RNA-responsive controllable pyroptosis system based on type iii-e crisper framework and application thereof

Abstract

The application discloses an RNA-responsive controllable cell pyroptosis system based on a III-E type CRISPR framework and application thereof, and relates to the technical field of biology. The RNA-responsive controllable cell pyroptosis system comprises an endonuclease Cas7-11, a protease Csx29, an effector protein and crRNA; the crRNA is used for specifically recognizing target RNA; the effector protein comprises GSDMs-N protein, a linker protein and GSDMs-C protein; the linker protein is cut by the protease Csx29; the linker protein is cut by the protease Csx29; the linker protein is a Csx30 protein with an amino acid sequence as shown in SEQ ID NO. 3; or is a truncated protein obtained by truncating the Csx30 protein. The application discloses a system capable of specifically responding to target RNA and inducing target cell pyroptosis, and corresponding mRNA and effector protein, and can be widely applied to different RNA differential disease treatments. The application lays a foundation for the treatment of RNA differential diseases and provides an important technical basis for promoting the application of the DAMAGE system in the clinic.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of biotechnology, in particular to an RNA-responsive controllable cell pyroptosis system based on a type III-E CRISPR framework and its application. BACKGROUND

[0002] Prokaryotes have a variety of defense systems against foreign genetic elements, including CRISPR and CRISPR-associated protein (Cas) systems. Although the main function of the CRISPR-Cas system is to provide adaptive immunity through RNA-guided DNA or RNA nuclease activity, other proteins with genetic association with CRISPR sites have been found, such as the type III CRISPR-associated proteinase (CASP) system. Previous studies have found that a series of proteins in the III-E CRISPR system (Cas7-11) locus have complex interactions. Cas7-11 can recognize target RNA complementary to crRNA and activate a protease activity, which is a member of the CHAT family containing a tetrapeptide repeat sequence, Csx29 (also known as TPR-CHAT), and the activated Csx29 specifically cleaves a protein called Csx30 in the same locus, with a cleavage site between Csx30 427-429, thereby achieving signal transmission from RNA to protease.

[0003] Pyroptosis refers to a cell lysis death caused by pathogen infection or endogenous danger signals. This process is an important natural immune response of the body, with the immunological characteristics of "heat". Pyroptosis triggers a strong inflammatory response by releasing inflammatory cytokines and danger signals, while activating the immune system, and ultimately eliminating harmful cells. The execution of pyroptosis is mediated by the gasdermin (GSDM) family of proteins, including GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB59. Except for DFNB59, all GSDM family members have similar double-domain features: the amino-terminal domain (NTD) can be inserted into the cell membrane to form oligomers and create pores, leading to membrane lysis and release of cell contents, thereby triggering pyroptosis; the carboxy-terminal domain (CTD) has an inhibitory effect on the pro-pyroptotic activity of NTD. As an inflammatory response, pyroptosis significantly stimulates the body's immune system by releasing inflammatory cytokines and danger signals. Therefore, pyroptosis has shown great potential in the field of cancer (tumor) treatment. However, pyroptosis involves complex signaling pathways, which makes it very difficult to actively induce and controllably regulate it for research and clinical treatment. SUMMARY

[0004] The application provides an RNA-responsive controllable cell pyroptosis system based on a type III-E CRISPR framework and application thereof, which can be used as a treatment strategy for the treatment of all RNA differential diseases.

[0005] The application provides an RNA-responsive controllable cell pyroptosis system based on a type III-E CRISPR framework, which comprises endonuclease Cas7-11 or a gene fragment expressing the endonuclease Cas7-11, protease Csx29 or a gene fragment expressing the protease Csx29, effector protein or a gene fragment expressing the effector protein, and crRNA; the crRNA is used for specific recognition of target RNA; and the effector protein comprises GSDMs-N protein, linker protein and GSDMs-C protein.

[0006] The amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 5 and SEQ ID NO. 6; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 7 and SEQ ID NO. 8; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 9 and SEQ ID NO. 10; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 11 and SEQ ID NO. 12; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 13 and SEQ ID NO. 14; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 15 and SEQ ID NO. 16; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 17 and SEQ ID NO. 18; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 19 and SEQ ID NO. 20; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 21 and SEQ ID NO. 22; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 23 and SEQ ID NO. 24; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 25 and SEQ ID NO. 26; or, the amino acid sequences of the GSDMs-N protein and the GSDMs-C protein are respectively shown as SEQ ID NO. 27 and SEQ ID NO. 28.

[0007] The linker protein is cleaved by the protease Csx29; the linker protein is a Csx30 protein with an amino acid sequence shown as SEQ ID NO. 3; or, is a truncated protein obtained by truncating the Csx30 protein.

[0008] Further, the truncated protein is obtained by truncating the amino acids at positions 250-565 of the amino acid sequence of the Csx30 protein;

[0009] or, is obtained by truncating the amino acids at positions 397-565 of the amino acid sequence of the Csx30 protein;

[0010] or, is obtained by truncating the amino acids at positions 407-565 of the amino acid sequence of the Csx30 protein;

[0011] or, is obtained by truncating the amino acids at positions 412-565 of the amino acid sequence of the Csx30 protein;

[0012] or, is obtained by truncating the amino acids at positions 417-565 of the amino acid sequence of the Csx30 protein;

[0013] or, is obtained by truncating the amino acids at positions 407-560 of the amino acid sequence of the Csx30 protein;

[0014] or, is obtained by truncating the amino acids at positions 417-560 of the amino acid sequence of the Csx30 protein.

[0015] Further, the above-mentioned system is a recombinant vector obtained by recombining the sequences of the endonuclease Cas7-11, the protease Csx29, and the effector protein on the same plasmid vector.

[0016] Further, the target RNA includes RNA of viral origin, or RNA of a genetically mutated pathogenic cell, or p16 INK4a / p21 CIP1 mRNA of a senescent cell.

