Composition and method for specifically killing target cells according to intracellular nucleic acid specificity

Abstract

The invention relates to the technical field of biology, in particular to a composition aiming at specifically killing target cells, which comprises a substance capable of identifying specific RNA (Ribonucleic Acid) or DNA (Deoxyribose Nucleic Acid) in the target cells, and nuclease capable of causing a non-specific DNA fracture'side cutting effect 'and activated after the target RNA or DNA is identified. According to the invention, a certain target cell can be ingeniously and specifically killed or inhibited by utilizing the side cutting effect, namely the side cutting effect of nuclease is activated by utilizing RNA (Ribonucleic Acid) or DNA (Deoxyribose Nucleic Acid) of the target cell, a large number of DNA fractures are caused in the target cell, and the purposes of inhibiting the growth of the target cell and even killing the target cell are achieved.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a composition and an application method for achieving specific killing of target cells by non-specifically cutting DNA through the side-cutting effect of gene editing technology. Background Art

[0002] Being able to specifically kill cells with certain nucleic acid characteristics will be of great value. For example, in plants, after specifically killing cells infected by pathogens, it is expected to improve the survival status of plants, which has certain application value in agriculture and medicine; in microbial communities such as bacteria or fungi, specifically killing bacteria or fungi infected by viruses is expected to improve the overall state of the flora, which also has certain application prospects in the field of fermentation engineering, or in the field of health issues related to the maintenance of normal flora in animals and humans; in animals, specifically killing cells infected by pathogens, or specifically killing target cells is expected to improve the health level of animals; in humans, specifically killing cells infected by pathogens, or specifically killing a certain type of cells or specifically killing target cells is expected to improve the health level of humans. However, there is currently no method to specifically kill a certain cell. Not only is there no method to specifically kill a certain cell in the field of plant or animal research, there is also no good way to do it in human diseases, for example, chemotherapy drugs can kill both target cells and normal cells. Radiotherapy relies on rays to interrupt DNA and cause cell death, but rays can interrupt DNA in both target cells and normal cells, so they cannot specifically kill target cells.

[0003] Is it possible to cause a large number of nucleic acid breaks only in target cells, resulting in specific killing of target cells? This is a question creatively raised by the inventors.

[0004] Gene editing technology allows people to cut, modify, and edit precise sites of DNA or RNA in cells. Existing gene editing technologies include: zinc finger nuclease (ZFN), transcription activator-like effector (TALEN), clustered regularly interspaced short palindromic repeats (CRISPR), Argonaute (Ago) protein-dependent gene editing systems, etc. Gene editing technology has broad application prospects.

[0005] Gene editing technology is like a pair of precise "scissors" that can accurately cut DNA or RNA at specific sites. If DNA is cut, double-strand breaks (DSBs) can be generated. In higher eukaryotes, DSBs are repaired through nonhomologous end joining (NHEJ) and homologous recombination (HR) repair pathways. If the CRISPR-Cas9 gene editing technology is used to precisely cut only the DNA specific to the target cell while inhibiting the above-mentioned DNA repair pathway, can the target cell be specifically killed? This is a concept of specific killing of target cells proposed by the inventor. And experiments have confirmed that this concept can indeed specifically kill cancer cells. Thus, a meaningful invention was formed: a method for specifically killing cancer cells through gene editing technology combined with DNA damage repair inhibitors (202210619622.7). The relevant results were published in Molecular Cancer in August 2022 (IF: 37.3, see literature: Jiang J, Chen Y, Zhang L, et al. i-CRISPR: a personalized cancer therapy strategy through cutting cancer-specific mutations. Mol Cancer. 2022; 21(1): 164. Published 2022Aug 16. doi: 10.1186 / s12943-022-01612-x).

[0006] Specifically, the above invention proposes a method for specifically killing cancer cells, which uses gene editing technology to cause DNA breaks that are unique to cancer cells (normal cells do not have these DNA cut points), and at the same time combines a DNA damage repair inhibitor (i) to inhibit DNA repair in cancer cells, leading to specific cancer cell death.

[0007] This invention has certain application prospects in the treatment of cancer, but if it is extended to other fields, such as specific killing of plant cells, animal cells, and microbial cells, the actual application value is often significantly reduced, because to achieve the killing efficiency at the application level, too many "scissors" need to be introduced into the cells, the cost is too high, and the delivery uniformity, efficiency and other indicators are difficult to control. In the above invention (202210619622.7), to produce a broken nucleic acid, a gRNA (or crRNA) is required to identify the target nucleic acid. However, our experiments found that the cell killing effect produced by a small amount of nucleic acid breakage is limited, resulting in more DNA breakpoints in the cell, and the higher the efficiency of cell killing (the experimental results in our article published in Molecular Cancer also illustrate this point). Since the previous invention only cuts the identified target, each "gene editing scissors" can only produce one cutting point (without considering copy number variation), so we need to introduce multiple "gene editing scissors" in our experiment. Even if it is applied to cancer treatment, the convenience and cost of the above invention (202210619622.7) in future practical applications will be significantly increased, and the number of mutations that can be used to distinguish cancer cells from normal cells in each patient is also relatively high (too few mutations that can be cut can only cause too few DNA breaks, and enough breaks require enough mutations). Therefore, for individual patients, high-throughput sequencing is often required first, and then many sites are selected to design and prepare many "custom scissors". In this way, it is impossible to prepare in advance some mutations that are highly present in a wide range of cancer patients on a large scale, and the application cost is relatively high.

[0008] If a small number of "gene editing scissors" can produce many DNA breakpoints, it is expected to significantly improve the efficiency of specific killing of target cells. This is not only of great significance for achieving specific killing of plant cells, animal cells, microbial cells and other broader fields, but also has important value for the application of specific killing of target cells or infected cells in the field of human health.

[0009] Therefore, the inventor creatively proposed another concept of specifically killing target cells: using the "paratriptic effect" of gene editing technology to create a large number of DNA breakpoints in the target cells.

[0010] Gene editing technology can accurately identify and cut target DNA or RNA. However, not all gene editing technologies only cut the identified target nucleic acid. Many gene editing tools also have a "by-cutting effect", that is, once the nuclease is activated, in addition to specifically cutting the target nucleic acid, it can also non-specifically cut other surrounding nucleic acid substances, which is also called the "trans-cutting" effect.

[0011] For example, Cas12a2, a CRISPR nuclease that was only reported by Science in 2023, can be activated by target RNA in bacteria. Once activated, in addition to having a cutting effect on the target RNA, it also has cutting activity for RNA that is not a target (no complementary sequence to it), double-stranded DNA, and single-stranded DNA. That is, once Cas12a2 and the corresponding crRNA encounter a target RNA that can be recognized by crRNA, the DNA and RNA around Cas12a2 will be "indiscriminately cut." It causes a large amount of broken DNA and RNA. For bacteria, this non-specific cutting of DNA and RNA will lead to abortive infection of bacteria, which means that a single virus-infected bacterium enters dormancy or death, thereby preventing the virus from spreading throughout the bacterial population (see literature: Dmytrenko O, Neumann GC, Hallmark T, et al. Cas12a2 elicits abortive infection through RNA-triggered destruction of dsDNA. Nature. 2023; 613(7944): 588-594. doi: 10.1038 / s41586-022-05559-3).

[0012] There are many similar examples. For example, after Cas12 is activated by the target DNA, its "bypass effect" can be activated, indiscriminately cutting the surrounding single-stranded DNA (see literature: Rananaware SR, Vesco EK, Shoemaker GM, et al. Programmable RNA detection with CRISPR-Cas12a. Nat Commun. 2023; 14(1): 5409. Published 2023Sep 5. doi: 10.1038 / s41467-023-41006-1). After being activated by target DNA or RNA, certain Argonaute (Ago) proteins can also indiscriminately cleave surrounding DNA (see literature: Prostova M, Kanevskaya A, Panteleev V, et al. DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity. Nat Microbiol. 2024; 9(5): 1368-1381. doi: 10.1038 / s41564-024-01654-5; Lu X, Xiao J, Wang L, Zhu B, Huang F. The nuclease-associated short prokaryotic Argonaute system nonspecifically degrades DNA upon activation by target recognition. Nucleic Acids Res. 2024; 52(2): 844-855. doi: 10.1093 / nar / gkad1145). It is believed that with the further development of the natural CRISPR system by humans, as well as the optimization and improvement of the existing CRISPR system such as mutation screening, or the transformation of technologies such as zinc finger nucleases (ZFNs), transcription activator-like effectors (TALENs), and Argonaute (Ago) protein-based gene editing systems, more gene editing technology systems with specific "bypass effects" may appear in the future.

[0013] This "paranoia effect" of the gene editing system has been used to detect target nucleic acids, and our invention uses the "paranoia effect" of gene editing elements to cause more DNA breaks, specifically killing target cells over a wide range, which has certain innovation and application prospects. Summary of the invention

[0014] The purpose of the present invention is to provide a composition and method for activating the "bypass effect" of gene editing technology based on the specificity of intracellular nucleic acids to achieve specific killing of target cells, and to provide a reference for better treatment of cancer.

[0015] The inventors imagined that if a large number of DNA breaks could be caused only in the target cells, it would be possible to achieve specific killing or inhibition of the target cells. Therefore, taking advantage of the fact that many gene editing tools have a "bystander effect" and non-specifically cut surrounding DNA, it was proposed to introduce a gene editing system with the above characteristics into the target cells that can recognize RNA or DNA that is only present in the target cells. When the system encounters the target RNA or DNA, its "bystander effect" is activated, which can cause a large amount of DNA in the target cells, achieving the purpose of inhibiting the growth of the target cells or even killing the target cells.

