A synthetic system for RNA-responsive pyroptosis based on type III-E CRISPR nuclease-protease

Domain: neuroscience
Published: 2026-02-10
Editorial outcome: major_revision

Abstract
Pyroptosis plays a crucial role in immune defense against infections and endogenous threats by eliminating harmful cells and modulating the immune response through inflammation. However, the natural activation of pyroptosis involves intricate signaling pathways, posing significant challenges for its artificial manipulation in research and therapies. Here, we present DAMAGE (Death Manipulation Gene), an innovative system that integrates gasdermins within the type III-E CRISPR framework, enabling the specific recognition of target RNA (tgRNA) and triggering pyroptosis. This mechanism allows DAMAGE to selectively identify and eliminate virus-infected, cancerous, and senescent cells, all of which exhibit altered RNA transcriptomes. Additionally, DAMAGE exhibits considerable promise as a platform for mRNA-LNP therapy. Our study highlights the potential of this CRISPR-based system in the controllable induction of pyroptosis, offering an innovative therapeutic strategy for treating RNA-heterogeneous diseases.

Introduction
Pyroptosis is a lytic cell death induced by pathogen infection or endogenous challenge, playing a pivotal role in immune defense1–3. This process is primarily executed by members of the gasdermin family proteins (GSDMs), which include GSDMA, GSDMB, GSDMC, GSDMD, GSDME, and DFNB594. All GSDMs except DFNB59 possess a conserved two-domain structure: the N-terminal domain (NTD) interacts with the cell membrane to oligomerize and form pores, leading to membrane lysis and pyroptosis, while the C-terminal domain (CTD) inhibits NTD activity5–8. Pyroptosis is characterized as a “hot” reaction in immunology, eliciting a robust inflammatory response by releasing inflammatory cytokines and danger-associated molecular patterns (DAMPs), effectively activating the immune system4,9–11. Consequently, several studies have explored the therapeutic potential of pyroptosis in cancer treatment, yielding encouraging results11–13. However, the intricate signaling pathways involved in pyroptosis complicate efforts to induce this process through natural signaling interventions14–16. To tackle this challenge, we strive to develop a synthetic biology system that can respond to specific signals and controllably induce pyroptosis.

To counteract exogenous genetic elements, prokaryotes possess a wide range of defense mechanisms, among which the CRISPR-Cas system is especially prominent17,18. Although the CRISPR-Cas system primarily functions in adaptive immunity through RNA-guided cleavage of nucleic acids, growing research attention has shifted toward auxiliary proteins encoded adjacent to CRISPR loci—termed the CRISPR-associated protease (CASP) system19,20. Recent studies have uncovered complex interactions among diverse proteins within the type III-E CASP system, establishing a foundation for its practical application. The type III-E effector Cas7-11 (also known as gRAMP) is a single-protein effector composed of four Cas7 domains and one Cas11 domain21,22. Cas7-11 specifically recognizes tgRNA that is complementary to CRISPR RNA (crRNA; CR), subsequently activating the activity of a protease from the CHAT family of tetrapeptide repeats (Csx29, or TPR-CHAT)23–27. The activated Csx29 specifically cleaves the protein Csx30 at amino acids 427 to 429, facilitating the signal transduction from RNA to protease23,25,26.

In view of this signal conversion mechanism, we hypothesize that Csx30 can serve as a linker between the NTD of GSDMs (GSDMs-N) and the CTD of GSDMs (GSDMs-C) to construct a fusion protein, named GSDMs-Csx30. The Cas7-11–crRNA complex identifies tgRNA and subsequently activates Csx29. This activation leads to the cleavage of GSDMs-Csx30 by Csx29, resulting in the release of GSDMs-N and ultimately triggering pyroptosis. Herein, we introduce an innovative system designed to detect tgRNA and actively induce pyroptosis, referred to as DAMAGE (Death Manipulation Gene). DAMAGE specifically targets and eliminates various transcriptome-altered cells, such as virus-infected, cancerous, and senescent cells. Furthermore, DAMAGE demonstrates the potential to be transcribed into mRNA and delivered in vivo via Lipid Nanoparticles (LNPs) for therapeutic applications. Overall, our study establishes an ingenious method for targeting RNA-heterogeneous diseases, which holds great promise as an innovative strategy for their treatment.

Results
The construction and functional validation of DAMAGE
Type III-E CASP system contains a variety of proteins, including Cas7-11, Csx29, Csx30, Csx31, and RpoE21–27. We constructed expression plasmids derived from the type III-E CASP loci of Desulfonema ishimotonii, including Cas7-11, Csx29, and Csx30. Additionally, a nuclease-inactive Cas7-11 variant (dCas7-11; D429A/D654A) and a protease-inactive Csx29 variant (dCsx29; H615A/C658A) (Supplementary Fig. 1a) were also generated. The interactions among Cas7-11, Csx29, and Csx30 indicated a significant functional interrelationship between these proteins (Supplementary Fig. 1b). The predicted structure generated by AlphaFold2 reveals that Csx30 comprises an NTD (amino acids 1 to 377) and a CTD (amino acids 419 to 565), which are connected by a long, flexible linker region (amino acids 378 to 418) (Supplementary Fig. 1c). Based on these findings, we performed a series of truncation assays and found that the CTD of Csx30 alone is sufficient for cleavage by activated Csx29 (Supplementary Fig. 1d–h). These results support a role for Csx30 as a linker within an “X-Csx30-Y” architecture, enabling the fusion of two distinct protein domains and their integration into the type III-E CRISPR system.

Consequently, we developed the DAMAGE (Death Manipulation Gene) system (Fig. 1a), which is designed to respond to specific tgRNA and induce pyroptosis. It consists of five essential components: Cas7-11, Csx29, GSDMs-Csx30, crRNA, and tgRNA, each of which plays a critical role in the functionality of the system. The core of DAMAGE lies in the GSDMs-Csx30 effector, wherein Csx30 functions as a linker bridging GSDMs-N and GSDMs-C. As GSDMD is one of the most extensively studied members of the GSDMs, we aligned amino acid sequences of murine GSDMD (mGSDMD) and human GSDMD (hGSDMD), and created a series of truncated GSDMD-Csx30 variants (Supplementary Fig. 2a) to determine the optimal length of the Csx30 linker. Through a series of screenings, D-X demonstrated tightly regulated pyroptotic activity upon tgRNA recognition and was identified as the optimal fusion protein (Supplementary Fig. 2b–f). Furthermore, shorter truncated forms of GSDMD-Csx30 induced irregular and unpredictable nonspecific pyroptosis (Supplementary Fig. 2g, h). These findings indicate that Csx30 407-565 (amino acids 407 to 565) may serve as the most suitable linker.