[0017] Still further, the virus includes respiratory syncytial virus, severe acute respiratory syndrome coronavirus 2, human papillomavirus, human immunodeficiency virus, or hepatitis B virus; and the genetically mutated pathogenic cell includes a pathogenic cell caused by a genetic mutation in the genomic sequence.

[0018] It is readily known that all cell pathogenesis caused by a genetic mutation in the genomic sequence, and further triggered diseases or conditions, including but not limited to cancer / tumor, can specifically trigger pyroptosis of the pathogenic cell by using the DAMAGE system of the present application.

[0019] Optionally, the genetically mutated pathogenic condition includes a genetically mutated cancer / tumor cell.

[0020] Further optionally, the genetically mutated cancer / tumor cell includes a KRAS gene-mutated cancer / tumor cell.

[0021] Further optionally, the type of single base mutation in the KRAS gene is that glycine at position 12 of the expressed protein of the KRAS gene is mutated into cysteine, aspartic acid, arginine, alanine, valine, or serine.

[0022] The locus of type III-E CRISPR system contains multiple proteins, including Cas7-11, Csx29 and Csx30, etc. The present application first cloned a tagged expression plasmid from the type III-E CRISPR system of D. sisimotonii, including Cas7-11-HA, Csx29-Myc and Csx30-Flag, and constructed Cas7-11 (dCas7-11, D429A / D654A) losing nuclease activity and Csx29 (dCsx29, C658A) losing protease activity, and designed crRNA complementary to the target RNA. Through the co-immunoprecipitation (CO-IP) experiment, the present application found that Cas7-11, Csx29 and Csx30 interacted with each other, and the interaction between them occurred in pairs. This indicates that the three proteins may have close functional association.

[0023] Subsequently, the present application used software Alphafold2 to predict the structure of Csx30, and the results showed that Csx30 was mainly composed of an N-terminal domain (1st to 377th amino acid site) and a C-terminal domain (419th to 565th amino acid site), and the two domains were connected by a long flexible domain (378th to 418th amino acid site). Based on this structural feature, the present application further designed a fusion protein of "X-Csx30-Y", in which Csx30 acts as a linker. Through Western Blot (WB) experiment, the results showed that Cas7-11 could recognize the target RNA complementary to crRNA, and thus cause the change of its conformation, thereby activating the protease activity of Csx29. The activated Csx29 could specifically cleave Csx30. Although dCas7-11 could also activate the protease activity of Csx29, but since dCas29 lacks protease activity, it cannot further cleave Csx30. These results show that the formation of ternary complex of Cas7-11, crRNA and target RNA, and the protease activity of Csx29, are necessary conditions for the cleavage of Csx30.

[0024] Full-length Csx30 contains 565 amino acids, which is too long as a linker. Therefore, the present application also carried out a series of truncation experiments on full-length Csx30. Finally, it was found that when Csx30 was truncated to Csx30 (417th to 560th amino acid site), it could still be cleaved by Csx29.

[0025] Based on the above information, this invention ultimately designed a system that combines the structural features of GSDM family proteins and III-E CRISPR, capable of recognizing and responding to target RNA, thereby specifically inducing pyroptosis in target cells. This system is called "Death Manipulation Gene (DAMAGE)," hereinafter referred to as the "DAMAGE system."

[0026] The core technology of the DAMAGE system lies in the design of the GSDMs-Csx30 effector protein, which uses Csx30 as a linker to connect GSDMs-N and GSDMs-C. This fusion protein retains the inhibitory effect of GSDMs-C on GSDMs-N, while simultaneously responding to target RNA by cleaving Csx30, thereby releasing the N-terminus of GSDMs and promoting pyroptosis. Therefore, the DAMAGE system recognizes target RNA through Cas7-11, activates the protease activity of Csx29, cleaves the GSDMs-Csx30 effector protein, and ultimately induces pyroptosis.

[0027] Based on the above-described mechanism of action, the DAMAGE system provided by this invention can precisely target and eliminate infected cells (such as RSV, HPV16, and HPV18), diseased cells with genetic mutations (such as cancer cells with gene mutations such as KRAS-G12C), and senescent cells with altered transcriptomes (such as p16 and p21). Therefore, all RNA-differential diseased cells, including but not limited to virus-infected cells, gene-mutated diseased cells, and senescent cells with altered transcriptomes, can be specifically eliminated by the DAMAGE system.

[0028] Therefore, based on different target RNAs, the DAMAGE system can specifically differentiate into DAMAGE-RSV, DAMAGE-HPV, DAMAGE-KRAS, and DAMAGE-Aging. Furthermore, this invention integrates the DAMAGE system into a single plasmid expression vector, named DAMAGE-Plus.

[0029] In summary, we have developed a DAMAGE system based on type III-E CRISPR technology that actively triggers pyroptosis in response to target RNA. This system can specifically kill virus-infected cells, diseased cells with single-base or multi-base mutations, gene insertions, gene deletions, and chromosomal aberrations, as well as senescent cells with transcriptome alterations. These results demonstrate a controllable pyroptosis mechanism based on CRISPR technology, laying the foundation for the treatment of RNA-differential diseases.

[0030] The present invention also provides a biomaterial, wherein the biomaterial is mRNA or an effector protein; the mRNA is transcribed from a DNA fragment corresponding to any of the above-mentioned RNA-responsive controllable pyroptosis systems based on the III-E CRISPR framework, and the effector protein is the effector protein in any of the above-mentioned systems.

[0031] Furthermore, the DAMAGE system provided by this invention can also transcribe the mRNA into corresponding mRNAs and deliver them effectively into the body through different forms and pathways to achieve the treatment of corresponding diseases. Similarly, effector proteins can also be delivered into the body in a similar manner to achieve the treatment of corresponding diseases.