[0016] The RNA or DNA that is only present in the target cell can be the nucleic acid material of the pathogen in the target cell after the target cell is infected by the pathogen, or it can be the DNA sequence site or RNA sequence site that is unique to the target cell caused by the mutation contained in the target cell itself, or it can be the RNA of the gene that is specifically highly expressed in the target cell, or the nucleic acid material with a high copy number that is only present in the target cell. In short, as long as it can provide target cell specificity, that is, the quantity or quality of this nucleic acid material in the target cell can be used to distinguish the target cell from other cells. Therefore, even if normal cells are introduced into this gene editing system, since there is no corresponding target DNA or RNA in normal cells, the "bypass effect" of indiscriminate cutting of the corresponding gene editing system will not be activated, and DNA will not be broken, so the cells can survive. In short, the introduction of a gene editing system that meets the above conditions is expected to achieve specific killing of target cells.

[0017] Therefore, the composition for specifically killing target cells involved in the present invention is a substance (such as crRNA) that can recognize RNA or DNA specific to target cells, and after recognizing the target RNA or DNA, activates a nuclease with a "bypass effect" that causes non-specific DNA breakage (such as Cas12a2, Cas12a, Cas12g, etc., or a nuclease with the above characteristics after modification). The method involved is a method of using the above-mentioned substances to specifically kill target cells.

[0018] Based on the above principles, the first aspect of the present invention provides a composition having the above characteristics, which may include the following specific types:

[0019] 1. A composition for the purpose of specifically killing target cells, the composition comprising: two components A and B, or a complex of A and B combined together.

[0020] A is a nuclease that can be activated by a specific sequence nucleic acid through B to have a "bystander effect" on DNA. The "bystander effect" on DNA is an effect of non-specific cutting of the DNA that the enzyme contacts;

[0021] B is a substance that can recognize nucleic acids of specific sequences in target cells but not nucleic acids in cells surrounding or nearby the target cells, and the substance can activate the non-specific "para-cleavage effect" of A on DNA after recognizing nucleic acids of specific sequences in the above target cells.

[0022] In a preferred embodiment,

[0023] A is a gene editing protein;

[0024] The B is a crRNA or gRNA used in conjunction with a gene editing protein in gene editing technology, and the crRNA or gRNA can bind to a specific sequence nucleic acid in the target cell and activate the non-specific "by-product effect" of A on DNA.

[0025] The gene editing proteins include wild-type, mutant, or gene editing protein homologs in other microorganisms.

[0026] The mutant gene editing protein includes a gene editing protein that is mutated from an existing gene editing protein that does not have a side-cutting effect, so that it has a non-specific "side-cutting effect" on DNA.

[0027] The gene editing protein homologs in other microorganisms include gene editing proteins that are homologous to known gene editing proteins and are discovered in microorganisms in the future and have non-specific "bypass effects" on DNA.

[0028] In another preferred embodiment, the gene editing protein is selected from the following group: Cas12a2, AbCas12a2, ca2Cas12a2, ca6Cas12a2, ca7Cas12a2, ca11Cas12a2, ca14Cas12a2, ca17Cas12a2, ca33Cas12a2, ca34Cas12a2, ca35Cas12a2, ca36Cas12a2, ca37Cas12a2, ca38Cas12a2, ca41Cas12a2, ca125Cas12a2, SuCas12a2, Cas12a, Cas12b, LbCas12a, AsCas12a, FnCas12a, AapCas12b, Cas13a, Cas13d, Cas9, ZFN, TALEN, NbaAgo-DREN, TmuRE-Ago, eAgo, pAgo or a combination of the above gene editing proteins.

[0029] The B is a crRNA or gRNA used in conjunction with a gene editing protein in gene editing technology, and the crRNA or gRNA can bind to a specific sequence nucleic acid in the target cell and activate the "bypass effect" of A.

[0030] 2. The "bystander effect" is activated by target cell-specific nucleic acid. After activation, it cuts DNA non-specifically and combines with substance "i" that can inhibit DNA damage repair, so that the damaged DNA cannot be repaired, thereby achieving a composition that specifically kills target cells, and its application in killing target cells.

[0031] The composition comprises three components: A, B and i, or "a complex of A and B combined together" + "i".

[0032] A is a nuclease that can be activated by RNA or DNA with a specific sequence through B. The "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts.

[0033] B is a substance that can recognize specific sequence RNA or DNA in the target cell but not recognize nucleic acids in cells surrounding or nearby the target cell, and this substance can activate the non-specific "bystander effect" of A on DNA after recognizing the specific sequence RNA in the above target cell.

[0034] "i" is a substance that can inhibit DNA damage repair, so that the damaged DNA cannot be repaired. "i" can be a compound, or siRNA, or an antibody, and is characterized by being able to inhibit the repair of DNA damage.

[0035] In a preferred embodiment,

[0036] The A is a gene editing protein.

[0037] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology (referred to as gRNA or gDNA in some technical contexts, and its characteristic is that it can recognize and bind to specific sequence nucleic acids in the target cells through base complementarity, and activate the "bystander effect" of the corresponding nuclease after recognition and binding). The crRNA can bind to the specific sequence RNA in the target cell and activate the non-specific "bystander effect" of A on DNA.

[0038] The "i" is an inhibitor of the DNA damage repair pathway.

[0039] In another preferred embodiment, the gene editing protein is selected from the following group: Cas12a2, AbCas12a2, ca2Cas12a2, ca6Cas12a2, ca7Cas12a2, ca11Cas12a2, ca14Cas12a2, ca17Cas12a2, ca33Cas12a2, ca34Cas12a2, ca35Cas12a2, ca36Cas12a2, ca37Cas12a2, ca38Cas12a2, ca41Cas12a2, ca125Cas12a2, SuCas12a2, Cas12g, Cas9, ZFN, TALEN, NbaAgo-DREN, TmuRE-Ago, eAGO, pAGO or a combination of the above gene editing proteins.

[0040] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology. The crRNA can bind to the specific sequence nucleic acid in the target cell and activate the non-specific "by-product effect" of A on DNA.

[0041] The i is a drug that inhibits DNA damage repair, including inhibitors that are currently known or will be developed to inhibit DNA damage repair, for example, inhibitors of the non-homologous endjoining (NHEJ) pathway, such as the DNA-PKcs inhibitor NU7441; or inhibitors of the homologous recombination (HR) pathway, such as the ATM inhibitor KU55933; or inhibitors that can inhibit both NHEJ and HR, such as wortmannin; including both combined use and single use.

[0042] 3. A composition in which the "bypass effect" is activated by target cell-specific RNA and achieves specific killing of target cells by non-specific cutting of DNA after activation, and its application in killing target cells.

[0043] The composition comprises two components, A and B, or a complex in which A and B are combined together.

[0044] A is a nuclease that can be activated by RNA of a specific sequence through B. The "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts.

[0045] B is a substance that can recognize specific sequence RNA in the target cell but not recognize nucleic acid in cells surrounding or nearby the target cell, and the substance can activate the non-specific "para-cutting effect" of A on DNA after recognizing the specific sequence RNA in the above target cell.

[0046] In a preferred embodiment,

[0047] The A is a gene editing protein.

[0048] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology. The crRNA can bind to the specific sequence RNA in the target cell and activate the non-specific "bypass effect" of A on DNA.

[0049] In another preferred embodiment, the gene editing protein is selected from the following group: Cas12a2, AbCas12a2, ca2Cas12a2, ca6Cas12a2, ca7Cas12a2, ca11Cas12a2, ca14Cas12a2, ca17Cas12a2, ca33Cas12a2, ca34Cas12a2, ca35Cas12a2, ca36Cas12a2, ca37Cas12a2, ca38Cas12a2, ca41Cas12a2, ca125Cas12a2, SuCas12a2, Cas12g or a combination thereof.

[0050] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology. The crRNA can bind to the specific sequence nucleic acid in the target cell and activate the non-specific "by-product effect" of A on DNA.

[0051] 4. The "bystander effect" is activated by target cell-specific RNA. After activation, it cuts DNA non-specifically and combines with substance "i" that can inhibit DNA damage repair, so that the damaged DNA cannot be repaired, thereby achieving a composition that specifically kills target cells, and its application in killing target cells.

[0052] The composition comprises three components: A, B and i, or "a complex of A and B combined together" + "i".

[0053] A is a nuclease that can be activated by RNA of a specific sequence through B. The "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts.

[0054] B is a substance that can recognize specific sequence RNA in the target cell but not recognize nucleic acid in cells surrounding or nearby the target cell, and the substance can activate the non-specific "para-cutting effect" of A on DNA after recognizing the specific sequence RNA in the above target cell.

[0055] "i" is a substance that can inhibit DNA damage repair, so that the damaged DNA cannot be repaired. "i" can be a compound, or siRNA, or an antibody, and is characterized by being able to inhibit the repair of DNA damage.

[0056] In a preferred embodiment,

[0057] The A is a gene editing protein.

[0058] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology. The crRNA can bind to the specific sequence RNA in the target cell and activate the non-specific "bypass effect" of A on DNA.

[0059] The "i" is an inhibitor of the DNA damage repair pathway.

