RNA‑responsive, controllable pyroptosis system based on type iii-e crispr framework, and use thereof

Abstract

Disclosed in the present invention are an RNA‑responsive, controllable pyroptosis system based on a type III-E CRISPR framework, and a use thereof. The RNA-responsive, controllable pyroptosis system comprises an endonuclease Cas7-11, a protease Csx29, an effector protein, and a crRNA; the crRNA is used for specifically recognizing a target RNA; the effector protein comprises a GSDMs-N protein, a linker protein, and a GSDMs-C protein; the linker protein is cleaved by the protease Csx29; the linker protein is a Csx30 protein having the amino acid sequence shown in SEQ ID NO: 3, or a truncated protein obtained by truncating the Csx30 protein.

Description

An RNA-responsive, controllable pyroptosis system based on the III-E CRISPR framework and its applications Technical Field

[0001] This invention relates to the field of biotechnology, and in particular to an RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework and its applications. Background Technology

[0002] Prokaryotes possess a variety of defense systems against foreign genetic elements, including the CRISPR and CRISPR-associated protein (Cas) systems. While the primary function of the CRISPR-Cas system is to provide adaptive immunity through RNA-guided DNA or RNA nuclease activity, other proteins genetically associated with CRISPR loci have been identified, such as the type III CRISPR-associated protease (CASP) system. Studies have revealed complex interactions among a series of proteins at the III-E CRISPR system (Cas7-11) locus. Cas7-11 can recognize target RNA complementary to crRNA and activate the activity of a protease, Csx29 (also known as TPR-CHAT), a member of the CHAT family containing a tetrapeptide repeat sequence. Activated Csx29 specifically cleaves a protein called Csx30 at the same locus, with the cleavage site between Csx30 427 and 429, thereby facilitating signal transduction from RNA to the protease.

[0003] Pyroptosis is the lytic death of cells caused by pathogen infection or endogenous danger signals. This process is an important innate immune response with a "hot" immunological characteristic. Pyroptosis triggers a strong inflammatory response by releasing inflammatory cytokines and danger signals, simultaneously activating the immune system and ultimately clearing 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 members of the GSDM family share a similar dual-domain feature: the N-terminal domain (NTD) can insert into the cell membrane, forming oligomers and creating pores, leading to membrane lysis and the release of cell contents, thereby initiating pyroptosis; the C-terminal domain (CTD) inhibits the pro-pyroptotic activity of the NTD. As an inflammatory response, pyroptosis significantly stimulates the body's immune system by releasing inflammatory cytokines and danger signals. Therefore, pyroptosis shows great potential in the field of cancer (tumor) treatment. However, pyroptosis involves complex signaling pathways, making it extremely difficult to actively induce and controllably regulate it for research and clinical treatment. Summary of the Invention

[0004] This invention provides an RNA-responsive, controllable pyroptosis system based on the III-E CRISPR framework and its application. This system can be used as a therapeutic strategy for the treatment of all RNA-differential diseases. Specifically, it is achieved through the following techniques.

[0005] This invention provides an RNA-responsive controlled pyroptosis system based on a type III-E CRISPR framework, comprising the nuclease Cas7-11 or a gene fragment expressing the nuclease 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 includes GSDMs-N protein, a linker protein, and GSDMs-C protein;

[0006] 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.

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

[0008] Furthermore, the truncated protein is obtained by truncating amino acids from position 250 to 565 of the amino acid sequence of the Csx30 protein;

[0009] Alternatively, it can be obtained by truncating amino acids from positions 397 to 565 of the amino acid sequence of the Csx30 protein;

[0010] Alternatively, it can be obtained by truncating amino acids from positions 407 to 565 of the amino acid sequence of the Csx30 protein;

[0011] Alternatively, it can be obtained by truncating amino acids from positions 412 to 565 of the amino acid sequence of the Csx30 protein;

[0012] Alternatively, it can be obtained by truncating amino acids from positions 417 to 565 of the amino acid sequence of the Csx30 protein;

[0013] Alternatively, it can be obtained by truncating amino acids from positions 407 to 560 of the amino acid sequence of the Csx30 protein;

[0014] Alternatively, it can be obtained by truncating amino acids from positions 417 to 560 of the amino acid sequence of the Csx30 protein.