[0032] The “different forms and pathways” claimed in this invention refer to commonly used forms and pathways that can effectively deliver mRNA into the body in current biotechnology or clinical practice.

[0033] Alternatively, mRNA can be delivered into the body as an LNP-mRNA drug via lipid nanoparticles (LNPs) for treatment. Alternatively, it can be delivered into the body via adenovirus vectors or recombinant viral vectors for treatment.

[0034] The present invention also provides an effector protein, which is the effector protein in any of the above-mentioned RNA-responsive controlled pyroptosis systems based on the III-E CRISPR framework.

[0035] The present invention also provides an application of any of the above-mentioned RNA-responsive controllable pyroptosis systems based on the III-E CRISPR framework, or the above-mentioned mRNA, or the above-mentioned effector proteins, specifically for the preparation of formulations for treating viral infectious diseases, gene-mutant diseased cells, or RNA-differential diseases.

[0036] Furthermore, the viral infectious diseases include respiratory syncytial virus, severe acute respiratory syndrome coronavirus type 2, human papillomavirus type 16, human papillomavirus type 18, human immunodeficiency virus, and hepatitis B virus; the gene-mutant lesion cells are cancers / tumors caused by gene mutations in the genome sequence; the RNA differential diseases are lesions caused by differences in epigenetic RNA transcriptomics due to factors such as aging, genetics, and radiation.

[0037] In this invention, the endonuclease Cas7-11, the protease Csx29, and the protein Csx30 can be synthesized using methods recognized in the art based on the sequence listing information provided by this invention, or can be artificially synthesized by a third party, or commercially available products can be purchased directly.

[0038] The “RNA differential disease” claimed in this invention refers to diseases that produce differences at the RNA level, including but not limited to differences in RNA structure caused by gene mutations and chromosomal aberrations, as well as differences in RNA abundance caused by complex factors such as aging, heredity and radiation.

[0039] For example, diseases caused by viral infections, or cancer / tumor cells caused by gene mutations, or aging / pathological cells, tissues and organs caused by changes in the RNA transcriptome.

[0040] The "gene mutation" claimed in this invention generally refers to changes in gene phenotype caused by various genetic factors such as mutations in the genome sequence, such as gene insertion, gene substitution or gene deletion, and chromosomal aberrations.

[0041] In terms of therapeutic applications, the RNA-responsive controlled pyroptosis system based on the CRISPR type III-E framework provided by this invention can specifically kill virus-infected cells (including all infectious viruses such as human papillomavirus, HIV, and hepatitis B virus), gene-mutated diseased cells (including but not limited to all gene-mutated cancers such as KRAS-G12C), and senescent cells with altered transcriptomes. Therefore, all RNA-differential diseases can be treated using the system of this invention.

[0042] In terms of therapeutic applications, the DAMAGE system provided by this invention can also be transcribed into corresponding mRNA and effectively delivered into the body as an mRNA drug through lipid nanoparticles (LNPs) and other forms and pathways, thereby achieving the treatment of corresponding diseases.

[0043] In broader applications, the DAMAGE system serves as an effective research tool capable of inducing pyroptosis at specific times and in specific tissue sites. By controlling RNA transcription or protein expression in any of its five components (e.g., the tetraribosome manipulator system), pyroptosis of target cells can be triggered at specific time points or in specific cells. DAMAGE's ability to target and eliminate cells expressing target RNA makes it ideal for treating persistent infections such as human papillomavirus (HPV), human immunodeficiency virus (HIV), and hepatitis B virus (HBV). DAMAGE technology enables precise identification at the single-base level. Therefore, all cancers caused by gene mutations can be effectively treated using the DAMAGE system, including cancers caused by a variety of factors such as single-base mutations, multi-base mutations, gene insertions, gene deletions, and chromosomal aberrations. Furthermore, the pyroptosis-promoting activity of the DAMAGE system is activated to varying degrees depending on the level of target RNA. Therefore, DAMAGE can selectively kill diseased cells and tissues with significant transcriptomic alterations, such as senescent cells, while having minimal impact on normal cells and tissues.

[0044] Compared with existing technologies, the advantages of this invention are: This invention discloses for the first time a programmable synthetic biology system capable of specifically responding to target RNA and inducing pyroptosis in target cells, while simultaneously providing corresponding mRNA and effector proteins. This system, the mRNA, and the effector proteins can be widely applied to the treatment of various RNA-dependent diseases, such as viral infections, genetically mutated diseased cells, and diseases with significant changes in the RNA transcriptome.

[0045] In principle, the DAMAGE system has broad application prospects and can treat all RNA differentially expressed diseases. This lays the foundation for the treatment of RNA differentially expressed diseases and provides important theoretical and technical basis for promoting the clinical application of the DAMAGE system. Attached Figure Description

[0046] Figure 1 This is a schematic diagram of the CASP site in strain III-E of *Desulfonema ishimotonii*. Nuclease-inactivated dCas7-11 (D429A / D654A); protease-inactivated dCsx29 (C658A).

[0047] Figure 2 The image shows the predicted structure of Csx30 by AlphaFold2. N represents the amino-terminal domain (NTD); C represents the carboxyl-terminal domain (CTD).

[0048] Figure 3This is a schematic diagram of the fusion protein X-Csx30-Y. X represents the molecular chaperone-mediated autophagy motif (CMA); Y represents the type VI CRISPR enzyme, RfxCas13d (CasRx).

[0049] Figure 4 The Cas7-11-Csx29 complex responds to target RNA cleavage of X-Csx30-Y. Equal amounts of X-Csx30-Y and crRNA were transfected into each group.

[0050] Figure 5 Proteolytic activity analysis of the Cas7-11-Csx29 complex against a series of truncated X-Csx30-Y segments.

[0051] Figure 6 This is a schematic diagram of the DAMAGE system in operation.

[0052] Figure 7 This is a schematic diagram of the overall technical roadmap for the DAMAGE system.

