under 200 nM Cas12i crRNA or 20 nM Cas12b sgRNA conditions. We also analyzed the activity of engineered PlmCasX. We found that the Cas12i variant N164Y+E176R+K238R+T447R+E563R+E323R+D362R (SED ID NO. 7) had the best trans-cleavage activity at 37 °C, while the Cas12b variant E475R+Q119F+E758R (SED ID NO. 13) had good trans-cleavage activity, and the engineered PlmCasX variant T26R+K610R+K808R (SED ID NO. 14-15) had good trans-cleavage activity (Figure 2).

[0148] Example 2. Probe sequence cleavage preference analysis of Cas12 family variants

[0149] To investigate the trans-cleavage activity of the Cas12 family for probe sequences, we first used the highly active Cas12i and Cas12b to analyze the preference for FQ-poly-7T, FQ-poly-7A, FQ-poly-7C, and FQ-poly-7G (SED IDNO. 28-31). We found that Cas12b and Cas12i exhibited better kinetics for cleavage of FQ-poly-7T and FQ-poly-7C, while the kinetic curve for poly-7A was more gradual, and there was almost no cleavage for FQ-poly-7G. Figure 3 When the positions of the original 5' FAM group and the 3' BHQ1 quencher group are reversed, i.e., the 5' end is modified with a BHQ1 group and the 3' end with a FAM fluorescent group, the cleavage signal of the probe shows an opposite cleavage trend (see...). Figure 4 ).

[0150] Example 3. Prediction analysis of Cas12 family modified phosphodiester bond probes

[0151] The applicant then considered further: since different Cas proteins may exhibit directional cleavage of probes modified with different groups, it was necessary to verify whether modifying the phosphodiester bonds inside the probe to prevent cleavage by Cas proteins while keeping the group positions unchanged would result in differences in cleavage between unmodified phosphodiester bonds at different positions. Therefore, experiments were conducted using FQ-poly-7T as a prototype. Since there are six phosphodiester bonds between the seven thymine groups, one of the six phosphodiester bonds was left unmodified while the other five were thiolated. The negative control involved modifying all six phosphodiester bonds. Therefore, we used phosphodiester bond-modified probes FQ-S-1-7T, FQ-S-2-7T, FQ-S-3-7T, FQ-S-4-7T, FQ-S-5-7T, FQ-S-6-7T, and FQ-S-7-7T (i.e., all phosphodiester bonds were phosphorylated, serving as a negative control) (SED ID NO. 28-34) as probes to analyze the probe cleavage direction preference of Cas12i and Cas12b. The results showed that Cas12i exhibited a gradual decrease in phosphodiester bond cleavage activity from the 5' to the 3' end; Cas12b exhibited a gradual increase in phosphodiester bond cleavage activity from the 5' to the 3' end. Figure 5 To broaden our understanding, we replaced the 5' FAM group with HEX (SED ID NO. 35-41), and we observed a similar trend. Figure 6We also replaced the sequence with a FAM-BHQ1 modified polyC probe (SED ID NO. 42-48), and we found a similar trend, indicating that this trend may be applicable as a probe for multiple detection methods. Figure 7 In addition, we also tried some proteins in the Cas12a family (SED ID NO. 16-22), but no new cleavage preferences were found. Figure 8 ).

[0152] Example 4. Multiplex detection of HPV16 and HPV18

[0153] To enable the application of multiplex detection of Cas12 family proteins in the laboratory, we selected two human papillomaviruses, HPV16 and HPV18, for typing. First, based on the E6 and E7 sequences of HPV16 and HPV18, we designed and screened gRNAs that were highly specific to each other. Figure 9 Then, we used the purified PCR product as an activator (to amplify the virus) to verify that the individual systems had good specificity under the action of 10 nM, 1 nM, 0.1 nM, and 0 nM activators, and that mixing them into one tube (a point of invention, two colors, which do not affect each other) is feasible. Figure 10 Therefore, we used the Cas12i probe for FAM fluorescence and the Cas12b probe for HEX fluorescence, thus mixing the two protein systems in one tube to achieve dual detection. We cultured three cell lines: SiHa (containing HPV16), HeLa (containing HPV18), and Farage. After extracting the genome and quantifying it using qPCR, we performed multiplex detection in one tube. We found that this system could detect 10 copies / µL of genome and perform genotyping. Figure 11 )