To develop additional GSDMs-Csx30 effectors, we performed a comparative analysis of amino acid sequences among various members of the GSDMs family. The results showed significant homology between GSDMs-N and GSDMs-C, while the linker region exhibited lower similarity (Supplementary Fig. 4a). We therefore inserted the Csx30 407-565 segment into the natural cleavage sites of GSDMs to create the full-length GSDMs-Csx30, termed GSDMs-Csx30-FL (A-FL to E-FL). GSDMs were further truncated at their conserved amino acid sites, and the linker region was replaced with Csx30 407-565 to generate the short-length GSDMs-Csx30, designated as GSDMs-Csx30-SL (A-SL to E-SL). Furthermore, the previously designed constructs D-X and D-Y were also included, incorporating an additional nuclear export signal (NES) sequence upstream of GSDMD-N, thereby distinguishing them from D-SL and D-FL. Meanwhile, we constructed a plasmid expressing GSDMs-Csx30-N (N) as the positive control to mimic the cleavage product of GSDMs-Csx30 generated by activated Csx29 (Fig. 1b–e). We found that the loss of nuclease activity in Cas7-11 had no significant effect on the activation of the DAMAGE system (Supplementary Fig. 1e, 2b). Therefore, to eliminate the possible interference caused by nuclease activity, this study mainly used dCas7-11 for subsequent experiments.

We initially evaluated the functionality of the DAMAGE system by employing Enhanced Green Fluorescent Protein (EGFP) as the target. The control groups (OFF) were transfected with a non-targeting crRNA (CR-NT), whereas the trigger groups (ON) were treated with a crRNA mixture (CR-Mix) specifically targeting EGFP mRNA (Supplementary Fig. 3a–c and Supplementary Table 1). Compared to the OFF groups, GSDMs-Csx30 in the ON groups exhibited significant cleavage and subsequent induction of pyroptosis. In these ON groups, cell membrane lysis released intracellular contents into the supernatant, leading to decreased levels of Cas7-11, Csx29, and GSDMs-Csx30 in the cell lysate, whereas the cleavage-generated GSDMs-Csx30-N could be detected (Supplementary Fig. 4b, c). Following the release of cellular contents, the full-length C-terminal Flag-tagged GSDMs-Csx30 and the cleaved GSDMs-Csx30-C, were pulled down from the culture medium via immunoprecipitation (IP) by Flag-tagged beads (Supplementary Fig. 4c). Compared to the OFF group, the two forms above showed a significantly increase in the supernatant of the ON group, especially pronounced in A-SL and A-FL. The results demonstrate that the ON group displayed significantly distinct trends compared to the OFF group, with performance closely aligning with the N+ positive control. This was reflected in elevated lactate dehydrogenase (LDH) release (Fig. 1b), a reduction in the EGFP+ live cell population (Fig. 1c) accompanied by a rise in Propidium Iodide (PI) positive dead cells (PI+), and diminished cell viability (Fig. 1d and Supplementary Fig. 12a). The ON group also displayed distinct morphological features of pyroptosis, such as cellular swelling, plasma membrane blebbing, rupture, and the release of intracellular contents (Fig. 1e, f and Supplementary Fig. 5a, b). All these results demonstrated the occurrence of pyroptosis upon triggering of DAMAGE. To further evaluate the specificity of DAMAGE, we co-cultured EGFP- and mCherry-expressing cells and transfected them with DAMAGE targeting EGFP mRNA (DAMAGE-EGFP). The results demonstrated a significant reduction in green fluorescence, while red fluorescence remained unchanged (Fig. 1g). All these findings indicated that DAMAGE can selectively eliminate target cells without affecting non-target cells.

In conclusion, the DAMAGE system demonstrates a robust capacity to respond to tgRNA and induce highly specific pyroptosis in target cells. These findings indicate that the DAMAGE system drives tgRNA-dependent pyroptosis activation for achieving clearance of targeted cell populations.

DAMAGE responds to RSV infection and induces pyroptosis
When viruses invade host cells, they produce exogenous RNA that is markedly distinct from the host transcriptome. This discrepancy offers a promising opportunity for DAMAGE to selectively target and eliminate virus-infected cells. In this study, we utilized human respiratory syncytial virus (RSV) as a model virus, which is a negative-sense, single-stranded RNA virus that belongs to the Pneumoviridae family28. The RSV genome contains 10 genes that encode 11 proteins. The mRNAs encoding these proteins are sequentially transcribed from the non-segmented genome, resulting in a progressive decline in transcriptional efficiency.

Consequently, we selected the RSV nucleocapsid protein (RSV-N) for its high transcription level as the tgRNA to detect RSV, and developed DAMAGE-RSV to specifically target and eliminate RSV-infected cells (Fig. 2a). Through a preliminary and relatively comprehensive screening, the effectors GSDMB/C-Csx30 (B-FL, B-SL, C-FL, C-SL) demonstrated superior lethality, consistent with the previous findings, and B-SL was selected for subsequent assay due to its low nonspecific pyroptotic activity (Fig. 2b and Supplementary Fig. 6a). In both plasmid transfection and viral infection, DAMAGE-RSV was activated upon detection of RSV-N mRNA, resulting in efficient cleavage of GSDMs-Csx30 effectors (Supplementary Fig. 6c) and the occurrence of pyroptosis (Fig. 2c) along with obvious morphological changes (Fig. 2e). The tagged proteins, including dCas7-11-HA, Csx29-Myc, RSV-N-Myc, GSDMs-Csx30-Flag, and the cleaved GSDMs-Csx30-C-Flag, were successfully pulled down from the culture medium (Fig. 2d and Supplementary Fig. 6d, e). Furthermore, a reduction in cell viability was observed in the ON groups (Supplementary Fig. 6b and 12b), which provides additional evidence supporting the efficacy of DAMAGE. These results indicate that in the presence of RSV-N mRNA, whether transcribed from either exogenous transfection or viral infection, can be detected by DAMAGE-RSV to induce efficient pyroptosis, leading to cellular membrane lysis and the release of intracellular components.

To further assess the sensitivity of DAMAGE-RSV, we conducted a series of gradient assays using negative quadratic dilution under both exogenous transfection and viral infection conditions. We observed that as the dilution factor increased, the pyroptotic activity of GSDMs-Csx30 progressively diminished. The cleavage efficiency of GSDMs-Csx30 decreased in response to reduced levels of tgRNA (Supplementary Fig. 7a–d), leading to a gradual decline in the PI positivity (Fig. 2f, g and Supplementary Fig. 6f, g) and a corresponding reduction in LDH release (Fig. 2f, g and Supplementary Fig. 6h, i). These findings highlight the remarkable sensitivity of DAMAGE in detecting minimal levels of tgRNA and its efficacy in triggering pyroptosis.