[0060] In another preferred embodiment, the gene editing protein is selected from the following group: Cas12a2, AbCas12a2, ca2Cas12a2, ca6Cas12a2, ca7Cas12a2, ca11Cas12a2, ca14Cas12a2, ca17Cas12a2, ca33Cas12a2, ca34Cas12a2, ca35Cas12a2, ca36Cas12a2, ca37Cas12a2, ca38Cas12a2, ca41Cas12a2, ca125Cas12a2, SuCas12a2, Cas12g, etc., or a combination of the above gene editing enzymes.

[0061] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology. The crRNA can bind to the specific sequence nucleic acid in the target cell and activate the non-specific "by-product effect" of A on DNA.

[0062] The i is a drug that inhibits DNA damage repair, including inhibitors that are currently known or will be developed to inhibit DNA damage repair, for example, inhibitors of the non-homologous endjoining (NHEJ) pathway, such as the DNA-PKcs inhibitor NU7441; or inhibitors of the homologous recombination (HR) pathway, such as the ATM inhibitor KU55933; or inhibitors that can inhibit both NHEJ and HR, such as wortmannin; including both combined use and single use.

[0063] 5. A composition in which the "bystander effect" is activated by target cell-specific DNA and achieves specific killing of target cells by non-specific cutting of DNA after activation, and its application in killing target cells.

[0064] The composition comprises two components, A and B, or a complex in which A and B are combined together.

[0065] A is a nuclease that can be activated by DNA with a specific sequence through B. The "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts.

[0066] B is a substance that can recognize specific sequence DNA in target cells but not nucleic acids in cells surrounding or nearby the target cells, and the substance can activate the non-specific "parasitic effect" of A on DNA after recognizing the specific sequence DNA in the above target cells.

[0067] In a preferred embodiment,

[0068] The A is a gene editing protein.

[0069] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology. The crRNA can bind to the specific sequence DNA in the target cell and activate the non-specific "bypass effect" of A on DNA.

[0070] In another preferred embodiment, the gene editing protein is selected from the following group: NbaAgo-DREN, TmuRE-Ago, eAGO, pAGO, Cas12a2, AbCas12a2, ca2Cas12a2, ca6Cas12a2, ca7Cas12a2, ca11Cas12a2, ca14Cas12a2, ca17Cas12a2, ca33Cas12a2, ca34Cas12a2, ca35Cas12a2, ca36Cas12a2, ca37Cas12a2, ca38Cas12a2, ca41Cas12a2, ca125Cas12a2, SuCas12a2, Cas12g or a combination thereof.

[0071] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology. The crRNA can bind to the specific sequence nucleic acid in the target cell and activate the non-specific "by-product effect" of A on DNA.

[0072] 6. The "para-cutting effect" is activated by the target cell-specific DNA. After activation, it cuts DNA non-specifically and combines with a substance "i" that can inhibit DNA damage and repair, so that the damaged DNA cannot be repaired, thereby achieving a composition that specifically kills the target cells, and its application in killing target cells.

[0073] The composition comprises three components: A, B and i, or "a complex of A and B combined together" + "i".

[0074] A is a nuclease that can be activated by DNA with a specific sequence through B. The "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts.

[0075] B is a substance that can recognize specific sequence DNA in target cells but not nucleic acids in cells surrounding or nearby the target cells, and the substance can activate the non-specific "parasitic effect" of A on DNA after recognizing the specific sequence DNA in the above target cells.

[0076] "i" is a substance that can inhibit DNA damage repair, so that the damaged DNA cannot be repaired. "i" can be a compound, or siRNA, or an antibody, and is characterized by being able to inhibit the repair of DNA damage.

[0077] In a preferred embodiment,

[0078] The A is a gene editing protein.

[0079] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology. The crRNA can bind to the specific sequence DNA in the target cell and activate the non-specific "bypass effect" of A on DNA.

[0080] The "i" is an inhibitor of the DNA damage repair pathway.

[0081] In another preferred embodiment, the gene editing protein is selected from the following group: NbaAgo-DREN, TmuRE-Ago, eAGO, pAGO, Cas12a2, AbCas12a2, ca2Cas12a2, ca6Cas12a2, ca7Cas12a2, ca11Cas12a2, ca14Cas12a2, ca17Cas12a2, ca33Cas12a2, ca34Cas12a2, ca35Cas12a2, ca36Cas12a2, ca37Cas12a2, ca38Cas12a2, ca41Cas12a2, ca125Cas12a2, SuCas12a2, Cas12g, etc., or a combination of the above gene editing enzymes.

[0082] The B is the crRNA used in conjunction with the gene editing protein in gene editing technology. The crRNA can bind to the specific sequence nucleic acid in the target cell and activate the non-specific "by-product effect" of A on DNA.

[0083] The i is a drug that inhibits DNA damage repair, including inhibitors that are currently known or will be developed to inhibit DNA damage repair, for example, inhibitors of the non-homologous endjoining (NHEJ) pathway, such as the DNA-PKcs inhibitor NU7441; or inhibitors of the homologous recombination (HR) pathway, such as the ATM inhibitor KU55933; or inhibitors that can inhibit both NHEJ and HR, such as wortmannin; including both combined use and single use.

[0084] 7. A composition that specifically kills target cells by relying on the DNA side-cutting effect of Cas12a2.

[0085] The composition comprises two components, A and B, or a complex in which A and B are combined together.

[0086] A is the gene editing protein Cas12a2.

[0087] Cas12a2 has a nuclease that can be activated by specific sequence nucleic acids through B to produce a "side-cutting effect" on DNA.

[0088] B is a crRNA that can recognize specific sequence nucleic acids in target cells but not nucleic acids in cells around or near the target cells, and the crRNA can activate the non-specific "bypass effect" of Cas12a2 on DNA after recognizing the specific sequence nucleic acids in the above-mentioned target cells.

[0089] 8. A composition for specifically killing target cells by relying on Ago protein with DNA cleavage effect, the composition comprising: two components A and B, or a complex of A and B combined together;

[0090] A is an Ago protein with DNA cleavage effect, such as TmuRE-Ago protein and NbaAgo-DREN;

[0091] Ago proteins with DNA cleavage effects have nucleases that can be activated by specific sequence nucleic acids through B to produce "cleavage effects" on non-specific DNA;

[0092] B is a gRNA or gDNA that can recognize nucleic acids of specific sequences in target cells but not nucleic acids in cells surrounding or nearby the target cells, and the gRNA or gDNA can activate the "bypass effect" of the Ago protein with a DNA bypass effect after recognizing the nucleic acids of specific sequences in the above-mentioned target cells, and perform non-specific cutting on the DNA encountered.

[0093] The second aspect of the present invention provides the use of the composition having the above characteristics in killing target cells through the para-segmentation effect.

[0094] The third aspect of the present invention provides a method for using the composition having the above characteristics in killing target cells.

[0095] S1. Determine at least one DNA or RNA sequence that is unique to the target cell and different from that in normal cells;

[0096] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0097] It should be noted that:

[0098] The target cell-specific RNA or DNA substance of the present invention can be obtained by high-throughput or low-throughput sequencing; for cells infected by known pathogens, the target cell-specific RNA or DNA substance can also be obtained through information such as sequence databases of known pathogens. The specific RNA can be a specific RNA site due to mutation, or a specific highly expressed RNA (that is, there is no difference in RNA sequence between the target cell and normal cells, but the expression level in the target cell is significantly higher). The specific nucleic acid in the target cell can be a pathogen nucleic acid infected by a pathogen, and the specific DNA in the target cell can be a specific DNA site due to mutation (mutation includes point mutation, translocation mutation, insertion or deletion mutation, gene fusion, etc., as long as the mutation is sufficient to distinguish the target cell from the normal cell, it is considered as a specific DNA in the target cell), or a specific high-copy DNA (that is, there is no difference in DNA sequence between the target cell and the normal cell, but the copy number in the target cell is significantly higher), and it can also be due to the superposition of sequence mutation and copy number variation, that is, the corresponding DNA site sequence is only in the target cell, and the copy number of the DNA segment where the sequence is located is also changed.

[0099] The nucleic acid material capable of identifying the specific nucleic acid in the target cell of the present invention can be prepared and stored in advance on a large scale according to the universal or known sequence of the pathogen of the infected cell, and the material of the specific RNA or DNA in the target cell can be prepared and stored in advance on a large scale according to the mutation sites or specific highly expressed RNA in the target cell, or can be prepared by personalized design after sequencing the cancer tissue or cells of the patient. In short, this material can specifically identify the specific RNA or DNA in the target cell.

[0100] The nuclease with "side-cutting effect" described in the present invention may be a nuclease with "side-cutting effect" derived from a CRISPR system (e.g., Cas12 family, Cas12a, Cas12g, Cas12a2), or a nuclease with "side-cutting effect" derived from an Ago system and a complex with nuclease activity (e.g., NbaAgo-DREN, TmuRE-Ago, eAgo, pAgo), or a nuclease that does not have a "side-cutting effect" but has a "side-cutting effect" after modification (e.g., a mutant Cas9-Mut, or Cas9+ with an added domain, Cas12-mut, Cas12+ or Cas12-mut, Cas12+, or Cas13-mut, Cas13+, or a mutant eAgo, a mutant pAgo, a linked Other proteins make substances with nucleic acid-activatable "bypass effect" properties), and can also be bypass effect modified nucleases based on zinc finger nucleases (ZFNs) and transcription activator-like effectors (TALENs). In short, nucleases that have the ability to be activated by specific RNA or DNA in target cells to non-specifically cut DNA are included in the scope of the present invention. The gene editing system can be directly synthesized, or it can be a vector that can be introduced into cells to generate an effective editing system, such as a plasmid, RNA, virus (adenovirus, lentivirus), etc. In nature, or through artificial modification, nucleases that can activate the "bypass effect" of DNA or RNA can also be obtained, and the "bypass effect" of this nuclease can cut DNA after being activated. This nuclease specifically kills target cells through the bypass effect and also belongs to the scope of the present invention.