[0154] Example 5. Dual detection of SARS-CoV-2

[0155] For the SARS-CoV-2 virus, different subtypes exhibit varying virulence, leading to differing future response strategies; therefore, subtyping becomes increasingly important. We screened detection sites in the S gene of the novel coronavirus genome (…). Figure 12 Subsequently, we screened isothermal amplification primers for RT-LAMP based on these highly efficient novel coronavirus corresponding sites (SED ID NO. 23-27). We performed in vitro transcription and purification of RNA, followed by quantification using qPCR. Finally, we serially diluted the RNA template and found that a single tube of multiplex nucleic acid detection achieved good sensitivity. Figure 13 (This can be referred to as the "one-pipe method").

[0156] Example 6. Establishment of a Visualized Dual Detection System

[0157] We established a visualized dual detection platform using the wild-type genome of the novel coronavirus and the genome of XBB1.5 as examples. Lateral flow chromatography is an important carrier method for test strips. By designing the test strips based on the two genomes, we selected the corresponding sgRNAs screened in Example 5 for detection, and used probes (SED ID NO. 49-50) of FAM-TTTTTTT-Biotin and / or FAM-CCCCCCC-Biotin for indication. The results are shown in Figure (…). Figure 14 The two bands are arranged as follows: the upper band is the T band (test band), which indicates a positive result; the lower band is the C band (control band), which indicates a valid result. Therefore... Figure 14 The pilot test strips showed perfect genotyping detection, and sensitivity tests demonstrated good sensitivity. Figure 15 (This can be referred to as the "test strip method"). Another visualization method is fluorescence or direct colorimetric observation. The distance between modifications of FAM-BHQ1 itself affects spectral changes; therefore, we increased the probe concentration to a final concentration of 25µM for visual genotyping detection. The results are shown in the figure. Figure 16 As shown in the four images of the visible light spectrum, positive results appear yellow (top left and bottom right images of the four small visible light images), while negative results appear red (bottom left and top right images of the four small visible light images). Under UV irradiation, positive results showed luminescence (top left and bottom right images of the four small UV images), while negative results did not (bottom left and top right images of the four small UV images). All results showed good typing and high sensitivity. Figure 17 ).

[0158] Although embodiments of the present invention have been described above in conjunction with the accompanying drawings, the present invention is not limited to the specific embodiments and application fields described above. The specific embodiments described above are merely illustrative and instructive, and not restrictive. Those skilled in the art can make many other forms based on the guidance of this specification and without departing from the scope of protection of the claims of the present invention, and all of these are within the scope of protection of the present invention.

[0159] Table 1. Sequence listing of the amino acids involved in this invention.

[0160]

[0161]

[0162]

[0163]

[0164]

[0165] Table 2. Nucleic acid DNA sequences to be protected:

[0166]

[0167] Table 3. Modification probes to be protected (where * indicates thio-modified phosphodiester bonds):

[0168]

Claims

1. A method for non-disease diagnosis that simultaneously detects two or more target genetic materials present in a biological sample, wherein, The method includes: Add two or more CRISPR-Cas systems capable of targeting two or more of the target genetic material respectively, and probe molecules corresponding to the CRISPR-Cas systems to the biological sample. When any of the Crispr-Cas systems encounters its target genetic material, the Cas enzyme in the Crispr-Cas system can be activated by its target genetic material, thereby trans-cleaving its corresponding probe molecule. The presence of the target genetic material in the biological sample is determined by detecting the fluorescence signal or colorimetric result of the trans-cleaved probe molecule. The Crispr-Cas system is of two types: a first system and a second system; the first system is a Crispr-Cas12i system, and the second system is a Crispr-Cas12a system or a Crispr-Cas12b system. The probe molecules are of two types, namely a first probe molecule and a second probe molecule, and the structures of the first probe molecule and the second probe molecule are respectively selected from the FQ-poly-7T or FQ-poly-7C sequence. The first probe molecule has only one unmodified phosphodiester bond, which is located between the m-th nucleotide and the (m+1)-th nucleotide, and the second probe molecule has only one unmodified phosphodiester bond, which is located between the n-th nucleotide and the (n+1)-th nucleotide, where m is less than n; Where m is 1, 2, or 3, and n is 4, 5, or 6; The modification mentioned therein is a thiomodification; After modification, the first system showed a gradual decrease in phosphodiester bond cleavage activity of the probe from the 5' to the 3' end; the second system showed a gradual increase in phosphodiester bond cleavage activity of the probe from the 5' to the 3' end. The probe molecule has a fluorescence-quenching group linked to its respective repeating nucleotide sequence; the fluorescence-quenching group is selected from two or more of the following: FAM-BHQ1, HEX-BHQ1, Rox-BHQ2, TET-Dabcyl, FAM-Dabcyl, FAM-BHQ2, Cy3-BHQ2, Cy5-BHQ2, JOE-BHQ1.