[0053] Figure 8 This is a schematic diagram of the structure of crRNA-Mix.

[0054] Figure 9 A schematic diagram of the design of the GSDMs-Csx30 effector protein.

[0055] Figure 10 To characterize the pyroptosis-promoting activity of the DAMAGE system using immunoblotting experiments.

[0056] Figure 11 To characterize the specificity of the pyroptosis activity of the DAMAGE system using fluorescence microscopy.

[0057] Figure 12 This is a schematic diagram of the DAMAGE-RSV system.

[0058] Figure 13 To analyze the ability of the DAMAGE-RSV system to induce pyroptosis in target cells under RSV-N plasmid transfection and RSV virus infection using Western blotting experiments.

[0059] Figure 14 Analysis of RSV-N plasmid concentration gradient for the DAMAGE-RSV system.

[0060] Figure 15 This study analyzed the RSV virus infection concentration gradient in the DAMAGE-RSV system. Virus concentration was expressed as the multiplicity of infection (MOI).

[0061] Figure 16This is a schematic diagram of the DAMAGE-HPV system.

[0062] Figure 17 To analyze the ability of DAMAGE-HPV to recognize HPV16 / 18-E6 / E7 using immunoblotting experiments.

[0063] Figure 18 To analyze the ability of DAMAGE-HPV to recognize HPV18-E6 / E7 mRNA transcribed from the HeLa cell line genome using Western blotting assay.

[0064] Figure 19 To characterize the specificity of the pyroptosis activity of the DAMAGE-HPV system using fluorescence microscopy.

[0065] Figure 20 An ATP-based cell viability assay was performed on the DAMAGE-HPV system under time gradient conditions.

[0066] Figure 21 Homology comparison for KRAS-G12 mutation.

[0067] Figure 22 To conduct preliminary validation of the DAMAGE-KRAS system through immunoblotting experiments.

[0068] Figure 23 A schematic diagram of the design of KRAS-G12C crRNA.

[0069] Figure 24 PI staining combined with flow cytometry analysis results for all KRAS-G12C crRNAs.

[0070] Figure 25 The results of the LDH release assay for all KRAS-G12C crRNAs were analyzed.

[0071] Figure 26 The results are from flow cytometry analysis of KRAS-G12C crRNA-23.

[0072] Figure 27 To perform fluorescence microscopy analysis of the DAMAGE-KRAS system in stably transfected cell lines 293T-EGFP-KRAS-G12C and 293T-mCherry-KRAS-WT, and to verify its specificity in recognizing target cells.

[0073] Figure 28 For LDH release assay and pyroptosis cell statistical analysis. Experimental treatment and... Figure 27 same.

[0074] Figure 29This is a schematic diagram of the DAMAGE-Aging system.

[0075] Figure 30 For screening of crRNAs that target and recognize p16 / p21 mRNA, analysis was performed using Western blotting.

[0076] Figure 31 Analysis of the characteristics of pyroptosis induced by the DAMAGE-Aging system. PI positivity rate heatmap.

[0077] Figure 32 Analysis of the characteristics of pyroptosis induced by the DAMAGE-Aging system. LDH release rate bar chart.

[0078] Figure 33 Endogenous p16 mRNA in 293T cells can directly activate the DAMAGE-Aging system.

[0079] Figure 34 The p16-KO cell line lost its ability to endogenously activate the DAMAGE-Aging effect. Fluorescence microscopy combined with PI positivity rate determination was used.

[0080] Figure 35 To analyze the ability of the p16-KO cell line to lose the endogenous activation of the DAMAGE-Aging effect by flow cytometry.

[0081] Figure 36 MS37452 treatment suppressed endogenous activation of DAMAGE-Aging.

[0082] Figure 37 This is a schematic diagram of the DAMAGE-Plus system.

[0083] Figure 38 To analyze the pyroptosis-promoting activity of DAMAGE-Plus using immunoblotting experiments.

[0084] Figure 39 To analyze the pyroptosis-promoting activity of DAMAGE-Plus cells by flow cytometry. PI staining indicates cells undergoing pyroptosis.

[0085] Figure 40 The pyroptosis-promoting activity of all target RNAs was characterized by the DAMAGE-Plus system using immunoblotting experiments.

[0086] Figure 41 To observe the specific killing ability of the DAMAGE-Plus system on target cells in HeLa-EGFP and HeLa-mCherry stably transfected cell lines using fluorescence microscopy.

[0087] Figure 42 The DAMAGE-Plus system was analyzed by PI staining combined with flow cytometry in mRNA form.

[0088] Figure 43 An experiment was conducted to measure LDH release from the DAMAGE-Plus system in mRNA form. Detailed Implementation

[0089] The technical solution of the present invention will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.

[0090] Example 1: Construction of an RNA-responsive controlled pyroptosis system (DeathManipulation Gene, DAMAGE) based on the III-E CRISPR framework.

[0091] The CRISPR system of type III-E contains multiple proteins, including Cas7-11, Csx29, Csx30, Csx31, and CASP-σ. In this embodiment, tagged expression plasmids, specifically Cas7-11-HA, Csx29-Myc, and Csx30-Flag, were cloned from the type III-E CRISPR system of the marine anaerobic bacterium *Desulfonema ishimotonii*. The amino acid sequences of Cas7-11-HA are shown in SEQ ID NO.1, Csx29-Myc in SEQ ID NO.2, and Csx30-Flag in SEQ ID NO.3. This embodiment also constructed Cas7-11 (dCas7-11, D429A / D654A) and Csx29 (dCsx29, C658A) without nuclease activity and without protease activity, respectively. The results are as follows: Figure 1 As shown.

[0092] This embodiment also designed a crRNA complementary to the target RNA, the nucleotide sequence of which is shown in SEQ ID NO. 29-45.