Since RSV predominantly infects respiratory epithelial cells, A549 cells, a human lung adenocarcinoma epithelial cell line, are extensively employed as a well-established in vitro model for the study of RSV infection. We performed transfection and infection experiments in A549 cells and observed consistent experimental outcomes (Fig. 2h). At the morphological level, the DAMAGE system successfully converts RSV-induced apoptosis into pyroptosis, thereby achieving efficient clearance of infected cells (Fig. 2i).

These findings demonstrate that DAMAGE can accurately identify “non-self” tgRNA originating from viruses or other pathogens and eliminate infected cells without adversely impacting normal cells. Moreover, DAMAGE exhibited high sensitivity in identifying these RNA signatures. This discovery provides pioneering strategies for combating viral infections by distinguishing the unique RNA profiles of viruses.

DAMAGE selectively targets and eliminates HPV-infected cervical cancer cells
Persistent infection with high-risk human papillomavirus (HPV) is recognized as the primary causative factor in the development of cervical cancer29,30. The integration of HPV DNA into the host genome results in the persistent expression of viral oncoproteins, particularly E6 and E7, making them pivotal targets for therapeutic vaccine development against HPV-associated malignancies31. Notably, the HeLa cell line, one of the most extensively utilized models in scientific research, originates from cervical cancer cells caused by HPV18 infection and accompanied by subsequent genomic integration. Therefore, we selected HeLa cells as a model system to examine the specific cytotoxic effects of DAMAGE on cervical cancer.

The validation experiments were initially conducted in HEK293T cells to evaluate the ability of DAMAGE to specifically target and recognize the E6 and E7 mRNA transcripts of HPV16/HPV18. The results indicated that the DAMAGE system can be specifically activated upon recognition of HPV mRNA (Supplementary Fig. 8a–c), leading to the induction of pyroptosis and a significant decrease in cell viability (Supplementary Fig. 12e-h). Consequently, we have developed DAMAGE-HPV, a system specifically designed to target and eliminate HPV infected cells. Through initial validation using individual crRNAs (CR1 to CR5) in HeLa cells, we found that endogenous HPV18-E6/E7 mRNA, transcribed from the HeLa genome, is sufficient to activate DAMAGE-HPV and induce pyroptosis (Fig. 3b). To further enhance the targeting capability of crRNA for HPV RNAs, we adopted a previously established strategy (Supplementary Fig. 3c) by fusing five individual target sites for each protein, constructing CR-E6, CR-E7, as well as a CR-E6+E7 combining the effective target sites from both E6 and E7. These constructs were used for subsequent experiments. We next evaluated the endogenous triggering-induced lethality of all effectors in HeLa cells (Supplementary Fig. 8d–g). The fluorescence intensity of EGFP and cell viability were assessed following co-transfection of cells with EGFP and DAMAGE-HPV to evaluate the extent of pyroptosis (Fig. 3c, d and Supplementary Fig. 8i). Consistent with previous findings in HEK293T cells, B-FL and C-FL exhibited the highest cytotoxicity in HeLa cells and were therefore selected as the primary effectors for further investigation.

With increasing transfection duration, both the PI-positive rate and LDH release levels gradually increased, peaking at 36 h post-transfection of DAMAGE-HPV (Fig. 3e–g). To visualize the effects of DAMAGE-HPV, we transfected HeLa cells with DAMAGE-HPV and co-transfected EGFP as a fluorescent marker to observe the morphological changes. Compared to the OFF groups (CR-NT), all three ON groups (CR-E6, CR-E7, and CR-E6+E7) significantly induced pyroptosis, along with a notable decrease in green fluorescence (Fig. 3h). To further investigate the recognition specificity of DAMAGE-HPV under endogenous activation conditions, we selected the HPV18-negative cervical cancer cell line C33-A as a negative control. In the absence of endogenous HPV18 mRNA, DAMAGE-HPV remains inactive in C33-A cells, preserving their viability and structural integrity. In contrast, HPV18-positive HeLa cells display pronounced pyroptotic morphological changes, accompanied by a significant decrease in fluorescence intensity (Fig. 3i).

These findings demonstrate that DAMAGE can specifically sense endogenous viral RNA transcribed by integrated viral genomic and induce pyroptosis in infected cells. This suggests that DAMAGE holds promise as a therapeutic strategy not only for acute infections like RSV, but also for chronic viral infections, such as HPV, HBV, and HIV.

DAMAGE identifies single nucleotide mutations and specifically triggers pyroptosis in KRAS-mutant cancer cells
KRAS is one of the most prevalent oncogenes, with mutations present in ~10% of all cancers32. Several mutations have been identified at the 12th amino acid Gly in KRAS, including G12C, G12D, G12R, G12A, G12S, and G12V33–35. Despite being identified more than four decades ago, it is only recently that inhibitors like sotorasib, which is a covalent binder of the KRAS-G12C mutation, have been approved for clinical application36.

Notably, significant base mutations exist in the mRNA between KRAS wild-type (KRAS-WT) and KRAS mutants (KRAS-mut), which facilitates the development of specifically targeted therapies. To evaluate the capability of DAMAGE in identifying single nucleotide mutations, we aligned the sequences of KRAS-WT and KRAS-mut. Two crRNAs were initially designed for each KRAS-mut variant, featuring spacers that perfectly match the KRAS-mut sequences while containing a single nucleotide mismatch relative to KRAS-WT (Supplementary Fig. 9a). We observed that the ON group transfected with KRAS-mut exhibited significant D-X cleavage, whereas the OFF group transfected with KRAS-WT showed negligible cleavage (Supplementary Fig. 9b), indicating that DAMAGE can discriminate single-nucleotide variants. Based on these findings, we have developed DAMAGE-KRAS, a targeted system for the detection of KRAS single nucleotide mutations.

To further enhance recognition accuracy, we focused on KRAS-G12C and designed 24 distinct crRNAs targeting KRAS-G12C by varying the position of the mutant nucleotide within the 24-bp spacer region of the crRNA (Fig. 4a). We systematically evaluated the capacity of these 24 crRNAs to detect single nucleotide mutations in KRAS-G12C. The results revealed that crRNA-16, crRNA-23, and crRNA-24 exhibited superior performance, with crRNA-23 displaying the highest specificity in discriminating KRAS-G12C from KRAS-WT (Fig. 4b–e and Supplementary Fig. 9c–g). To investigate the impact of endogenous KRAS mutations, HEK293T and HeLa cells stably expressing mCherry-tagged KRAS-WT or EGFP-tagged KRAS-G12C were subsequently constructed. In the presence of crRNA-23, DAMAGE-KRAS markedly induced pyroptosis in EGFP-KRAS-G12C stable cell lines, whereas no significant effect was observed in mCherry-KRAS-WT stable cell lines (Fig. 4f–h and Supplementary Fig. 9h). Time-dependent ATP-based cell viability assays further demonstrated that crRNA-16 and crRNA-23 significantly reduced cell viability in the KRAS-G12C groups compared to the KRAS-WT controls (Supplementary Fig. 12i, j). In addition, we systematically screened the six KRAS-mut subtypes and identified the optimal crRNAs to specifically target the mRNAs of these KRAS-mut (Fig. 4i and Supplementary Fig. 9i). Moreover, utilizing the NCI-H23 cells that endogenously harbor the KRAS-G12C mutation (Fig. 4j) and the A549 cells carrying the KRAS-G12S mutation (Fig. 4k), we demonstrated that endogenous KRAS-mut mRNA can effectively activate the DAMAGE-KRAS system, thereby indicating its potential applicability in clinical therapy.