[0101] In summary, the composition of our invention comprises:

[0102] A substance that can recognize specific DNA or RNA sites in the target cell (such as crRNA, gRNA or sgRNA), and after recognizing the target DNA or RNA, activates its "bypass effect" to cut the DNA nuclease.

[0103] The invented method is to introduce the above substances into target cells for specifically killing the target cells.

[0104] This composition includes a recognition unit and a cleavage unit. The recognition unit recognizes RNA or DNA that is only present in the target cell, such as the crRNA in Example 1; the cleavage unit is a unit that can non-specifically cleave DNA or RNA after being activated by the recognition unit, such as Cas12a2 in Example 1.

[0105] The site recognized by the recognition unit can be RNA or DNA that is recognized to be present only in the target cells, such as widely reported mutant genes, highly expressed RNA, or it can be RNA or DNA that is unique to the corresponding individual target cells determined according to sequencing results in personalized treatment. For example, after sequencing, it is found that a certain gene has a mutation in the target cells of the corresponding individual, so the crRNA designed for the mutation is also within the protection scope of the present invention.

[0106] The sequencing technology mentioned above refers to all methods that can directly or indirectly determine the whole or part of the DNA sequence in the patient's target cells, such as whole genome sequencing, whole exome sequencing, first-generation sequencing, second-generation sequencing, third-generation sequencing, RNA sequencing, single-cell sequencing, spatial transcriptome sequencing, etc.

[0107] The composition of the above invention is introduced into the target cell for the method of specifically killing the target cell. The related gene editing system can be directly synthesized or can be a vector that can generate an effective editing system by the cell after being introduced into the cell, such as plasmid, RNA, virus (adenovirus, lentivirus), RNA protein complex, etc. The vector introduced into the cell can be a virus, a nano-drug-encapsulated expression vector, a nano-drug-encapsulated RNA protein complex, etc.

[0108] Being able to specifically kill cells with certain nucleic acid characteristics will be of great value. For example, in plants, after specifically killing cells infected by pathogens, it is expected to improve the survival status of plants, which has certain application value in agriculture and medicine; in microbial communities such as bacteria or fungi, specifically killing bacteria or fungi infected by viruses is expected to improve the overall state of the flora, which also has certain application prospects in the field of fermentation engineering, or in the field of health issues related to the maintenance of normal flora in animals and humans; in animals, specifically killing cells infected by pathogens, or specifically killing target cells is expected to improve the health level of animals; in humans, specifically killing cells infected by pathogens, or specifically killing a certain type of cells or specifically killing target cells is expected to improve the health level of humans. However, there are currently no materials and methods that use the differences in nucleic acid substances between cells to specifically kill cells with certain nucleic acid characteristics. Gene editing technology can accurately identify and shear target DNA or RNA, however, not all gene editing technologies only cut target nucleic acids, and many gene editing tools also have "side cutting effect", that is, once the nuclease is activated, in addition to specific cutting of target nucleic acids, it can also non-specifically cut other nucleic acid substances around, such as Cas13, Cas12a2, etc. "Side cutting effect" is a shortcoming for precise gene editing, but the present invention can cleverly specifically kill or inhibit certain target cells using this "side cutting effect": that is, the "side cutting effect" of nuclease activated by RNA or DNA only in target cells is used to cause a large number of DNA breaks in target cells, so as to inhibit the growth of target cells and even kill target cells. The material of specific killing of target cells involved in the present invention is a material (such as crRNA) that can identify RNA or DNA specific to target cells, and a nuclease (such as Cas13, Cas12a2) with "side cutting effect" of non-specific cutting DNA after being activated, and the method involved is to use the above-mentioned material for the method of specific killing of target cells.

[0109] The present invention can achieve specific killing of cells with certain nucleic acid sequence characteristics, which is difficult to achieve with other methods at present.

[0110] A method that can also achieve a similar purpose is also invented by the inventor in the early stage, which uses the cis-cleavage activity of gene editing to cause specific breakage of the target DNA, and at the same time adds a DNA damage repair inhibitor (i) to kill the target cells (cancer cells) (patent application number: 202210619622.7, related results were published in Molecular Cancer in August 2022, IF: 37.3.)

[0111] Compared with the applicant's aforementioned invention (202210619622.7), the most fundamental difference between the present invention and the applicant's aforementioned invention (202210619622.7) is that the new invention utilizes the side-cutting effect of gene editing to cause non-specific DNA breakage. However, this non-specific DNA cutting is activated by specific DNA sequences, while the invention 202210619622.7 relies on the cis-cleavage activity of gene editing to cause specific breakage of the target DNA.

[0112] The present invention has the following more significant advantages:

[0113] 1. It is more efficient because the side cutting effect is a non-specific cutting of the DNA encountered, so many DNA breakpoints can be generated, which is more efficient.

[0114] 2. DNA damage repair inhibitors (i) are not necessarily required to achieve efficient killing of target cells. The fundamental reason is that the side cutting effect is a non-specific cutting of the encountered DNA, so there will be many DNA breakpoints that can be generated, and the efficiency is higher. Therefore, even if the DNA damage repair inhibitor (i) is not added, there are too many broken DNA in the cells, and the cells cannot be repaired, and the cells will still die. Of course, the present invention can also be used together with the DNA damage repair inhibitor (i) to further improve the killing efficiency.

[0115] 3. Specific killing of target cells can be achieved at a lower cost. In our previous invention (202210619622.7), a gRNA (or crRNA) is required to identify the target nucleic acid in order to produce a broken nucleic acid. However, our experiments have found that the cell killing effect produced by a small amount of nucleic acid breaks is limited, resulting in more DNA breakpoints in cells, and the higher the efficiency of cell killing (the experimental results in our article published in Molecular Cancer also illustrate this point). Since the previous invention only cuts the identified target, each "gene editing scissors" can only produce one cutting point (without considering copy number variation), so we need to introduce multiple "gene editing scissors" in our experiment. Even if it is applied to cancer treatment, the above invention (202210619622.7) will face problems such as low convenience and high cost in future practical applications, and it also has high requirements for the number of mutations that can be used to distinguish cancer cells from normal cells in each patient (too few mutations that can be cut can only cause too few DNA breaks, and enough breaks require enough mutations). Therefore, for individual patients, high-throughput sequencing is often required first, and then many sites are selected to design and prepare many "customized scissors". This makes it impossible to carry out large-scale preparation in advance for some mutations that are highly present in a wide range of cancer patients, and the application cost is relatively high.

[0116] The present invention can introduce a small amount of "gene editing scissors" to produce many DNA breakpoints, which can not only significantly improve the efficiency of specific killing of target cells, but also significantly reduce the amount of "gene editing scissors" introduced, so that the cost is significantly reduced. Moreover, a large number of "gene editing scissors" can be prepared in batches for commonly used cancer mutations, and the cost is further reduced. Moreover, for cells with clear specific sequences in target cells (such as infected cells), it is not necessary to prepare "gene editing scissors" personalized for mutations in cancer cells like killing cancer cells, but a large number of "gene editing scissors" can be prepared in batches according to the pathogen inhibition sequence in the infected cells, just like preparing medicines and storing them for standby use. Due to the scale effect, the cost is further greatly reduced.

[0117] 4. Wider application scope. Also due to the low cost, this invention can not only be used to kill diseased cells in humans and play a role in the field of human health, but can also be used in a wider range of fields such as specific killing of plant cells, animal cells, microbial cells, etc., and play a wider role. BRIEF DESCRIPTION OF THE DRAWINGS

[0118] Figure 1 . Schematic diagram of the present invention for specific killing of target cells;

[0119] Figure 2 . Schematic diagram of the composition of the present invention used for specific killing of target cells in Examples 1-13;

[0120] Figure 3 .The effect diagram of the specific killing effect of the composition of the present invention on PANC-1 pancreatic cancer cells in Example 1. Figure 3 A is the result of detecting cell survival after the composition targeting PANC-1 pancreatic cancer cells was introduced into PANC-1 pancreatic cancer cells. Figure 3 B is the result of detecting cell survival after the composition targeting PANC-1 pancreatic cancer cells was introduced into human normal cell line HEK-293 cells.

[0121] Figure 4 .The effect diagram of the composition of the present invention in Example 2 on the specific killing of HEPG2 liver cancer cells. Figure 4 A is the result of detecting cell survival after the composition targeting HEPG2 liver cancer cells was introduced into HEPG2 liver cancer cells. Figure 4 B is the result of detecting cell survival after the composition targeting HEPG2 liver cancer cells was introduced into human normal cell line HEK-293 cells.

[0122] Figure 5 .The effect diagram of the composition of the present invention in Example 3 on the specific killing of liver cancer cells.

[0123] Figure 6.A diagram showing the specific killing effect of the composition of the present invention on hepatocytes infected with HBV in Example 4.

[0124] Figure 7 .A diagram showing the specific killing effect of the composition of the present invention on cells infected with HPV in Example 5.

[0125] Figure 8 .Another combination form of the composition of the present invention in Example 6 is used to show the specific killing effect of cells infected with HPV.

[0126] Fig. 9 .Another combination form of the composition of the present invention in Example 7 is used to show the specific killing effect of virus-infected pig cultured cells.