[0015] Furthermore, the above system is a recombinant vector obtained by recombining the sequences expressing the nuclease Cas7-11, the protease Csx29, and the effector protein on the same plasmid vector.

[0016] Furthermore, the target RNA includes viral RNA, RNA from gene-mutated diseased cells, or p16 RNA from senescent cells. INK4a / p21 CIP1 mRNA.

[0017] Furthermore, the virus includes respiratory syncytial virus, severe acute respiratory syndrome coronavirus type 2, human papillomavirus, human immunodeficiency virus, or hepatitis B virus; the mutant lesion cells include lesion cells caused by gene mutations in the genome sequence.

[0018] It is readily apparent 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.

[0019] Optionally, the genetic mutation condition includes genetic mutation cancer / tumor cells.

[0020] Further, optionally, the genetically mutated cancer / tumor cells include cancer / tumor cells with KRAS gene mutations.

[0021] Further optionally, 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.

[0022] The CRISPR type III-E system contains multiple proteins, including Cas7-11, Csx29, and Csx30. This invention first cloned tagged expression plasmids, including Cas7-11-HA, Csx29-Myc, and Csx30-Flag, from the CRISPR type III-E system of *D. sisimotonii*. Nuclease-inactive Cas7-11 (dCas7-11, D429A / D654A) and protease-inactive Csx29 (dCsx29, C658A) were constructed, and a crRNA complementary to the target RNA was designed. Through co-immunoprecipitation (CO-IP) experiments, this invention revealed that Cas7-11, Csx29, and Csx30 interact, and these interactions occur in pairs. This suggests a potentially close functional relationship among these three proteins.

[0023] Subsequently, the present invention used the Alphafold2 software to predict the structure of Csx30. The results showed that Csx30 mainly consists of an N-terminal domain (amino acid sites 1 to 377) and a C-terminal domain (amino acid sites 419 to 565), which are connected by a long flexible domain (amino acid sites 378 to 418). Based on this structural feature, the present invention further designed an "X-Csx30-Y" fusion protein, in which Csx30 acts as a linker. Western blotting (WB) experiments showed that Cas7-11 can recognize target RNA complementary to crRNA, thus causing a conformational change and activating the protease activity of Csx29. The activated Csx29 can specifically cleave Csx30. Although dCas7-11 can also activate the protease activity of Csx29, dCas29 lacks protease activity and therefore cannot cleave Csx30. These results indicate that the formation of the ternary complex of Cas7-11, crRNA, and target RNA, as well as the protease activity of Csx29, are necessary conditions for Csx30 cleavage.

[0024] The full-length Csx30 contains 565 amino acids, which is too long as a linker. Therefore, this invention also conducted a series of truncation experiments on the full-length Csx30. It was finally found that when Csx30 was truncated to Csx30 (amino acid sites 417 to 560), 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 variations, 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 is a schematic diagram of the CASP sites of type III-E in Desulfonema ishimotonii strain. Nuclease-inactivated dCas7-11 (D429A / D654A); protease-inactivated dCsx29 (C658A).

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

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

[0049] Figure 4 shows the Cas7-11-Csx29 complex responding to target RNA cleavage of X-Csx30-Y. Each group was transfected with equal amounts of X-Csx30-Y and crRNA.

[0050] Figure 5 shows the proteolytic activity analysis of the Cas7-11-Csx29 complex on a series of truncated X-Csx30-Y.

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

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

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

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

[0055] Figure 10 shows the pyroptosis activity of the DAMAGE system characterized by immunoblotting.

[0056] Figure 11 shows the specificity of the pyroptosis activity of the DAMAGE system characterized by fluorescence microscopy.

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

[0058] Figure 13 shows the ability of the DAMAGE-RSV system to induce pyroptosis in target cells under RSV-N plasmid transfection and RSV virus infection, respectively, by immunoblotting experiments.