2. The method for non-disease diagnosis according to claim 1, wherein, The CRISPR-Cas system includes a Cas enzyme or a nucleic acid capable of expressing a Cas enzyme, and a guide molecule capable of specifically binding to the target genetic material. The guide molecules are sgRNA and / or crRNA.

3. The method for non-disease diagnosis according to claim 1, wherein, The two or more target genetic materials are genetic materials from different biological samples or different genetic materials from the same biological sample.

4. The method for non-disease diagnosis according to claim 1, wherein, The first probe molecule and the second probe are both FQ-poly-7C or FQ-poly-7T sequences.

5. The method for non-disease diagnosis according to claim 4, wherein, The Crispr-Cas12b is an engineered Cas12b that has undergone E475R+Q119F+E758R substitution relative to its wild type; while the Crispr-Cas12i is an engineered Cas12i that has undergone N164Y+E176R+K238R+T447R+E563R+E323R+D362R substitution relative to its wild type; wherein, the engineered Cas12i is shown as SEQ ID NO:7, and the engineered Cas12b is shown as SEQ ID NO:

13.

6. A kit for simultaneously detecting two or more target genetic materials carried by a biological sample, wherein, The kit includes two or more CRISPR-Cas systems that target the target genetic material, and probe molecules that can be trans-cleaved by the CRISPR-Cas system and emit fluorescent signals after the CRISPR-Cas system is activated by the target genetic material. The Crispr-Cas system is of two types: a first system and a second system; the first system is a Crispr-Cas12i system, and the second system is a Crispr-Cas12a system or a Crispr-Cas12b system. The probe molecules are of two types, namely a first probe molecule and a second probe molecule, and the structures of the first probe molecule and the second probe molecule are respectively selected from the FQ-poly-7T or FQ-poly-7C sequence. The first probe molecule has only one unmodified phosphodiester bond, which is located between the m-th nucleotide and the (m+1)-th nucleotide, and the second probe molecule has only one unmodified phosphodiester bond, which is located between the n-th nucleotide and the (n+1)-th nucleotide, where m is less than n; Where m is 1, 2, or 3, and n is 4, 5, or 6; The modification mentioned therein is a thiomodification; After modification, the first system showed a gradual decrease in phosphodiester bond cleavage activity of the probe from the 5' to the 3' end; the second system showed a gradual increase in phosphodiester bond cleavage activity of the probe from the 5' to the 3' end. The probe molecule has a fluorescence-quenching group linked to its respective repeating nucleotide sequence; the fluorescence-quenching group is selected from two or more of the following: FAM-BHQ1, HEX-BHQ1, Rox-BHQ2, TET-Dabcyl, FAM-Dabcyl, FAM-BHQ2, Cy3-BHQ2, Cy5-BHQ2, JOE-BHQ1.

7. The kit according to claim 6, wherein, The CRISPR-Cas system includes a Cas enzyme or a nucleic acid capable of expressing a Cas enzyme, and a guide molecule capable of specifically binding to the target genetic material. The guide molecules are sgRNA and / or crRNA.

8. The kit according to claim 6, wherein, The two or more target genetic materials are genetic materials from different biological samples or different genetic materials from the same biological sample.

9. The kit according to claim 6, wherein, The first probe molecule and the second probe are both FQ-poly-7C or FQ-poly-7T sequences.

10. The kit according to claim 9, wherein, The Crispr-Cas12b is an engineered Cas12b that has undergone E475R+Q119F+E758R substitution relative to its wild type; while the Crispr-Cas12i is an engineered Cas12i that has undergone N164Y+E176R+K238R+T447R+E563R+E323R+D362R substitution relative to its wild type; wherein, the engineered Cas12i is shown as SEQ ID NO:7, and the engineered Cas12b is shown as SEQ ID NO:13.

Images