[0127] Fig.10 .Another combination form of the composition of the present invention in Example 8 is used to show the specific killing effect of hepatocytes infected with HBV.

[0128] Fig.11 .Another combination form of the composition of the present invention in Example 9 is used to show the specific killing effect of liver cancer cells.

[0129] Fig.12 .A diagram showing the specific killing effect of the composition of the present invention in Example 10 on multiple cancer cell types containing the same mutation.

[0130] Fig.13 .Another combination form of the composition of the present invention in Example 11 is used to show the specific killing effect of liver cancer cells.

[0131] Fig.14 .Another combination form of the composition of the present invention in Example 12 is used to show the specific killing effect of hepatocytes infected with HBV. DETAILED DESCRIPTION

[0132] The specific implementation methods provided by the present invention are described in detail below in conjunction with examples.

[0133] The nuclease with "bypass effect" is characterized in that it can be activated by a nucleic acid (target) that is complementary to crRNA. After activation, not only the site of the nuclease that cuts the target nucleic acid is activated, but also its "bypass effect" cutting site is activated, and the "bypass effect" cutting site can cut the DNA encountered.

[0134] The crRNA that can recognize nucleic acids unique to the target cells (such as mutant DNA or RNA in cancer cells, viral DNA or RNA in virus-infected cells) and the nuclease with a "bystander effect" are introduced into the target cells. The unique nucleic acids in the target cells will activate the "bystander effect" cutting sites of the nuclease, and the "red scissors will open" to cut the DNA encountered. These broken DNAs will cause cell death, just like being exposed to radiation.

[0135] Even if the corresponding crRNA and nuclease combination enters normal cells, since there is no nucleic acid that can be recognized by crRNA in normal cells, the nuclease cannot be activated ("red scissors are closed"), and DNA will not be broken, and the cells can survive. ( Figure 1 )

[0136] This method can achieve specific killing of target cells with certain nucleic acid sequence information characteristics, and has certain application prospects.

[0137] Example 1: Using Cas12a2 nuclease + crRNA to specifically kill pancreatic cancer cells

[0138] S1. Determine at least one RNA sequence that is unique to the target cell and different from that in normal cells;

[0139] In this case, through whole genome sequencing we know that there are many DNA mutations in PANC-1 pancreatic cancer cells, which lead to many RNAs containing mutations.

[0140] The specific RNA mutations and crRNA design sequences selected in this example in PANC-1 pancreatic cancer cells are as follows:

[0141]

[0142] These crRNAs cannot recognize RNA in any normal cells, but can recognize RNA containing mutant sequences that are unique to PANC-1 cancer cells.

[0143] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0144] The composition selected in this example for the purpose of specifically killing target cells includes two components: A and B.

[0145] A is Cas12a2

[0146] B is a crRNA that can recognize a unique RNA sequence in PANC-1 pancreatic cancer cells.

[0147] This example uses a CRISPR nuclease such as Cas12a2, which can be activated by RNA (target) complementary to crRNA. After activation, not only the site of Cas12a2 that cuts the target RNA is activated, but also its "bypass effect" cutting site is activated, which can cut the RNA and single-stranded DNA and double-stranded DNA encountered.

[0148] B is specifically a crRNA that can recognize the unique RNA in PANC-1 pancreatic cancer cells in the above table.

[0149] The above A+B composition is introduced into the target cells (PANC-1 pancreatic cancer cells), and the unique RNA in the target cells activates the "side-cutting effect" cleavage site of Cas12a2, and the "red scissors are turned on" to cut the RNA and single-stranded DNA and double-stranded DNA encountered. These broken RNA and DNA will cause cell death, just like encountering radiation exposure. Even if the corresponding crRNA and Cas12a2 composition enters normal cells, since there is no RNA that can be recognized by crRNA in normal cells, Cas12a2 cannot be activated ("red scissors are turned off"), and will not cause the breakage of RNA and DNA, and the cells can survive.

[0150] In practice, the above A+B composition was introduced into target cells (PANC-1 pancreatic cancer cells) and normal cells (HEK293 cells), and then cell death was detected.

[0151] The experimental results show that our strategy can significantly kill PANC-1 cells. Figure 3 As shown:

[0152] The CCK-8 experiment detected the survival of the pancreatic cancer PANC-1 cells and the normal human cell line HEK-293 after the composition of the present invention for PANC-1 cells was introduced into each cell. It can be seen that the A+B composition has a significant specific killing effect on PANC-1 cells, but has no significant effect on HEK293 cells.

[0153] Example 2: Using Cas12a2 nuclease + crRNA to specifically kill liver cancer cells

[0154] S1. Determine at least one RNA sequence that is unique to the target cell and different from that in normal cells;

[0155] In this example, we obtained the mutation information of DNA and RNA in HepG2 liver cancer cells by analyzing the tumor database website CCLE (https: / / depmap.org / portal / cell_line / ACH-000979?tab=mutation).

[0156] The unique RNA mutations and crRNA design sequences selected in this example for HEPG2 liver cancer cells are as follows:

[0157]

[0158] These crRNAs cannot recognize RNA in any normal cells, but can recognize RNA containing mutation sequences that are unique to HEPG2 liver cancer cells.

[0159] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0160] The composition selected in this example for the purpose of specifically killing target cells includes two components: A and B.

[0161] A is Cas12a2

[0162] B is a crRNA that can recognize the unique RNA sequence in HEPG2 liver cancer cells.

[0163] This example uses a CRISPR nuclease such as Cas12a2, which can be activated by RNA (target) complementary to crRNA. After activation, not only the site of Cas12a2 that cuts the target RNA is activated, but also its "bypass effect" cutting site is activated, which can cut the RNA and single-stranded DNA and double-stranded DNA encountered.

[0164] B is specifically a crRNA that can recognize the unique RNA in HEPG2 liver cancer cells in the above table.

[0165] The above A+B composition is introduced into the target cells (HEPG2 liver cancer cells), and the unique RNA in the target cells activates the "side-cutting effect" cleavage site of Cas12a2. The "red scissors are open" can cut the RNA and single-stranded DNA and double-stranded DNA encountered. These broken RNA and DNA will cause cell death, just like encountering radiation exposure. Even if the corresponding crRNA and Cas12a2 composition enters normal cells, since there is no RNA that can be recognized by crRNA in normal cells, Cas12a2 cannot be activated ("red scissors are closed"), and will not cause RNA and DNA breaks, and cells can survive.

[0166] In practice, the above A+B composition was introduced into target cells (HEPG2 liver cancer cells) and normal cells (HEK293 cells), and then cell death was detected.

[0167] The experimental results show that our strategy can significantly kill HEPG2 liver cancer cells. Figure 4As shown:

[0168] The CCK-8 experiment detected the survival of the respective cells after the composition of the present invention targeting HEPG2 liver cancer cells was introduced into pancreatic cancer HEPG2 liver cancer cells and human normal cell line HEK-293. It can be seen that the A+B composition has a significant specific killing effect on HEPG2 liver cancer cells, but has no significant effect on HEK293 cells.

[0169] Example 3: Using Cas12a2 nuclease + crRNA combination to specifically kill liver cancer cells

[0170] S1. Determine at least one RNA sequence that is unique to the target cell and different from that in normal cells;

[0171] In this case, we analyzed the TCGA tumor database to obtain high-frequency mutation information in the liver cancer sample population and found that some mutations appeared frequently in different patient samples. The combination of these mutations has the prospect of preparative use.

[0172] In order to expand the scope of application, we selected a crRNA combination for the high-frequency RNA mutations that may be unique to these liver cancer cells. These mutations may or may not appear in some liver cancer cells. In this way, as long as there is even one mutation point in the cell, it can be used to kill cancer cells. This is a strategy to improve the efficiency of the response. That is, for some high-frequency mutations, a crRNA composition is prepared in advance. When one of these mutations exists in cancer cells, the crRNA composition can identify cancer cells and non-cancerous cells.

[0173] The mutant RNA selected in this example and the corresponding crRNA that recognizes the RNA are as follows:

[0174]

[0175] These crRNAs cannot recognize RNA in any normal cells, but may recognize RNA containing mutation sequences that are unique to liver cancer cells.

[0176] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0177] The composition selected in this example for the purpose of specifically killing target cells includes two components: A and B.

[0178] A is Cas12a2

[0179] B is a crRNA that can recognize unique RNA sequences in liver cancer cells.

[0180] This example uses a CRISPR nuclease such as Cas12a2, which can be activated by RNA (target) complementary to crRNA. After activation, not only the site of Cas12a2 that cuts the target RNA is activated, but also its "bypass effect" cutting site is activated, which can cut the RNA and single-stranded DNA and double-stranded DNA encountered.

[0181] B is specifically a crRNA that can recognize the unique RNA in HEPG2 liver cancer cells in the above table.

[0182] The above A+B composition is introduced into the target cell (HEP3B liver cancer cell), and the unique RNA in the target cell activates the "side-cutting effect" cleavage site of Cas12a2, and the "red scissors are turned on" to cut the RNA and single-stranded DNA and double-stranded DNA encountered. These broken RNA and DNA will cause cell death, just like encountering radiation exposure. Even if the corresponding crRNA and Cas12a2 composition enters normal cells, since there is no RNA that can be recognized by crRNA in normal cells, Cas12a2 cannot be activated ("red scissors are turned off"), and will not cause the breakage of RNA and DNA, and the cells can survive.

[0183] In practice, the above A+B composition was introduced into target cells (HEP3B liver cancer cells) and normal cells (HEK293 cells), and then cell death was detected.