[0059] Figure 14 shows the concentration gradient analysis of RSV-N plasmid in the DAMAGE-RSV system.

[0060] Figure 15 shows the RSV virus infection concentration gradient analysis of the DAMAGE-RSV system. Virus concentration is expressed as the multiplicity of infection (MOI).

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

[0062] Figure 17 shows the ability of DAMAGE-HPV to recognize HPV16 / 18-E6 / E7 by immunoblotting.

[0063] Figure 18 shows the ability of DAMAGE-HPV to recognize HPV18-E6 / E7 mRNA transcribed from the HeLa cell line genome, analyzed by immunoblotting.

[0064] Figure 19 shows the specificity of the pyroptosis activity of the DAMAGE-HPV system characterized by fluorescence microscopy.

[0065] Figure 20 shows an ATP-based cell viability assay performed using the DAMAGE-HPV system under time gradient conditions.

[0066] Figure 21 shows the homology comparison of the KRAS-G12 mutation.

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

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

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

[0070] Figure 25 shows the experimental analysis results of LDH release assay for all KRAS-G12C crRNAs.

[0071] Figure 26 shows the flow cytometry analysis results of KRAS-G12C crRNA-23.

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

[0073] Figure 28 shows the LDH release assay and pyroptosis cell statistical analysis. The experimental treatment was the same as in Figure 27.

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

[0075] Figure 30 shows the screening of crRNAs that target and recognize p16 / p21 mRNA. Analysis was performed using Western blotting.

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

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

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

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

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

[0081] Figure 36 shows that MS37452 treatment inhibited endogenous activation of DAMAGE-Aging.

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

[0083] Figure 38 shows the pyroptosis-promoting activity of DAMAGE-Plus analyzed by immunoblotting.

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

[0085] Figure 40 shows the pyroptosis-promoting activity of all target RNAs using the DAMAGE-Plus system via immunoblotting.

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

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

[0088] Figure 43 shows the LDH release assay performed on 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 (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) lacking nuclease activity and Csx29 (dCsx29, C658A) lacking protease activity. The results are shown in Figure 1.

[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] In this embodiment, AlphaFold2 was used 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), which are connected by a long flexible region (amino acids 378-418), as shown in Figure 2.

[0094] Based on these findings, this embodiment designed a fusion protein X-Csx30-Y, in which Csx30 serves as the linker protein, as shown in Figure 3. When Cas7-11 recognizes a target RNA complementary to the crRNA, it activates the proteolytic activity of Csx29, leading to the specific cleavage of Csx30 in X-Csx30-Y. 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 Figure 4.

[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 study show that the Csx30 fragment with only amino acids 417 to 560 retained can still be cleaved by activated Csx29, as shown in Figure 5.

[0098] Therefore, this embodiment developed an RNA-responsive controlled pyroptosis system (Death Manipulation Gene, DAMAGE) based on the III-E CRISPR framework, as shown in Figures 6 and 7. Simultaneously, this embodiment also constructed a crRNA-Mix capable of simultaneously transcribing five crRNAs, as shown in Figure 8. The core of DAMAGE is the design of the GSDMs-Csx30 effector protein, where Csx30 acts as a linker 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, when the target RNA (tgRNA) is present, DAMAGE recognizes the target RNA through 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 (shown in SEQ ID NO.3) as linker proteins, designing a total of 12 GSDMs-Csx30 effector proteins, as illustrated in Figure 9.

[0101] The specific truncation method is as follows: (1) GSDMs-N protein of GSDMA-Csx30-FL is shown as SEQ ID NO.5, and GSDMs-C protein is shown as 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 with tagged antibodies, as shown in Figure 10.

[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, as shown in Figure 11. This observation indicates that DAMAGE has a strong targeted cell-killing ability.

[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 in Figure 12. Immunoblotting experiments showed 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 shown in Figure 13.

[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 shown in Figures 14 and 15.

[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 mainly 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 developed DAMAGE-HPV, which specifically targets HPV mRNA. A schematic diagram is shown in Figure 16.

[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 shown in Figure 17.