Our findings demonstrate that the position of single nucleotide mutations within the crRNA spacer significantly influences the pyroptotic activity of DAMAGE. Following systematic optimization, DAMAGE achieves effective and precise detection of single nucleotide variants, underscoring its potential for therapeutic applications in a wide range of cancers driven by genetic alterations, including those involving BRAF, NRAS, and TP53. This category encompasses single- or multi-base substitutions, insertions or deletions, as well as chromosomal rearrangements.

DAMAGE targets p16/p21 mRNA and selectively eliminates senescent cells
Cellular senescence is a stress response that prevents the proliferation of aged or damaged cells. However, when these senescent cells accumulate in tissues over time, they may contribute to age-related decline and disease progression37. This process is triggered by the activation of retinoblastoma (RB) and p53 tumor suppressor networks, resulting in the transcription upregulation of cyclin-dependent kinase inhibitors (CDKIs), such as CDKN1A (p21CIP1; p21) and CDKN2A (p16INK4a; p16), which are commonly used as senescence markers38.

To selectively target senescent cells, we designed crRNAs for p16 and p21, including five individual crRNAs and a five-in-one crRNA-Mix, thereby establishing DAMAGE-Aging (Fig. 5a and Supplementary Table 1). Compared to the OFF groups (CR-NT), transient transfection of DAMAGE-Aging into HEK293T cells resulted in significant pyroptosis in the ON groups (p16/p21 CR-Mix), as evidenced by marked GSDMs-Csx30 cleavage, elevated LDH release, and an increased number of PI-labeled dead cells (Fig. 5b, c and Supplementary Fig. 10a, b). As the duration of transfection increased, the proportion of PI-positive dead cells rose progressively and cell viability declined in the ON groups, whereas no significant changes were observed in the OFF groups, even after 48 h (Supplementary Fig. 10c and 12c, d).

However, it was unexpected to observe that the abundant endogenous mRNAs of p16 and p21 in HEK293T cells (Fig. 5d, e and Supplementary Fig. 10d) and HeLa cells (Supplementary Fig. 11a–c) were capable of activating DAMAGE-Aging and inducing pyroptosis, especially p16. Therefore, we established a HEK293T p16 knockout (HEK293T-p16-KO) cell line and successfully isolated a monoclonal cell line in which p16 mRNA was no longer detectable by p16 crRNA-5 (p16-CR5) (Supplementary Fig. 3a, b). Next, we introduced DAMAGE-Aging into HEK293T wild-type (HEK293T-WT) and HEK293T-p16-KO cells, along with EGFP as the fluorescent marker. In the ON groups (p16-CR5), HEK293T-WT cells exhibited a significant pyroptosis phenotype, marked by increased PI-positive dead cells and reduced green fluorescence. In contrast, HEK293T-p16-KO cells showed no significant pyroptotic response and resembled the OFF groups (CR-NT) (Fig. 5f–i). Consistent results were also observed in the HeLa-p16-KO cell line (Supplementary Fig. 11d, e). These results demonstrate that p16-KO effectively suppresses endogenous p16 mRNA-mediated DAMAGE-Aging activation, further confirming its target specificity.

To further assess the ability of DAMAGE-Aging to identify changes in RNA transcript levels, we modulated p16 and p21 mRNA translation using pharmacological agents. We found that MS37452, a small-molecule inhibitor of p16 mRNA translation, partially attenuated DAMAGE-Aging-induced pyroptosis, resulting in an increased proportion of EGFP-positive viable cells (Fig. 5j and Supplementary Fig. 10e). Furthermore, to inversely validate whether upregulation of p16/p21 mRNA could enhance the pyroptotic activity of DAMAGE-Aging, cells were treated with etoposide (2 μM) or doxorubicin (100 nM), both function as transcriptional activators of p16 and p21. These treatments significantly enhanced DAMAGE-Aging-induced pyroptosis, as indicated by increased PI-positive cells, elevated LDH release, and characteristic pyroptotic morphology (Fig. 5k and Supplementary Fig. 10f, 11g).

Based on the experimental results above and the gradient experiments illustrated in Fig. 2, we have confirmed that DAMAGE can effectively detect differences at the mRNA level. By specifically targeting highly expressed mRNAs in senescent cells, such as p16 and p21, DAMAGE-Aging shows a great potential for selectively eliminating senescent cells and offers an innovative strategy for targeted senotherapies.

The potential of DAMAGE in mRNA-LNP therapeutic applications
Given the proven efficacy of mRNA vaccines in combating the coronavirus disease 2019 (COVID-19) pandemic, this innovative platform has garnered considerable attention for its potential application in developing effective therapeutics for a range of infectious diseases and oncological conditions30,39,40. In this study, we attempt to transcribe DAMAGE into mRNA in vitro and deliver it utilizing Lipid Nanoparticles (LNPs) to explore its potential for mRNA-LNP therapeutic applications.

Originally, DAMAGE consisted of five components: Cas7-11, Csx29, GSDMs-Csx30, crRNA, and tgRNA, each integrated into separate plasmids. To simplify the DAMAGE system, we cloned Cas7-11, Csx29, and GSDMs-Csx30 into a single plasmid and renamed it DAMAGE-Plus (Fig. 6a). We initially transfected all the DAMAGE-Plus plasmid into HEK293T and HeLa cells. The results demonstrated that DAMAGE-Plus functioned in a similar trend to the original DAMAGE and even showed a lower level of nonspecific pyroptosis. Among them, DAMAGE-Plus B-FL performed the best and was chosen for subsequent experiments (Fig. 6b and Supplementary Fig. 13a). Consistent with prior findings, DAMAGE-Plus effectively targeted all previously investigated tgRNAs and induced significant pyroptosis (Supplementary Fig. 13b–d). Furthermore, DAMAGE-Plus can accurately detect tgRNAs, such as EGFP mRNA and HPV18-E7 mRNA, thereby specifically inducing pyroptosis in targeted cells without affecting non-target cells. This functional specificity has been confirmed through cell co-culture assays (Fig. 6c, d and Supplementary Fig. 13e, f) and live-cell imaging (Supplementary Fig. 15 and Supplementary Movie 1–6). We also verified the recognition capability for single-nucleotide mutation of DAMAGE-Plus in HEK293T-KRAS-G12C stable cell lines, which was consistent with the results presented in Fig. 4 (Supplementary Fig. 13g, h).