[0184] The experimental results show that our strategy can significantly kill liver cancer cells. Figure 5 As shown:

[0185] The CCK-8 experiment detected the survival of the respective cells after the composition of the present invention targeting HEP3B liver cancer cells was introduced into pancreatic cancer HEP3B liver cancer cells and human normal cell line HEK-293. It can be seen that the A+B composition has a significant specific killing effect on liver cancer cells, but has no significant effect on HEK293 cells.

[0186] Example 4: Using Cas12a2 nuclease + crRNA to specifically kill hepatitis B virus-infected hepatocytes

[0187] S1. Determine at least one RNA sequence that is unique to the target cell and different from that in normal cells;

[0188] In this case, the sequence of hepatitis B virus (HBV) is known. We obtained the mRNA sequence of the HBV expression gene through NCBI and designed and prepared crRNA for it.

[0189] The mutant RNA selected in this example and the corresponding crRNA that recognizes the RNA are as follows:

[0190]

[0191] This crRNA cannot recognize RNA in any normal cells, but it can recognize RNA derived from the HBV virus that is unique to HEPG2 liver cancer cells containing the HBV virus.

[0192] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0193] The composition selected in this example for the purpose of specifically killing target cells includes two components: A and B.

[0194] A is Cas12a2

[0195] B is a crRNA that can recognize the unique RNA sequence in HEPG2 liver cancer cells.

[0196] This example uses a CRISPR nuclease such as Cas12a2, which can be activated by RNA (target) complementary to crRNA. After activation, not only the site of Cas12a2 that cuts the target RNA is activated, but also its "bypass effect" cutting site is activated, which can cut the RNA and single-stranded DNA and double-stranded DNA encountered.

[0197] B is specifically a crRNA that can recognize the unique RNA in HEPG2 liver cancer cells in the above table.

[0198] The above A+B composition is introduced into the target cell (HEPG2 liver cancer cells transfected with the nucleic acid sequence of HBV contained in the above cells), and the unique RNA in the target cell activates the "side-cutting effect" cleavage site of Cas12a2, and the "red scissors are open", which can cut the RNA and single-stranded DNA and double-stranded DNA encountered. These broken RNA and DNA will cause cell death, just like encountering radiation exposure. Even if the corresponding crRNA and Cas12a2 composition enters normal cells, since there is no RNA that can be recognized by crRNA in normal cells, Cas12a2 cannot be activated ("red scissors are closed"), and will not cause the breakage of RNA and DNA, and the cells can survive.

[0199] In practice, the above A+B composition is introduced into target cells (HEPG2 liver cancer cells transfected with the nucleic acid sequence expressing HBV contained in the above cells) and then the cell death is detected.

[0200] The experimental results show that our strategy can significantly kill HEPG2 liver cancer cells containing HBV nucleic acid. Figure 6 shown.

[0201] Example 5: Using Cas12a2 nuclease + crRNA to specifically kill cells infected with human papillomavirus

[0202] S1. Determine at least one RNA sequence that is unique to the target cell and different from that in normal cells;

[0203] In this case, the sequence of human papillomavirus (HPV) is known. We obtained the mRNA sequence of HPV-expressed genes from NCBI and designed and prepared crRNA for it.

[0204] The mutant RNA selected in this example and the corresponding crRNA that recognizes the RNA are as follows:

[0205]

[0206] This crRNA cannot recognize RNA in any normal cells, but can recognize RNA derived from HPV virus-specific HEK293 cells containing HPV virus.

[0207] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0208] The composition selected in this example for the purpose of specifically killing target cells includes two components: A and B.

[0209] A is Cas12a2

[0210] B is a crRNA that can recognize a unique RNA sequence in HEK293 cells.

[0211] This example uses a CRISPR nuclease such as Cas12a2, which can be activated by RNA complementary to crRNA. After activation, not only the site of Cas12a2 that cuts the target RNA is activated, but also its "bypass effect" cutting site is activated. The "bypass effect" cutting site can cut the RNA and single-stranded DNA and double-stranded DNA encountered.

[0212] B is specifically a crRNA that can recognize the unique RNA in the infected HEK293 cells in the above table.

[0213] The above-mentioned A+B composition is introduced into the target cells (HEK293 cells transfected with the nucleic acid sequence contained in the HPV expressing the above-mentioned cells), and the unique RNA in the target cells activates the "side-cutting effect" cleavage site of Cas12a2, and the "red scissors are open", which can cut the RNA and single-stranded DNA and double-stranded DNA encountered. These broken RNA and DNA will cause cell death, just like encountering radiation exposure. Even if the corresponding crRNA and Cas12a2 composition enters normal cells, since there is no RNA that can be recognized by crRNA in normal cells, Cas12a2 cannot be activated ("red scissors are closed"), and will not cause the breakage of RNA and DNA, and the cells can survive.

[0214] In practice, the above A+B composition is introduced into target cells (HEK293 cells transfected with the nucleic acid sequence expressed by HPV in the above cells) and then the cell death is detected.

[0215] The experimental results show that our strategy can significantly kill HEK293 cells. Figure 7 shown.

[0216] Example 6: Using Cas12a2 nuclease + crRNA + i to specifically kill cells infected with human papillomavirus

[0217] S1. Determine at least one RNA sequence that is unique to the target cell and different from that in normal cells;

[0218] In this case, the sequence of human papillomavirus (HPV) is known. We obtained the mRNA sequence of HPV-expressed genes from NCBI and designed and prepared crRNA for it.

[0219] The mutant RNA selected in this example and the corresponding crRNA that recognizes the RNA are as follows:

[0220]

[0221] This crRNA cannot recognize RNA in any normal cells, but can recognize RNA derived from HPV virus-specific HEK293 cells containing HPV virus.

[0222] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0223] The composition selected in this example for the purpose of specifically killing target cells includes three components: A, B and i.

[0224] A is Cas12a2

[0225] B is a crRNA that can recognize a unique RNA sequence in HEK293 cells.

[0226] This example uses a CRISPR nuclease such as Cas12a2, which can be activated by RNA complementary to crRNA. After activation, not only the site of Cas12a2 that cuts the target RNA is activated, but also its "bypass effect" cutting site is activated. The "bypass effect" cutting site can cut the RNA and single-stranded DNA and double-stranded DNA encountered.

[0227] B is specifically a crRNA that can recognize the unique RNA in the infected HEK293 cells in the above table.

[0228] i is a DNA damage repair inhibitor (in this example, we added NU7441, an inhibitor of DNA-PKcs that inhibits the NHEJ pathway, and KU55933, an ATM inhibitor of the HR pathway).

[0229] The above-mentioned A+B+i composition is introduced into the target cells (HEK293 cells transfected with the nucleic acid sequence contained in the HPV expressing the above-mentioned cells), and the unique RNA in the target cells will activate the "side cutting effect" cutting site of Cas12a2, and the "red scissors are open", which can cut the RNA and single-stranded DNA and double-stranded DNA encountered. These broken DNAs cannot be repaired and will cause cell death, just like encountering radiation exposure. Even if the corresponding crRNA and Cas12a2 composition enters normal cells, since there is no RNA that can be recognized by crRNA in normal cells, Cas12a2 cannot be activated ("red scissors are closed"), and will not cause the breakage of RNA and DNA, and the cells can survive.

[0230] In practice, the above A+B+i composition is introduced into target cells (HEK293 cells transfected with the nucleic acid sequence expressed by HPV in the above cells) and then cell death is detected.

[0231] The experimental results show that our strategy can significantly kill HEK293 cells. Figure 8 shown.

[0232] Example 7: Using Cas12a2 nuclease + crRNA + i to specifically kill pig cultured cells infected with the virus

[0233] S1. Determine at least one RNA sequence that is unique to the target cell and different from that in normal cells;

[0234] In this example, we constructed a lentivirus that can express green fluorescent protein (EGFP) in pig cells after infection, and divided the cultured pig testicular cells (ST) into two groups, one group was not infected with the virus, and the other was infected with the above virus. Then, crRNA was designed for the mRNA of EGFP expressed by the virus.

[0235] In this example, the partial RNA sequences that specifically exist in cells after virus infection and the selected crRNA sequences that specifically identify the infected cells are as follows:

[0236]

[0237] This crRNA cannot recognize RNA in any normal cells, but can recognize RNA of lentivirus origin unique to pig testicular cells (ST) containing RNA carried by lentivirus.

[0238] S2. Introducing the above composition that can recognize RNA sequences specific to target cells into target cells.

[0239] The composition selected in this example for the purpose of specifically killing target cells includes three components: A, B and i.

[0240] A is Cas12a2

[0241] B is a crRNA that can recognize a specific RNA sequence in pig testicular cells (ST) infected with a lentivirus.

[0242] This example uses a CRISPR nuclease such as Cas12a2, which can be activated by RNA complementary to crRNA. After activation, not only the site of Cas12a2 that cuts the target RNA is activated, but also its "bypass effect" cutting site is activated. The "bypass effect" cutting site can cut the RNA and single-stranded DNA and double-stranded DNA encountered.

[0243] B is specifically a crRNA that can recognize the unique RNA in the infected pig testicular cells (ST) cells in the above table.

[0244] i is a DNA damage repair inhibitor (in this example, we added NU7441, an inhibitor of DNA-PKcs that inhibits the NHEJ pathway, and KU55933, an ATM inhibitor of the HR pathway).