[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. Immunoblotting experiments demonstrated that HPV18-E6 / E7 mRNA transcribed from the HeLa genome was sufficient to activate DAMAGE-HPV and induce pyroptosis in HeLa cells. The results are shown in Figure 18.

[0125] In this embodiment, EGFP was transfected into HeLa cells as a fluorescent marker, and DAMAGE-HPV was co-transfected simultaneously. Cellular morphological changes were observed. Compared to 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 EGFP green fluorescence. The results are shown in Figure 19.

[0126] In this study, 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. The results are shown in Figure 20.

[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 those of KRAS-WT. A schematic diagram is shown in Figure 21.

[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 target cells. The results are shown in Figure 22.

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

[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 nucleotide mutations 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 shown in Figures 24, 25, and 26.

[0135] To investigate whether endogenous KRAS mutations play the same role, two stable 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 shown in Figures 27 and 28.

[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. A schematic diagram is shown in Figure 29.

[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 a decrease in the expression levels of Cas7-11 and Csx29 proteins, indicating pyroptosis. The results are shown in Figure 30.

[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 exhibited significant pyroptosis compared to the OFF- group, specifically manifested as increased PI positivity and LDH release rates. The results are shown in Figures 31 and 32.

[0142] This embodiment found that endogenous p16 in cells is sufficient to activate DAMAGE and induce strong pyroptosis. The results are shown in Figure 33.

[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 showed only mild cell death, similar to the OFF- group. The results are shown in Figures 34 and 35.

[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 shown in Figure 36.

[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. A schematic diagram is shown in Figure 37.

[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 activity when not triggered. The results are shown in Figures 38 and 39.

[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. The results are shown in Figure 40.

[0151] In this embodiment, 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. The results are shown in Figure 41.

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

[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 includes GSDMs-N protein, a linker protein, and GSDMs-C protein; 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 the Csx30 protein with the amino acid sequence shown in SEQ ID NO.3; or, it is a truncated protein obtained by shortening the Csx30 protein.

2. The RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 1, characterized in that, The truncated protein is obtained by truncating amino acids from position 250 to 565 of the amino acid sequence of the Csx30 protein. Alternatively, it can be obtained by truncating amino acids from positions 397 to 565 of the amino acid sequence of the Csx30 protein; Alternatively, it can be obtained by truncating amino acids from positions 407 to 565 of the amino acid sequence of the Csx30 protein; Alternatively, it can be obtained by truncating amino acids from positions 412 to 565 of the amino acid sequence of the Csx30 protein; Alternatively, it can be obtained by truncating amino acids from positions 417 to 565 of the amino acid sequence of the Csx30 protein; Alternatively, it can be obtained by truncating amino acids from positions 407 to 560 of the amino acid sequence of the Csx30 protein; Alternatively, it can be obtained by truncating amino acids from positions 417 to 560 of the amino acid sequence of the Csx30 protein.

3. 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.

4. The RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 1, characterized in that, The target RNA includes viral RNA, RNA from genetically modified diseased cells, or p16 RNA from senescent cells. INK4a / p21 CIP1 mRNA.

5. The RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 4, 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.

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

7. The RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 6, 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.

8. A biomaterial, characterized in that, The biological material is mRNA or an effector protein; the mRNA is transcribed from the DNA fragment corresponding to the RNA-responsive controlled pyroptosis system based on the III-E CRISPR framework as described in any one of claims 1-7, and the effector protein is the effector protein in the RNA-responsive controlled pyroptosis system based on the III-E CRISPR framework as described in any one of claims 1-7.

9. The application of an RNA-responsive controllable pyroptosis system based on a type III-E CRISPR framework as described in any one of claims 1-7, or the biomaterial described in claim 8, characterized in that, It is used to prepare preparations for treating viral infectious diseases, gene-mutated diseased cells, or RNA-differential diseases.

10. The application of the RNA-responsive controllable pyroptosis system based on the III-E CRISPR framework according to claim 9, characterized in that, 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 disease cells are disease cells caused by gene mutations in the genome sequence; the RNA differential diseases are diseases caused by differences in RNA transcriptomics due to aging, heredity, and radiation.

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