Next, we performed in vitro transcription of all components of DAMAGE-Plus into RNA, and then co-transfected these RNAs into HEK293T cells. In the ON groups, DAMAGE-Plus in RNA form demonstrated the ability to effectively recognize all tgRNAs and induce pyroptosis (Fig. 6e, f and Supplementary Fig. 14a, b). Furthermore, we validated whether DAMAGE could be delivered in the form of LNP-encapsulated mRNA (mRNA-LNP). Considering that an excessively large size of DAMAGE-Plus would reduce the efficiency of in vitro transcription and complicate LNP encapsulation. We divided DAMAGE-Plus into two components, Cas7-11 and Csx29-B-FL, which were transcribed separately in vitro (Supplementary Fig. 14c–e). Meanwhile, mRNA-LNP was synthesized to evaluate the therapeutic potential of DAMAGE (Supplementary Fig. 14f, g). In HEK293T cells, whether in the form of mRNA (Supplementary Fig. 14h) or mRNA-LNP (Supplementary Fig. 14i), DAMAGE was found to effectively recognize tgRNA and trigger pyroptosis upon activation. Furthermore, both mRNA transfection (Fig. 6g, h and Supplementary Fig. 14j) and mRNA-LNP delivery (Fig. 6i and Supplementary Fig. 14k) of the DAMAGE system in HeLa cells could also lead to specific recognition of endogenous HPV18-E7 mRNA and efficient induction of pyroptosis.

These findings demonstrate that DAMAGE can be transcribed into mRNA in vitro and delivered in the form of mRNA-LNP, underscoring its potential as an mRNA therapeutic for in vivo delivery and treatment, with promising prospects for translation into clinical application.

Methods
Cells
The HEK293T (CRL-1573, ATCC), A549 (CCL-185, ATCC), HeLa and C33-A (obtained from Dr. Zheng Hu, Wuhan University) cells were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM, 11995065, Gibco) supplemented with 10% fetal bovine serum (FBS, 086150, WISENT) and 1% Penicillin-Streptomycin (15140-122, Gibco). The NCI-H23 cells (GDC0651, CCTCC) were cultured in RPMI Medium 1640 basic (C11875500BT, Gibco) supplemented with 10% FBS and 1% Penicillin-Streptomycin. Cells used in experiments were from passages fewer than 30. All cell cultures were maintained at 37°C in a humidified incubator with 5% CO2.

Plasmid construction
The Cas7-11, Csx29, and Csx30 genes were cloned from the DiCas7-11 full locus plasmid (172501, Addgene), and the fragments were ligated into restriction enzyme-digested expression vectors using T4 DNA ligase (RK21501, ABclonal). The crRNAs were designed using the Cas13 design online platform, selecting the five top-ranked, non-overlapping complementary spacers (https://cas13design.nygenome.org/)41. For the construction of GSDMs-Csx30, gene fragments of all human GSDMs were obtained through gene synthesis (Tsingke Biotech). The GSDMs-Csx30 gene fragments were constructed by bridge PCR and cloned into the vector via homologous recombination by Hieff Clone Universal II One Step Cloning Kit (10923ES20, Yeasen). All inserted fragments have been validated through DNA sequencing to ensure their accuracy and reliability.

Plasmid transfection
HEK293T or HeLa cells were seeded in a 12-well plate, a 24-well plate or a 96-well plate and then incubated overnight to reach a density of approximately 70% before transfection. The five components of DAMAGE: dCas7-11, Csx29, GSDMs-Csx30, crRNA, and tgRNA were maintained at a mass ratio of 1:1:1:1:1. A total of 2 μg of plasmid was transfected into each well of a 12-well plate, 1 μg into each well of a 24-well plate and 0.5 μg into each well of a 96-well plate, with the same ratio maintained. When DAMAGE was constructed in a single plasmid, 1.2 μg DAMAGE-Plus plasmid, 0.4 μg tgRNA plasmid and 0.4 μg crRNA plasmid were transfected into each well of a 12-well plate. For endogenous trigger experiments, the tgRNA plasmid was omitted. For transfection of HEK293T or HeLa cells in a 12-well plate, all the plasmids were diluted in 50 μL Opti-MEM (31985-070, Gibco) for every 1 μg of plasmid transfection added with 1 μL of Neofect™ DNA transfection reagent (TF20121201, NEOFECT). For transfection of A549, NCI-H23 and C33-A cells, Lipofectamine™ 3,000 Transfection Reagent (L3000075, Invitrogen) was employed in accordance with the manufacturer’s instructions. Equal amounts of DNA were added to each well.

Virus infection
HEK293T, HeLa or A549 cells were seeded in a 12-well plate and incubated overnight until they reached a density of ~70% before viral infection. In the infection procedure, the culture medium was removed, and the cells were washed with PBS (SH30256.01, Cytiva) and then exposed to DMEM (11995065, Gibco) containing RSV (strain A2) for 2 h with different MOI values. Subsequently, the medium was replaced with fresh DMEM supplemented with 4% FBS, followed by transfection.

Cell co-culture assay
In the co-culture assay of transient transfection, HEK293T or HeLa cells were divided into two groups and seeded in a 12-well plate at the same density. After overnight culture, one group was transfected with mCherry, while the other was transfected with EGFP. Simultaneously, both groups were transfected with DAMAGE targeting EGFP mRNA. 8 h post transfection, the two groups of cells were mixed at a 1:1 ratio. The OFF groups transfected with CR-NT were employed as a negative control, as they did not trigger DAMAGE activation. For the stable cell lines, the same amount of HEK293T-mCherry-KRAS-WT and HEK293T-EGFP-KRAS-G12C, or HeLa-mCherry-KRAS-WT and HeLa-EGFP-KRAS-G12C, or HeLa-EGFP and HeLa-mCherry were collected and seeded in a 12-well plate. The cells were transfected with the corresponding plasmids after overnight culture.

mRNA production and transfection
DAMAGE-Plus was cloned into a DNA backbone featuring a T7 promoter, and the plasmid was linearized through restriction endonucleases after purification for mRNA production. Then, the mMESSAGE mMACHINE™ T7 ULTRA Transcription Kit (AM1345, Invitrogen) was employed to transcribe DAMAGE-Plus mRNA with a 5’ cap structure and a 3’ polyA tail in vitro. To generate RNA for tgRNA and crRNA, PCR was utilized to amplify them from plasmids, with a T7 promoter incorporated at the 5’ end. The resulting DNA amplicons were then subjected to in vitro transcription using the TranscriptAid T7 High Yield Transcription Kit (K0441, Invitrogen). Finally, all these RNA products were purified with the MEGAclear™ Transcription Clean-Up Kit (AM1908, Invitrogen), followed by transfection executed using Lipofectamine™ MessengerMAX™ Transfection Reagent (LMRNA008, Invitrogen) in accordance with the manufacturer’s instructions. The components: DAMAGE-Plus, crRNA and tgRNA were transfected at molar ratio of 1:2:1.