[0245] The above-mentioned A+B+i composition is introduced into the target cell (pig testicular cell (ST) cell transfected with the nucleic acid sequence contained in the HPV expressing the above-mentioned cell), and the unique RNA in the target cell activates the "side cutting effect" cleavage site of Cas12a2, and the "red scissors are turned on", and the RNA and single-stranded DNA and double-stranded DNA encountered can be cut. These broken DNAs cannot be repaired and will cause cell death, just like encountering radiation exposure. Even if the corresponding crRNA and Cas12a2 composition enters normal cells, since there is no RNA that can be recognized by crRNA in normal cells, Cas12a2 cannot be activated ("red scissors are turned off"), and will not cause the breakage of RNA and DNA, and the cells can survive.

[0246] In practice, the above A+B+i composition is introduced into target cells (pig testicular cells (ST) cells transfected with nucleic acid sequences expressing HPV contained in the above cells) and then cell death is detected.

[0247] The experimental results show that our strategy can significantly kill virus-infected pig testicular cells (ST) without killing cells that are not infected by the virus. Fig. 9 shown.

[0248] Example 8: Using TmuRE-Ago protein + gRNA + i to specifically kill hepatitis B virus-infected liver cells

[0249] S1. Determine at least one DNA sequence site that is unique to the target cell and different from that in normal cells;

[0250] In this example, the sequence of hepatitis B virus (HBV) is known. We obtained the HBV sequence from NCBI and designed and prepared gRNA for it.

[0251] The DNA recognition sites selected in this example and the corresponding gRNAs that recognize the DNA are as follows:

[0252]

[0253]

[0254] This gRNA cannot recognize DNA in any normal cells, but can recognize HBV virus-derived DNA that is unique to HEPG2 liver cancer cells containing HBV virus.

[0255] S2. Introduce the above composition that can recognize a specific DNA sequence in the target cell into the target cell.

[0256] The composition selected in this example for the purpose of specifically killing target cells includes three components: A, B and i.

[0257] A is the TmuRE-Ago protein complex

[0258] This example uses the TmuRE-Ago protein complex, which has the characteristic of being activated by target DNA that is complementary to the gRNA. The "bystander effect" cleavage site of the activated TmuRE-Ago protein complex is activated, and the "bystander effect" cleavage site can cut the single-stranded DNA and double-stranded DNA encountered.

[0259] B is a gRNA that can recognize a unique DNA sequence in HEPG2 cells infected with hepatitis B virus.

[0260] i is a DNA damage repair inhibitor (in this example, we added NU7441, an inhibitor of DNA-PKcs that inhibits the NHEJ pathway, and KU55933, an ATM inhibitor of the HR pathway).

[0261] The above A+B+i composition is introduced into the target cells (cells infected with HBV, which will transcribe RNA containing HBV specificity). The specific DNA in the target cells will activate the "bystander effect" cleavage site of the TmuRE-Ago protein complex, which can non-specifically cut the single-stranded DNA and double-stranded DNA encountered. These broken DNAs cannot be repaired and will cause cell death, just like encountering radiation exposure. Even if the corresponding A+B+i composition enters normal cells, since there is no DNA that can be recognized by gRNA in normal cells, the TmuRE-Ago protein complex cannot be activated, and DNA breakage will not be caused, and the cells can survive.

[0262] In practice, the above A+B+i composition is introduced into target cells (HEPG2 liver cancer cells transfected with nucleic acid sequences expressing HBV contained in the above cells) and control cells (HEPG2 cells not infected with HBV), and then cell death is detected.

[0263] The experimental results showed that our strategy can significantly kill HEPG2 liver cancer cells containing HBV nucleic acid, but has no obvious killing effect on HEPG2 cells that are not infected with HBV nucleic acid. Fig.10 shown.

[0264] Example 9: Using TmuRE-Ago protein + gRNA combination + i to specifically kill liver cancer cells

[0265] S1. Determine at least one DNA sequence that is unique to the target cell and different from that in normal cells;

[0266] In this case, we analyzed the TCGA tumor database to obtain high-frequency mutation information in the liver cancer sample population and found that some mutations appeared frequently in different patient samples. The combination of these mutations has the prospect of preparative use.

[0267] In order to expand the scope of application, we select a gRNA combination for the high-frequency DNA mutations that may be unique to these liver cancer cells. These mutations may or may not appear in some liver cancer cells. In this way, as long as there is even one mutation point in the cell, it can be used to kill cancer cells. This is a strategy to improve the efficiency of the response. That is, for some high-frequency mutations, a gRNA combination is prepared in advance. When one of these mutations exists in cancer cells, the gRNA combination can identify cancer cells and non-cancerous cells.

[0268] The mutant DNA selected in this example and the corresponding gRNA that recognizes the DNA are as follows:

[0269]

[0270] These gRNAs cannot recognize DNA in any normal cells, but may recognize DNA containing mutation sequences that are unique to liver cancer cells.

[0271] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0272] The composition selected in this example for the purpose of specifically killing target cells includes three components: A, B and i.

[0273] A is the TmuRE-Ago protein complex

[0274] This example uses the TmuRE-Ago protein complex, which has the characteristic of being activated by DNA complementary to the gRNA. The "bystander effect" cleavage site of the activated TmuRE-Ago protein complex is activated, and the "bystander effect" cleavage site can cut the single-stranded DNA and double-stranded DNA encountered.

[0275] B is a gRNA that can recognize unique DNA sequences in liver cancer cells.

[0276] i is a DNA damage repair inhibitor (in this example, we added wortmannin, an inhibitor that can inhibit both the NHEJ pathway and the HR pathway).

[0277] The above A+B+i composition was introduced into target cells and normal cells (HEK293 cells), and then cell death was detected.

[0278] The experimental results show that our strategy can significantly kill liver cancer cells. Fig.11 As shown:

[0279] The CCK-8 experiment detected the survival of the respective cells after the composition of the present invention targeting HEP3B liver cancer cells was introduced into pancreatic cancer HEP3B liver cancer cells and human normal cell line HEK-293. It can be seen that the A+B+i composition has a significant specific killing effect on liver cancer cells, but has no significant effect on HEK293 cells.

[0280] Example 10: Using Cas12a2 nuclease + crRNA + i to specifically kill cancer cells containing KRAS mutations

[0281] S1. Determine at least one RNA sequence that is unique to the target cell and different from that in normal cells;

[0282] In this case, we know that KRAS is a gene that mutates at a high frequency in many tumors, so KRAS mutations can be seen in many cancer cells. We selected one of the mutant forms of KRAS mRNA as the RNA to distinguish cancer cells from normal cells.

[0283] By analyzing the TCGA tumor database, we obtained high-frequency mutation information in liver cancer sample groups and found that some mutations appeared frequently in different patient samples. The combination of these mutations has the prospect of pre-preparation. That is, gRNA or crRNA for many high-frequency mutation genes is prepared in advance and stored appropriately. When the corresponding mutation does exist in a certain cancer cell, the corresponding pre-prepared gRNA or crRNA can be selected for treatment. In this example, we take a mutation of KRAS as an example to illustrate:

[0284] The specific design sequence is as follows:

[0285]

[0286] These guide RNAs cannot recognize RNA in any normal cells, but may recognize RNA that is unique to cancer cells that contain the mutant KRAS.

[0287] After searching the literature, we found that the human pancreatic cancer cell PANC-1 contained the mutated KRAS. To confirm the universality of this strategy, we also overexpressed the mutant KRAS in liver cancer cells HEPG2, lung cancer cells A549, and colorectal cancer cells HCT116. Then, the composition was introduced into these cells and control cells HEK293 cells.

[0288] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0289] The composition selected in this example for the purpose of specifically killing target cells includes three components: A, B and i.

[0290] A is Cas12a2

[0291] This example uses a CRISPR nuclease such as Cas12a2, which can be activated by RNA complementary to crRNA. After activation, not only the site of Cas12a2 that cuts the target RNA is activated, but also its "bypass effect" cutting site is activated. The "bypass effect" cutting site can cut the RNA and single-stranded DNA and double-stranded DNA encountered.

[0292] B is a crRNA that can recognize the RNA sequence of mutant KRAS unique to cancer cells.

[0293] i is a DNA damage repair inhibitor (in this example, we added worumannin, an inhibitor that can inhibit both the NHEJ pathway and the HR pathway).

[0294] The above A+B+i composition was introduced into target cells and normal cells (HEK293 cells), and then cell death was detected.

[0295] The experimental results showed that our strategy can significantly kill cancer cells containing mutant KRAS mRNA. Fig.12 As shown:

[0296] CCK-8 experiments detected that after adding crRNA and Cas12a2 virus expressing mRNA targeting KRAS mutation and the combination of i, the viability of normal cells (HEK293 cells) was not significantly affected, while the viability of human pancreatic cancer cells PANC-1, liver cancer cells HEPG2, lung cancer cells A549, and colorectal cancer cells HCT116 containing KRAS mutation mRNA was significantly reduced.

[0297] Example 11: Specific killing of liver cancer cells using NbaAgo-DREN protein complex + gRNA composition + i

[0298] S1. Determine at least one DNA sequence that is unique to the target cell and different from that in normal cells;

[0299] In this case, we analyzed the TCGA tumor database to obtain high-frequency mutation information in the liver cancer sample population and found that some mutations appeared frequently in different patient samples. The combination of these mutations has the prospect of preparative use.

[0300] In order to expand the scope of application, we selected a gRNA combination for the high-frequency DNA mutations that may appear in some liver cancer cells, or may not appear in some liver cancer cells. This way, as long as there is even one mutation point in the cell, it can be used to kill cancer cells. This is a strategy to improve the efficiency of the response. That is, for some high-frequency mutations, a gRNA combination is prepared in advance. When one of the mutations exists in the cancer cells, the gRNA combination can identify cancer cells and non-cancerous cells.