[0093] This embodiment uses AlphaFold2 to predict the structure of Csx30. The predicted structure shows that Csx30 includes an NTD (amino acids 1-377) and a CTD (amino acids 419-565), connected by a long flexible region (amino acids 378-418), as shown in the figure. Figure 2 As shown.

[0094] Based on these findings, this embodiment designed a fusion protein X-Csx30-Y, where Csx30 serves as the linker protein, and the results are as follows: Figure 3 As shown in the figure, when Cas7-11 recognizes a target RNA complementary to crRNA, it activates the proteolytic activity of Csx29, leading to the specific cleavage of Csx30 in the X-Csx30-Y region. Notably, dCas7-11 retains the ability to activate Csx29 to cleave Csx30, while protease-inactivated dCsx29 loses this ability. These results indicate that the complex formed by Cas7-11, crRNA, and target RNA, along with the proteolytic activity of Csx29, is crucial for the cleavage of Csx30, as shown in the figure. Figure 4 As shown.

[0095] The results of this study confirm that DAMAGE, derived from the CRISPR type III-E system and integrated with GSDMs, has the ability to respond to target RNA and control pyroptosis. DAMAGE consists of five basic components: Cas7-11, Csx29, GSDMs-Csx30, crRNA, and target RNA. Pyroptosis can only be induced when all five components are present simultaneously.

[0096] Since the full-length Csx30 consists of 565 amino acids, it is too long as a linker protein. Therefore, this embodiment conducted a series of truncation experiments on Csx30. The specific truncation methods are as follows: (1) The truncated protein is obtained by truncating the amino acids at positions 250-565 of the Csx30 protein; (2) The truncated protein is obtained by truncating the amino acids at positions 397-565 of the Csx30 protein; (3) The truncated protein is obtained by truncating the amino acids at positions 407-565 of the Csx30 protein; (4) The truncated protein is obtained by truncating the amino acids at positions 412-565 of the Csx30 protein; (5) The truncated protein is obtained by truncating the amino acids at positions 417-565 of the Csx30 protein; (6) The truncated protein is obtained by truncating the amino acids at positions 407-560 of the Csx30 protein; (7) The truncated protein is obtained by truncating the amino acids at positions 417-560 of the Csx30 protein.

[0097] The results of this embodiment indicate that the Csx30 fragment retaining only amino acids 417 to 560 can still be cleaved by activated Csx29, as shown in the following figures. Figure 5 As shown.

[0098] Therefore, this embodiment develops an RNA-responsive controlled pyroptosis (DAMAGE) system based on the III-E CRISPR framework, as shown in the schematic diagram. Figure 6 and Figure 7 As shown in the diagram. Simultaneously, this embodiment also constructed a crRNA-Mix capable of simultaneously transcribing five crRNAs, as illustrated in the diagram. Figure 8 As shown, the core of DAMAGE is the design of the GSDMs-Csx30 effector protein, where Csx30 acts as a linker protein between GSDMs-N and GSDMs-C. Under conditions where the entire effector protein remains intact, the GSDMs-Csx30 effector protein retains the inhibitory effect of GSDMs-C on GSDMs-N, preventing GSDMs-N-induced pyroptosis. However, in the presence of target RNA (tgRNA), DAMAGE recognizes the target RNA via the Cas7-11-crRNA complex, thereby activating the proteolytic activity of Csx29. This activation subsequently cleaves the GSDMs-Csx30 effector protein, ultimately inducing pyroptosis.

[0099] Example 2: Design of GSDMs-Csx30 effector protein

[0100] To develop more GSDMs-Csx30 effector proteins, this embodiment compared the amino acid sequences of different GSDMs. The results showed that the GSDMs-N and GSDMs-C domains had significant homology, while the similarity of the linker region between these two domains was low. Therefore, this embodiment selected amino acid sequences from positions 397 to 565, or from positions 407 to 565, of the Csx30 protein's amino acid sequence (shown in SEQ ID NO.3) as linker proteins, designing a total of 12 GSDMs-Csx30 effector proteins, as illustrated in the diagram below. Figure 9 As shown.

[0101] The specific truncation methods are as follows: (1) GSDMs-N protein of GSDMA-Csx30-FL is shown in SEQ ID NO.5, and GSDMs-C protein is shown in SEQ ID NO.6;

[0102] (2) The GSDMs-N protein of GSDMA-Csx30-SL is shown in SEQ ID NO.7, and the GSDMs-C protein is shown in SEQ ID NO.8;

[0103] (3) The GSDMs-N protein of GSDMB-Csx30-FL is shown in SEQ ID NO.9, and the GSDMs-C protein is shown in SEQ ID NO.10;

[0104] (4) The GSDMs-N protein of GSDMB-Csx30-SL is shown in SEQ ID NO.11, and the GSDMs-C protein is shown in SEQ ID NO.12;

[0105] (5) The GSDMs-N protein of GSDMC-Csx30-FL is shown in SEQ ID NO.13, and the GSDMs-C protein is shown in SEQ ID NO.14;

[0106] (6) The GSDMs-N protein of GSDMC-Csx30-SL is shown in SEQ ID NO.15, and the GSDMs-C protein is shown in SEQ ID NO.16;

[0107] (7) The GSDMs-N protein of GSDMD-Csx30-FL is shown in SEQ ID NO.17, and the GSDMs-C protein is shown in SEQ ID NO.18;

[0108] (8) The GSDMs-N protein of GSDMD-Csx30-SL is shown in SEQ ID NO.19, and the GSDMs-C protein is shown in SEQ ID NO.20;

[0109] (9) The GSDMs-N protein of GSDME-Csx30-FL is shown in SEQ ID NO.21, and the GSDMs-C protein is shown in SEQ ID NO.22;

[0110] (10) The GSDMs-N protein of GSDME-Csx30-SL is shown in SEQ ID NO.23, and the GSDMs-C protein is shown in SEQ ID NO.24;

[0111] (11) The GSDMs-N protein of GSDMD-Csx30-Y is shown in SEQ ID NO.25, and the GSDMs-C protein is shown in SEQ ID NO.26;

[0112] (12) The GSDMs-N protein of GSDMD-Csx30-X is shown in SEQ ID NO.27, and the GSDMs-C protein is shown in SEQ ID NO.28.