The preparation and delivery of mRNA-LNP
The two plasmids (Cas7-11 and Csx29-B-FL) were cloned into a DNA backbone featuring a T7 promoter respectively, serving as the template for in vitro RNA transcription. The mRNA-LNP synthesis was performed by GenScript Biotech Corporation with the molar ratio of Cas7-11: Csx29-B-FL: crRNA = 1:1:3, and the product was stored at −80°C until used. For the delivery experiment in cells, HEK293T or HeLa cells were seeded in a 12-well plate and cultured overnight to the density of 60%–70%. Next, the frozen mRNA-LNP was allowed to thaw on ice for 10 min and mixed by gentle inversion 3–5 times (vortexing was avoided). The thawed LNPs was then diluted in Opti-MEM to a final concentration of 1 μg mRNA per well and added to the culture medium. The plate was rocked gently for even distribution of the LNPs, and then incubated for 24 h prior to analysis.

Cell line engineering
For the construction of stable expression or CRISPR/Cas9 knockout cell lines, lentiviral plasmids carrying the desired gene or sgRNA were initially transfected into HEK293T cells along with the packaging plasmids pSPAX2 (12260, Addgene) and pMD2G (12259, Addgene) at a ratio of 3:2:1 for lentiviral packaging. The supernatants were collected 48 h post-transfection and filtered through a 0.45-μm filter to remove cellular debris, and the filtered supernatant was used to infect the indicated cells for another 48 h. The stable expression cells were selected with blasticidin (3513-03-9, Yeasen), while p16/p21-KO cells were generated under puromycin selection (540411, Sigma-Aldrich). Fluorescence-tagged stable lines were subsequently validated by fluorescence microscopy, while knockout efficiency was confirmed by immunoblotting34.

Reagent treatment
For MS37452 (HY-119344, MCE) treatment, concentration gradients of 0 (DMSO), 25, 50, and 100 μM were established. After cells adhesion, the compound was added for a 12 h treatment period, after which transfection was performed. The experiment included the following treatment groups: a DMSO control, and groups treated with 25, 50, or 100 μM MS37452. Cells in each group were transfected with the DAMAGE system, using either the non‑targeting CR‑NT (OFF groups) or the p16‑targeting crRNAs (p16‑CR5 or p16‑CR‑Mix; ON groups) constructs. For flow cytometry data analysis, the EGFP+ population in the CR-NT (OFF) in each treatment was set as 100%. The percentage of live cells was then calculated as the ratio of the EGFP+ cells in the p16-CR5 or p16-CR-Mix (ON) group to that in its corresponding CR-NT (OFF) group.

In the Etoposide (HY-13629, MCE) and Doxorubicin (HY-15142, MCE) treatment, the cells were seeded in a 12-well plate, and transfection was performed after the overnight culture. 8 h post transfection, the supernatant was replaced with fresh medium containing Etoposide (2 μM) or Doxorubicin (100 nM), and an equal amount of DMSO was added as a control. The samples were collected for assay after 36 h of treatment.

Antibodies
The antibodies used in the study are listed as follows: DYKDDDDK Tag (D6W5B) Rabbit mAb (14793S, Cell Signaling Technology), DYKDDDDK Tag (9A3) Mouse mAb (8146S, Cell Signaling Technology), Anti-HA-tag Mouse mAb (M180-3, MBL), Anti-Myc-tag Mouse mAb (M192-3, MBL) were utilized to detect labeled proteins. Gasdermin A (49307S), Gasdermin B (76439S), Gasdermin C (61921S), Gasdermin D (39754S), Gasdermin E (19453S) were used for the direct detection of expressed GSDMs (all from Cell Signaling Technology). Mouse monoclonal anti-RSV Nucleoprotein (N) (ab94806, Abcam), Mouse monoclonal anti-RSV Phosphoprotein (P) (ab94965, Abcam) and Mouse monoclonal anti-RSV M2-1 (ab94805, Abcam) were used to detect viral proteins. p16INK4A (E6N8P) Rabbit mAb (18769S, Cell Signaling Technology) and p21Waf1/Cip1 (12D1) Rabbit mAb (2947S, Cell Signaling Technology) were utilized to detect endogenous p16/p21. α-Tubulin Mouse mAb (AC012, ABclonal) and GAPDH Mouse mAb (AC033, ABclonal) were employed as loading controls. Goat anti-Mouse IgG (H+L) Highly Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 488 (A-11029, Invitrogen); Goat anti-Rabbit IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (A-11012, Invitrogen); and Goat anti-Mouse IgG (H+L) Cross-Adsorbed Secondary Antibody, Alexa Fluor™ 594 (A-11005, Invitrogen) were used for immunofluorescence assay in confocal microscopy.

Microscopy
Cells were seeded in a 12-well plate for transfection. After 24 h for HEK293T or 36 h for HeLa post-transfection, each well was stained with appropriate dye according to the recommended dilution ratio, conditions and time: PI (P3566, Invitrogen) at a ratio of 1:1000; Hoechst (C1029, Beyotime) at a ratio of 1:1000; or 2 drops of SYTOX Green (R37168, Invitrogen). Subsequently, cell morphology was observed and images were captured using a Leica DMi8 microscope. For confocal microscopy, cells were seeded in a 24-well plate with aseptic coverslips. 20 h post-transfection, the supernatant was removed and the cells were washed with PBS three times. The samples then fixed with 4% paraformaldehyde at room temperature for 20 min and permeabilized with 0.2% Triton X-100 (T109028, Aladdin) in PBS for 10 min. Subsequently, cells were blocked using a buffer containing 3% BSA (BioFrox, 4240GR500) for 30 min. They were then incubated with the primary antibody at 4°C overnight and followed by a fluorescent labeled secondary antibody at room temperature for 1 h. The nuclei were stained for 10 min using DAPI (D9542, Sigma-Aldrich) at 50 μg/mL. After three 5-min PBS washes, the coverslips were inverted onto glass slides with ProLong Diamond Antifade Mountant (P36970, Invitrogen) and stayed overnight in the dark before observation. The images were captured using a Zeiss LSM980 confocal microscope and analyzed by ZEN 2.3, and the scale bar was added using ImageJ software (Experimental Methodology Reference Zhou, B. et al.)42.