[0301] The mutant DNA selected in this example and the corresponding gRNA that recognizes the DNA are as follows:

[0302]

[0303] These gRNAs cannot recognize RNA in any normal cells, but may recognize RNA containing mutation sequences that are unique to liver cancer cells.

[0304] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0305] The composition selected in this example for the purpose of specifically killing target cells includes three components: A, B and i.

[0306] A is the NbaAgo-DREN protein complex

[0307] This example uses the NbaAgo-DREN protein complex, which can be activated by DNA complementary to the gRNA. After activation, the "bystander effect" cleavage site of the NbaAgo-DREN protein complex is activated, and the "bystander effect" cleavage site can cut the single-stranded DNA and double-stranded DNA encountered.

[0308] B is a gRNA that can recognize unique DNA sequences in liver cancer cells.

[0309] i is a DNA damage repair inhibitor (in this example, we added wortmannin, an inhibitor that can inhibit both the NHEJ pathway and the HR pathway).

[0310] The above A+B+i composition was introduced into target cells and normal cells (HEK293 cells), and then cell death was detected.

[0311] The experimental results show that our strategy can significantly kill liver cancer cells. Fig.13 As shown:

[0312] The CCK-8 experiment detected the survival of the respective cells after the composition of the present invention targeting HEP3B liver cancer cells was introduced into pancreatic cancer HEP3B liver cancer cells and human normal cell line HEK-293. It can be seen that the A+B+i composition has a significant specific killing effect on liver cancer cells, but has no significant effect on HEK293 cells.

[0313] Example 12: Using Cas12a nuclease + crRNA to specifically kill hepatitis B virus-infected hepatocytes

[0314] S1. Determine at least one DNA sequence that is unique to the target cell and different from that in normal cells;

[0315] In this case, the sequence of hepatitis B virus (HBV) is known. We obtained the HBV sequence from NCBI and designed and prepared crRNA for it.

[0316] The hepatitis B virus DNA sequence selected in this example and the corresponding crRNA that recognizes the RNA are as follows:

[0317]

[0318] This crRNA cannot recognize DNA in any normal cells, but it can recognize DNA derived from the HBV virus that is unique to HEPG2 liver cancer cells containing the HBV virus.

[0319] S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

[0320] The composition selected in this example for the purpose of specifically killing target cells includes two components: A and B.

[0321] A is Cas12a

[0322] B is a crRNA that can recognize a unique DNA sequence in HEPG2 liver cancer cells.

[0323] This example uses a CRISPR nuclease such as Cas12a, which can be activated by DNA (target) complementary to crRNA. After activation, not only the site of Cas12a that cuts the target DNA is activated, but also its "bypass effect" cutting site is activated, which can cut the single-stranded DNA encountered.

[0324] B is specifically a crRNA that can recognize the unique DNA in the HEPG2 cells infected with HBV in the above table.

[0325] The above A+B composition is introduced into the target cell (HEPG2 liver cancer cell transfected with the nucleic acid sequence of HBV contained in the above cell), and the unique DNA in the target cell activates the "side-cutting effect" cleavage site of Cas12a, and the "red scissors are open", which can cut the single-stranded DNA encountered. These broken DNAs will cause cell death, just like encountering radiation exposure. Even if the corresponding crRNA and Cas12a composition enters normal cells, since there is no DNA that can be recognized by crRNA in normal cells, Cas12a cannot be activated ("red scissors are closed"), and will not cause DNA breakage, and the cells can survive.

[0326] In practice, the above A+B composition is introduced into target cells (HEPG2 hepatocytes transfected with the nucleic acid sequence expressing HBV contained in the above cells) and control cells (HEPG2 cells not infected with HBV), and then cell death is detected.

[0327] The experimental results showed that our strategy can significantly kill HEPG2 liver cancer cells containing HBV nucleic acid, but has no significant killing effect on uninfected HEPG2 cells. Fig.14 shown.

[0328] The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims

1. A composition for the purpose of specifically killing target cells, characterized in that: The composition comprises: two components A and B, or a complex of A and B combined together; A is a nuclease that can be activated by a specific sequence nucleic acid through B to have a "bypass effect" on DNA; the "bypass effect" on DNA is an effect of non-specific cutting of the DNA that the enzyme contacts; B is a substance that can recognize nucleic acid with a specific sequence in the target cell but not nucleic acid in cells surrounding or near the target cell, and the substance can activate the non-specific "bystander effect" of A on DNA after recognizing the nucleic acid with a specific sequence in the above target cell.

2. A composition for the purpose of specifically killing target cells, characterized in that: The composition comprises: three components A, B and i, or "a complex of A and B combined together" + "i"; A is a nuclease that can be activated by RNA or DNA of a specific sequence through B to produce a "bystander effect"; the "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts; B is a substance that can recognize specific sequence RNA or DNA in the target cell but not nucleic acid in the cells around or near the target cell, and the substance can activate the non-specific "bystander effect" of A on DNA after recognizing the specific sequence RNA in the above target cell; "i" is a substance that can inhibit the repair of DNA damage, so that the damaged DNA cannot be repaired; "i" can be a compound, or it can be siRNA, or it can be an antibody, and its characteristic is that it can inhibit the repair of DNA damage.

3. A composition for the purpose of specifically killing target cells, characterized in that: The composition comprises: two components A and B, or a complex of A and B combined together; A is a nuclease that can be activated by RNA of a specific sequence through B to produce a "bystander effect"; the "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts; B is a substance that can recognize specific sequence RNA in the target cell but not recognize nucleic acids in cells surrounding or nearby the target cell, and the substance can activate the non-specific "parasitic effect" of A on DNA after recognizing the specific sequence RNA in the above target cell.

4. A composition for the purpose of specifically killing target cells, characterized in that: The composition comprises: three components A, B and i, or "a complex of A and B combined together" + "i"; A is a nuclease that can be activated by RNA of a specific sequence through B to produce a "bystander effect"; the "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts; B is a substance that can recognize RNA with a specific sequence in the target cell but not nucleic acids in cells surrounding or near the target cell, and the substance can activate the non-specific "parasitic effect" of A on DNA after recognizing the RNA with a specific sequence in the target cell; "i" is a substance that can inhibit the repair of DNA damage, so that the damaged DNA cannot be repaired; "i" can be a compound, or it can be siRNA, or it can be an antibody, and its characteristic is that it can inhibit the repair of DNA damage.

5. A composition for the purpose of specifically killing target cells, characterized in that: The composition comprises: two components A and B, or a complex of A and B combined together; A is a nuclease that can be activated by DNA with a specific sequence through B to produce a "bystander effect"; the "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts; B is a substance that can recognize specific sequence DNA in target cells but not nucleic acids in cells surrounding or nearby the target cells, and the substance can activate the non-specific "parasitic effect" of A on DNA after recognizing the specific sequence DNA in the above target cells.

6. A composition for the purpose of specifically killing target cells, characterized in that: The composition comprises: three components A, B and i, or "a complex of A and B combined together" + "i"; A is a nuclease that can be activated by DNA with a specific sequence through B to produce a "bystander effect"; the "bystander effect" is an effect of non-specific cutting of the DNA that the enzyme contacts; B is a substance that can recognize a specific sequence of DNA in the target cell but not recognize nucleic acids in cells surrounding or near the target cell, and the substance can activate the non-specific "parasitic effect" of A on DNA after recognizing the specific sequence of DNA in the target cell; "i" is a substance that can inhibit the repair of DNA damage, so that the damaged DNA cannot be repaired; "i" can be a compound, or it can be siRNA, or it can be an antibody, and its characteristic is that it can inhibit the repair of DNA damage.

7. A composition that specifically kills target cells by relying on the DNA side cutting effect of Cas12a2, characterized in that the composition comprises: two components A and B, or a complex in which A and B are combined together; A is the gene editing protein Cas12a2; Cas12a2 has a nuclease that can be activated by specific sequence nucleic acid through B to produce a "side-cutting effect" on DNA; B is a crRNA that can recognize a specific sequence nucleic acid in a target cell but not a substance that recognizes nucleic acids in cells around or near the target cell, and the crRNA can activate the non-specific "bypass effect" of Cas12a2 on DNA after recognizing the specific sequence nucleic acid in the above-mentioned target cell.

8. A composition that relies on Ago protein with DNA cleavage effect to specifically kill target cells, characterized in that: The composition comprises: two components A and B, or a complex of A and B combined together; A is the Ago protein with DNA cleavage effect; Ago proteins with DNA side-cutting effects have nucleases that can be activated by specific sequence nucleic acids through B to produce "side-cutting effects" on non-specific DNA; B is a gRNA or gDNA that can recognize a specific sequence nucleic acid in a target cell but not recognize nucleic acids in cells surrounding or near the target cell, and the gRNA or gDNA can activate the "bypass effect" of the Ago protein with a DNA bypass effect after recognizing the specific sequence nucleic acid in the above-mentioned target cell, and perform non-specific cutting on the encountered DNA.

9. Use of the composition as described in any one of claims 1 to 8 in killing target cells through the para-segmentation effect.

10. A method for using the composition according to any one of claims 1 to 8 to kill target cells, characterized in that: The following steps are involved: S1. Determine at least one DNA or RNA sequence that is unique to the target cell and different from that in normal cells; S2. Introduce the above-mentioned composition that can recognize a specific DNA or RNA sequence in the target cell into the target cell.

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