[0113] In this embodiment, enhanced green fluorescent protein (EGFP) was used as the target RNA to characterize and validate the functions of the aforementioned GSDMs-Csx30 effector proteins. The experimental group (ON+ group) was transfected with Cas7-11-HA, Csx29-Myc, GSDMs-Csx30-Flag, EGFP-crRNA-Mix, and EGFP-Myc. In contrast, the control group (OFF- group) lacked one of the five components of DAMAGE while keeping the other components unchanged.

[0114] The results showed that, compared with the OFF- group, the GSDMs-Csx30-Flag in the ON+ group was cleaved, leading to the release of GSDMs-Csx30-N, followed by pyroptosis. Western blot experiments revealed a significant decrease in the total expression levels of Cas7-11-HA and Csx29-Myc in the ON+ group cells. This reduction was attributed to cell rupture causing the release of cell contents into the culture medium, rendering them undetectable.

[0115] This embodiment also included an immunoprecipitation (IP) assay, in which released GSDMs-Csx30-Flag and cleaved GSDMs-Csx30-C-Flag were captured from the culture supernatant using magnetic beads tagged with antibodies. The results are as follows: Figure 10 As shown.

[0116] Cell morphology imaging showed that, similar to the positive control N+ group, the ON+ group exhibited significant pyroptosis, including cell swelling, rupture, and release of contents, accompanied by increased propidium iodide staining and a significant decrease in EGFP green fluorescence. The results are as follows: Figure 11 As shown in the figure. This observation indicates that DAMAGE has a strong ability to target and kill cells.

[0117] Example 3: DAMAGE responds to RSV infection and induces pyroptosis in target cells.

[0118] When viruses invade host cells, they produce exogenous RNA that differs from the host transcriptome. In this embodiment, RSV nucleocapsid protein (RSV-N) mRNA with high transcriptional levels was selected as the target RNA for RSV detection, and DAMAGE-RSV was constructed to specifically eliminate RSV-infected diseased cells. A schematic diagram is shown below. Figure 12 As shown in the figure. Immunoblotting experiments demonstrated that DAMAGE-RSV could sense RSV-N mRNA and actively induce pyroptosis in target cells during both plasmid transfection and viral infection. The results are as follows... Figure 13 As shown.

[0119] To evaluate the sensitivity of DAMAGE-RSV, this study performed RSV-N plasmid transfection and RSV infection on all GSDMs-Csx30 effector proteins (excluding D-SL and D-FL) under serial dilution conditions. The results showed that the PI positivity rate and LDH release rate gradually decreased with increasing dilution. These phenomena demonstrate that the pyroptosis-promoting activity of DAMAGE is positively correlated with the target RNA concentration. The results are as follows... Figure 14 and Figure 15 As shown.

[0120] Therefore, this embodiment demonstrates that DAMAGE can effectively identify target RNAs from viruses or other invasive pathogens, selectively eliminating infected cells without affecting normal cells. This discovery may provide a new strategy for combating viral infections.

[0121] Example 4: DAMAGE specifically targets and kills HPV-infected cervical cancer cells.

[0122] Persistent human papillomavirus (HPV) infection is closely associated with cervical cancer. Therapeutic vaccines against HPV-induced tumors primarily target the E6 and E7 oncoproteins, which are specifically expressed in tumor cells after HPV genomic DNA integrates into the host genome. Based on this, this embodiment develops DAMAGE-HPV, which specifically targets HPV mRNA. (See schematic diagram below.) Figure 16 As shown.

[0123] This embodiment first preliminarily verified the sensing ability of DAMAGE on E6 and E7 mRNA of HPV16 and HPV18 in the 293T cell line. The results showed that pyroptosis only occurred when the target RNA was present in the cell. The results are as follows... Figure 17 As shown.

[0124] HeLa cells are one of the most widely used cell lines in scientific research, derived from cervical cancer cells infected with HPV18 and integrated into the genome. Therefore, this study used HeLa cells as a model system to investigate the specific killing effect of DAMAGE on cervical cancer. Western blotting experiments demonstrated that HPV18-E6 / E7 mRNA transcribed from the HeLa genome was sufficient to activate DAMAGE-HPV and induce pyroptosis in HeLa cells. Results are as follows... Figure 18 As shown.

[0125] In this embodiment, EGFP was transfected into HeLa cells as a fluorescent marker, and DAMAGE-HPV was co-transfected. Cellular morphological changes were observed. Compared with the OFF- group (CR-NT), all three ON+ groups (CR-E6 / CR-E7 / CR-E6+E7) significantly induced pyroptosis in HeLa cells, specifically manifested as cell swelling, rupture, and release of contents, accompanied by a significant reduction in the green fluorescence of EGFP. Results are as follows... Figure 19 As shown.

[0126] In this embodiment, the cytotoxic effect of DAMAGE-HPV on all HPV targets was evaluated in the 293T cell line using an ATP-based cell viability assay. With prolonged transfection time, the cell viability of the ON+ group significantly decreased, approaching that of the positive control. Results are as follows... Figure 20 As shown.

[0127] In summary, this embodiment demonstrates that DAMAGE can effectively recognize viral RNAs, such as HPV, HIV, and HBV, that are integrated into and transcribed into the host genome, and specifically induce pyroptosis in virus-infected target cells. This highlights the powerful potential of DAMAGE in treating persistent viral infections.

[0128] Example 5: DAMAGE recognizes single nucleotide mutations and specifically triggers pyroptosis in KRAS-mutant cancer cells.