Live cell imaging
Live cell imaging was performed on a Zeiss LSM980 equipped with a stage-top incubator. The cells were cultured in a Φ20 mm TC-treated confocal dish (BS-20-GJM, Biosharp) for transfection. Live-cell imaging was initiated 12 h post-transfection, acquiring one frame every 5 min for a total of 100 frames. Each group was imaged under identical laser intensity settings. The movies were processed and laid out by ZEN 2.3, and the scale bar was added using ImageJ43.

Cell stain and flow cytometry
Cells seeded in a 12-well plate and transfected with plasmid or mRNA for 24–36 h were stained and harvested for flow cytometry. For PI (P3566, Invitrogen) staining, the dye was added to the cell supernatant at 1:1000 dilution and incubated at 37 °C for 30 min. All cells were collected for analysis. Floating cells in supernatant were first transferred to a 1.5 mL Eppendorf tube and pelleted by centrifugation (200×g, 5 min; Eppendorf 5424R). The remaining adherent cells were trypsinized and combined with the pelleted floating cells. Then, all cells were harvested by centrifugation at 200×g for 5 min. The pellet was resuspended in 1 mL PBS and analyzed using CytoFLEX flow cytometer (Beckman Coulter). Forward and side scatter were set as follows: 500/500 for HEK293T, 200/200 for HeLa, and 300/300 for A549 cells. Cellular fluorescence was detected using two main channels: FITC (for EGFP; excitation 488 nm, emission 525/40 nm; gain 5–10), PE (for PI, excitation 488 nm, emission 585/42 nm; gain 50–100) or PC5.5 channel (for PI, excitation 488 nm, emission 690/50; gain 50). For experiments involving two colors, single-color controls were used to establish the compensation matrix with the CytExpert software (Version 2.0.4.8). All the results were analyzed by FlowJo 10.10, using negative controls (unstained) to define positivity thresholds. The gray dashed line was employed to mark the fluorescence intensity threshold separating PI-negative viable cells (left) from PI-positive dead cells (right) in this study.

Cell viability and cytotoxicity experiments
LDH release was measured using the CytoTox 96® Non-Radioactive Cytotoxicity Assay (G1781, Promega) to evaluate the process of cell pyroptosis according to the manufacturer’s instructions. Cell culture supernatant was collected 24–48 h post transfection and centrifuged at 16000×g for 5 min to remove cell debris. For the assay, 50 μL supernatant of each sample was added to the wells of a 96-well plate, followed by the addition of an equal volume of reaction mix. The plate was gently shaken to mix the components and incubated in the dark for 20–30 min. The reaction was stopped by adding 50 μL stop solution, and the absorbance at 490 nm was measured using a Multi-function microplate reader (Molecular Devices, SpectraMax iD3). Each sample did 3 parallels. The LDH release rate was calculated according to the manufacturer’s instructions. To detect the cell viability, the ATP-based viability assements were conducted using the CellTiter-Glo® Luminescent Cell Viability Assay (G7571, Promega) according to the manufacturer’s instructions. 13,000 HEK293T cells were seeded per well of a 96-well plate and transfected at approximately 60% confluency. Following 24 h of incubation, 100 μL assay reagent was added to an equal volume of culture medium in each well. Plates were mixed on the orbital shaker (80 cycles per minute, 10 min), incubated for 10 min at room temperature, and then the luminescence was recorded through a Multi-function microplate reader. ATP-based cell viability was calculated as the luminescence of the transfected wells normalized to that of un-transfected controls (Opti-MEM only). Each sample did 3 parallels.

RNA extraction, reverse transcription PCR, and real-time PCR
Total RNA was extracted using Trizol reagent (15596026CN, Invitrogen) in accordance with the manufacturer’s protocol. 1 μg mRNA was used for reverse transcription with the ABScript II cDNA First-Strand Synthesis Kit (RK20400, ABclonal) to obtain cDNA. Subsequently, the expression level of RSV-N mRNA after gradient RSV-N transfection or RSV infection was quantified by real-time PCR using 2× Universal SYBR Green Fast qPCR Mix (RK21203, ABclonal). Moreover, the expression level of the target gene was normalized to that of GAPDH as the housekeeping gene.

Immunoblotting and supernatant immunoprecipitation
To investigate the effect of DAMAGE, cells were lysed in lysis buffer (150 mM NaCl, 50 mM Tris-HCl pH 7.4, 1% Triton X-100, 1 mM EDTA pH 8.0, 0.1% SDS) containing protease inhibitor cocktail. Proteins in the cell lysates were separated via SDS-PAGE and analyzed through immunoblotting. Given that the Flag tag is fused to the C-terminus of effectors, the detection of N-terminal cleavage products requires the usage of endogenous antibodies. For comprehensive detection of all effectors and their N-terminal fragments, the endogenous antibody anti-GSDMA/B/C/D/E were diluted to the recommended dilution ratio. To ascertain whether the cellular contents were released into the supernatant upon cell rupture, culture supernatant was collected and incubated with tag-coupled beads overnight at 4°C. The supernatant was subjected to three rounds of immunoprecipitation (IP). In the first round, the supernatant was incubated with Flag-tagged agarose beads (651503, Biolegend) overnight at 4°C, followed by centrifugation at 6000×g for 2 min. The supernatant was then transferred to a new tube, while the beads were washed three times with lysis buffer and subsequently denatured in 1× SDS-PAGE loading buffer for the immunoblotting assay. The supernatant underwent a second round of IP and was incubated with Myc-tagged magnetic beads (HY-K0206, MCE), following the same procedure as above. The third round of IP was performed with HA-tagged magnetic beads (HY-K0207, MCE), using identical steps.

Co-immunoprecipitation
The interaction between Cas7-11-HA, Csx29-Myc, and Csx30-Flag were investigated for three pairs: Cas7-11-HA and Csx29-Myc; Csx29-Myc and Csx30-Flag; Cas7-11-HA and Csx30-Flag. For the experiments of Cas7-11-HA and Csx29-Myc, cells in a 6-well plate were harvested and lysed 24 h post transfection. Lysates were incubated with HA-tagged beads at 4°C overnight to immunoprecipitate HA-tagged Cas7-11. Interaction was assessed by detecting co-precipitated Csx29-Myc in the denatured beads via immunoblotting. To further verify the interaction, reciprocal co-immunoprecipitation was performed using Myc-tagged beads to pull down Csx29-Myc and detect associated Cas7-11-HA. Given the nuclease activity of Cas7-11 and the proteolytic activity of Csx29, corresponding inactive variants (dCas7-11 and dCsx29) were also included in the assays. The other two interaction pairs were assayed in the same manner.