[0129] KRAS is one of the most common oncogenes. This embodiment uses the KRAS oncogene as a representative example of a gene-mutant diseased cell. It is readily understood that all cell diseases or symptoms caused by gene mutations in the genome sequence, including but not limited to cancer / tumors, can be specifically triggered by the DAMAGE system of this invention to induce pyroptosis in diseased cells.

[0130] Several amino acid mutations were found at the 12th amino acid (glycine) of KRAS, with common mutations including cysteine ​​(G12C), aspartic acid (G12D), arginine (G12R), alanine (G12A), valine (G12S), and serine (G12V). Therefore, significant base mutations exist in the mRNA between wild-type KRAS (KRAS-WT) and the KRAS mutant (KRAS-mut).

[0131] To evaluate the ability of DAMAGE to recognize gene mutations, this embodiment first performed sequence alignment on KRAS-WT and KRAS-mut. Two crRNAs were designed for each KRAS-mut, with their spacer sequences perfectly matching those of KRAS-mut, but exhibiting single-nucleotide mismatches with KRAS-WT. (See schematic diagram below.) Figure 21 As shown.

[0132] Western blot analysis showed that the ON+ group transfected with KRAS-mut exhibited significant DX cleavage compared to the OFF- group transfected with KRAS-WT. Therefore, this embodiment developed DAMAGE-KRAS to target and kill KRAS-mutant cells. Results are as follows... Figure 22 As shown.

[0133] To further enhance the specificity of DAMAGE-KRAS, this embodiment focuses on KRAS-G12C. By sequentially altering the position of KRAS mutant bases in the crRNA spacer region, this embodiment designed a total of 24 crRNAs targeting KRAS-G12C. (See schematic diagram below.) Figure 23 As shown.

[0134] This study used flow cytometry, LDH release assay, and Western blotting to evaluate the ability of these 24 KRAS-G12C-crRNAs to recognize single nucleotide mutations. The results showed that base mutation sites in the crRNA spacer region significantly affected the pyroptosis activity of DAMAGE-KRAS. crRNA-16, crRNA-23, and crRNA-24 showed excellent performance, with crRNA-23 exhibiting the strongest recognition specificity for KRAS-G12C. The results are as follows... Figure 24 , Figure 25 and Figure 26 As shown.

[0135] To investigate whether endogenous KRAS mutations play the same role, two stably transfected cell lines were constructed in this study. One line stably expressed mCherry-KRAS-WT as a control group, and the other stably transfected with EGFP-KRAS-G12C as an experimental group. In the presence of crRNA-23, DAMAGE-KRAS significantly induced pyroptosis in the stably transfected EGFP-KRAS-G12C cell line, while this effect was not observed in the stably transfected mCherry-KRAS-WT cell line. The results are as follows: Figure 27 and Figure 28 As shown.

[0136] This embodiment demonstrates that the DAMAGE system can effectively detect single nucleotide mutations, highlighting its potential application in treating cancers with gene mutations. Therefore, this embodiment concludes that DAMAGE may be suitable for treating a variety of cancers or other cellular diseases associated with gene mutations, including single or multibase mutations, gene insertions or deletions, and chromosomal aberrations.

[0137] Example 6: DAMAGE demonstrates the potential to selectively eliminate senescent cells.

[0138] Cellular senescence is a significant factor contributing to individual aging. In senescent cells, the transcription of cyclin-dependent kinase inhibitors (CDKIs) is enhanced, such as CDKN1A (p21). CIP1 / p21) and CDKN2A (p16) INK4a Therefore, the transcription and expression of p16 / p21 are often used as biomarkers for measuring cellular senescence.

[0139] To selectively target senescent cells, this embodiment designed crRNAs targeting p16 and p21, thereby constructing the DAMAGE-Aging system. By recognizing the differences in p16 and p21 mRNA transcription levels between senescent and normal cells, DAMAGE-Aging can specifically target and eliminate senescent cells. (See schematic diagram below.) Figure 29 As shown.

[0140] Western blot analysis showed that, compared with the OFF- group transfected with CR-NT, the ON+ group transfected with crRNA targeting p16 and p21 exhibited significant DX effector protein cleavage, accompanied by decreased expression levels of Cas7-11 and Csx29 proteins, indicating pyroptosis. Results are as follows... Figure 30 As shown.

[0141] This embodiment, through PI staining combined with flow cytometry analysis and LDH release assay, showed that among all GSDMs-Csx30 effector proteins, the ON+ group showed significant pyroptosis compared to the OFF- group, specifically manifested as increased PI positivity rate and LDH release rate. Results are as follows... Figure 31 and Figure 32 As shown.

[0142] This embodiment found that endogenous p16 in cells is sufficient to activate DAMAGE and induce strong pyroptosis. Results are as follows: Figure 33 As shown.

[0143] Therefore, this embodiment constructed a p16 knockout 293T cell line (293T-p16-KO) and successfully obtained a monoclonal cell line that cannot be recognized by p16 crRNA-5 (p16-CR5). In this embodiment, the DAMAGE-Aging system was transfected into 293T wild-type (293T-WT) and 293T-p16-KO cells, co-transfected with EGFP as a fluorescent label, and p16 mRNA was detected using p16-CR5. In 293T-WT cells, the ON+ group showed significant pyroptosis, with a marked decrease in green fluorescence. In contrast, the ON+ group of the 293T-p16-KO cell line only showed mild cell death, similar to the OFF- group. The results are as follows... Figure 34 and Figure 35As shown.

[0144] Furthermore, in this embodiment, cells were treated with the small molecule drug MS37452 to reduce the transcriptional level of p16 mRNA. The results showed that MS37452 treatment reduced DAMAGE-Aging-induced pyroptosis. The results are as follows... Figure 36 As shown.

[0145] Combined with the gradient experiments shown in Example 3, this example demonstrates that DAMAGE can effectively identify differences at the mRNA level. By specifically targeting highly expressed mRNAs such as p16 and p21 in senescent cells, DAMAGE-Aging shows the potential to selectively eliminate senescent cells and provides a new approach for anti-aging therapy.

[0146] Example 7: Delivery of the DAMAGE system in the form of mRNA-LNP therapy

[0147] This embodiment attempts to transcribe DAMAGE into mRNA in vitro and deliver it using lipid nanoparticles (LNPs). Initially, DAMAGE consists of five components: Cas7-11, Csx29, GSDMs-Csx30, crRNA, and target RNA, each integrated into a separate plasmid. To simplify the DAMAGE system, this embodiment clones Cas7-11, Csx29, and GSDMs-Csx30 into a single plasmid and renames it DAMAGE-Plus. This plasmid is used to transcribe a long mRNA, which is then translated into a large fusion protein.

[0148] Subsequently, the proteins were separated into three independent proteins through the autocatalytic cleavage activities of T2A and P2A. (See diagram below.) Figure 37 As shown.

[0149] This embodiment demonstrates through a series of experiments that the pyroptosis activity of DAMAGE-Plus is consistent with that of the original DAMAGE, and even exhibits better control over pyroptosis-promoting activity when not triggered. Results are as follows: Figure 38 and Figure 39 As shown.

[0150] This embodiment tested DAMAGE-Plus's ability to recognize all previously identified target RNAs. Western blotting experiments showed that DAMAGE-Plus retained its ability to recognize target RNAs and induce pyroptosis. Results are as follows... Figure 40 As shown.

[0151] In this example, a stable 293T-EGFP / mCherry transfected cell line was cultured and transfected with DAMAGE-Plus, which recognizes EGFP mRNA. Fluorescence microscopy showed that DAMAGE-Plus specifically induced pyroptosis in the stable 293T-EGFP transfected cells, but had no effect on the stable 293T-mCherry transfected cells. Results are as follows: Figure 41 As shown.

[0152] Finally, in this embodiment, all components of DAMAGE-Plus were transcribed in vitro and then delivered as mRNA into 293T cells. PI staining combined with flow cytometry analysis and LDH release assays both demonstrated that DAMAGE-Plus can be effectively delivered as mRNA, recognize all target RNAs, and induce pyroptosis. Results are as follows... Figure 42 and 43 As shown.

[0153] This embodiment demonstrates that DAMAGE can be transcribed into mRNA in vitro and delivered in the form of mRNA-LNP. This finding indicates that DAMAGE has the potential for in vivo delivery and treatment as an mRNA therapeutic agent, highlighting its promising clinical application prospects.

[0154] The above detailed embodiments describe the implementation of the present invention; however, the present invention is not limited to the specific details described in the above embodiments. Within the scope of the claims and technical concept of the present invention, various simple modifications and changes can be made to the technical solution of the present invention, and these simple modifications all fall within the protection scope of the present invention.

Claims

1. An RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework, characterized in that, It includes the endonuclease Cas7-11 or a gene fragment expressing the endonuclease Cas7-11, the protease Csx29 or a gene fragment expressing the protease Csx29, an effector protein or a gene fragment expressing the effector protein, and crRNA; the crRNA is used to specifically recognize target RNA; the effector protein is composed of GSDMs-N protein, a linker protein, and GSDMs-C protein linked together; The target RNA includes viral RNA, RNA from genetically modified diseased cells, or p16 RNA from senescent cells. INK4a / p21 CIP1 mRNA; The amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO. 5 and SEQ ID NO. 6, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO. 7 and SEQ ID NO. 8, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO. 9 and SEQ ID NO. 10, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO. 11 and SEQ ID NO. 12, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO. 13 and SEQ ID NO. 14, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO. 15 and SEQ ID NO. 16, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO. 17 and SEQ ID NO. 18, respectively. As shown in NO.18; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO.19 and SEQ ID NO.20, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO.21 and SEQ ID NO.22, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO.23 and SEQ ID NO.24, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO.25 and SEQ ID NO.26, respectively; or, the amino acid sequences of the GSDMs-N protein and GSDMs-C protein are shown in SEQ ID NO.27 and SEQ ID NO.28, respectively. The linker protein is cleaved by the protease Csx29; the linker protein is a truncated Csx30 protein with an amino acid sequence as shown in positions 407-565 of SEQ ID NO.

3.

2. The RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 1, characterized in that, The recombinant vector is obtained by recombining the sequences expressing the nuclease Cas7-11, the protease Csx29, and the effector protein onto the same plasmid vector.

3. The RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 1, characterized in that, The viruses include respiratory syncytial virus, severe acute respiratory syndrome coronavirus type 2, human papillomavirus, human immunodeficiency virus, or hepatitis B virus; the mutant disease cells include disease cells caused by gene mutations in the genome sequence.

4. The RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 3, characterized in that, The genetically mutated disease cells include genetically mutated cancer or tumor cells.

5. The RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 4, characterized in that, The genetically mutated cancer or tumor cells include cancer or tumor cells with KRAS gene mutations.

6. The RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 5, characterized in that, The type of single-base mutation in the KRAS gene is a mutation of glycine at position 12 in the expressed protein of the KRAS gene into cysteine, aspartic acid, arginine, alanine, valine, or serine.

7. A biomaterial, characterized in that, The biological material is mRNA or an effector protein; the mRNA is obtained by transcription from the DNA fragment corresponding to the recombinant vector of claim 2, and the effector protein is the effector protein in the RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework as described in any one of claims 1-5.

8. The application of an RNA-responsive controllable pyroptosis system based on a type III-E CRISPR framework as described in any one of claims 1-6, or the biomaterial described in claim 7, characterized in that, This preparation is used to treat viral infectious diseases; the viral infectious diseases are respiratory syncytial virus, human papillomavirus type 16, human papillomavirus type 18 or hepatitis B virus.

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