Protein structure analysis
The structures of Csx30 was predicted by AlphaFold2. UCSF ChimeraX and PyMOL were employed to visualize the predicted outcomes44–46.

EGFP fluorescence intensity detection
To assess the pyroptotic capacity of various DAMAGE effectors for endogenous activation in HeLa cells, EGFP fluorescence intensity was quantitatively measured as a proxy for cell viability. Cells were seeded in a 96-well plate at 13,000 cells per well and cultured overnight to allow attachment. Upon reaching ~70% confluency, cells were co-transfected with DAMAGE-HPV and EGFP, using endogenous HPV18-E7 mRNA as the tgRNA. As cell death occurs, the fluorescence diminishes, accompanied by a decrease in EGFP intensity and cell viability. Therefore, EGFP fluorescence intensity can serve as an indicator of cell viability. The transfected cells were incubated for 24–36 h, and EGFP fluorescence intensity was detected using a Multi-function microplate reader. Wells transfected with EGFP alone served as the positive control (set as 100%), while wells treated with Opti-MEM only were used as the negative control. The relative EGFP intensity of each well was calculated by the formula: Y% = (X − negative control)/(positive control − negative control).

Statistics and reproducibility
The sample sizes and analytical measures are indicated in the figure legends and method details. In all cases, data from at least three independent experiments were used. Excel (Microsoft), ImageJ, ZEN 2.3, LAS X (Version 3.7.4, Leica), FlowJo (Version 10.10, BD Biosciences), and GraphPad Prism (Version 10, Dotmatics) were used. To visually and statistically represent data, each flow cytometry plot displays 10,000 or 20,000 randomly selected events.

Statistical analyses were conducted using GraphPad Prism 10. Different types of t-test, one-way ANOVA test and two-way ANOVA test were utilized as appropriate for different experimental conditions, and details of the statistical analysis performed and p values were described in the figure legends. Datasets were considered significantly different if the p value was less than 0.05. All Western blotting experiments were repeated three times. All micrographs were repeated three times, with at least three random fields of view captured per experiment.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "A Synthetic System for RNA-Responsive Pyroptosis Based on Type III-E CRISPR Nuclease-Protease\nThis work by He and colleagues engineers the type III-E CRISPR protease system containing the RNA-guided RNA\nnuclease Cas7-11 and the CHAT family protease Csx29. Normally, this protease cleaves Csx30, and the main finding of this\nwork is that the authors engineer pore-forming gasdermin proteins to be responsive to Csx30 cleavage. This protein\nengineering allows for the induction of pyroptosis and cell death following recognition of target RNA programmed by the\nCas7-11 guide. The authors measure this response through several cell viability assays, including PI staining and lactate\ndehydrogenase release, to quantify perforated cells and compare non-target and targeting conditions.\nThe authors test numerous gasdermin genes to make them responsive to cleavage by the Csx29 protease by inserting a\nCsx30 linker, forming the basic components of the DAMAGE system. This system is then applied to detect different RNA\nfrom RSV (Fig. 2), HPV (Fig. 3), KRAS (Fig. 4), and miRNA (Fig. 5). This work aims to tackle the biological problem of how\none might use RNA signatures to control specific cells, here to induce cell death in response to specific RNA, which is\nan interesting and important problem.\nI commend the authors on their substantial efforts in testing these constructs and attempting to develop a new tool. However,\nI am not convinced that the system works well enough. Many of these components have been used before, and this work\ndoes not adequately address the critical issues of using the CRISPR protease system in human cells, particularly regarding\nactivity and specificity, especially in response to endogenously expressed genes.\nI have two primary concerns with this work.\nThe first is an issue with the presentation and clarity of the paper. I found the manuscript difficult to follow, and I believe that\ntoo much unnecessary data is presented, which obscures the main findings. This is a case where “less is more,” and the\nauthors could have selected the best DAMAGE construct from Fig. 1 and presented it more clearly and rigorously for the rest\nof the paper. To me it would have been more useful to optimize one construct rather than testing all the reporters in parallel\nwith multiple target RNAs. In my opinion, Figs. 2-6 are not that useful, as we know the system is programmable and can be\napplied to different targets.\nI also found that the text and methods lacked sufficient detail, making some experiments unclear. It is not always explicitly\nstated how the experiments were conducted, whether target genes were transfected or integrated, which promoters were\nused, and whether targets were overexpressed. Additionally, certain figure abbreviations do not seem to be explained in the\ntext or legends.\nThe second major concern is the practical utility of this system outside of overexpression experiments in HEK293 cells.\nMany core ideas of this paper—using the CRISPR protease in human cells, truncating Csx30 as a linker to trigger biological\nactivities, and engineering gasdermins for cleavage by other proteases—have already been explored. While this work\nextends these concepts in an interesting direction, it does not fundamentally advance our understanding of CRISPR\nprotease biology or inducible cell death circuits.\nI therefore disagree with the claim that this work demonstrates “considerable promise” for therapeutic applications. The PI-\npositive cell quantification in Fig. 1c shows an increase from 5% to 15-20%, and the ATP-based readout in Fig. 1e shows\nviability decreasing from 85-90% to 60-70%. As such, it seems to fall into a “proof of principle” zone but I am not sure this is\nsufficient for publication. In my opinion the authors would need to improve the system and show more compelling results\nbeyond HEK293 cells.\nOverall, while the study presents a potentially interesting application of previous work and attempts to tackle an important\nbiological problem, to me it does not sufficiently address key challenges or demonstrate a substantial advance.\nSpecific Comments:\n• The figure legends and text lack clarity in explaining the experiments and different conditions tested. For instance, the\nabbreviations in Fig. 1 (N- and N+) are not clearly defined. Some of the data presented feels a bit unnecessary and makes\nthe paper “heavy” and could be moved to supplementary materials or omitted for ease of reading.\n• Line 134: “All GSDMs-Csx30 effectors can respond to tgRNA and induce pyroptosis, but they display a distinct control over\npyroptotic activity.” However, D-SL and D-FL clearly do not respond to target RNA. Why do the authors make this claim and\ncontinue to test these constructs that do not seem promising for this application?\n• There appears to be selective presentation of different constructs across figures, which introduces confusion. For example,\nFig. 1f shows C-FL, Fig. 1g shows A-FL, and Fig. 2b switches to D-X. A more consistent focus on the best reporter construct\nwould improve the manuscript.\n• The positive control in Fig. 1e at ~48% is unclear. Additionally, where are the error bars indicating standard deviation\nbetween replicates?\n• A critical issue with CRISPR protease systems, as seen with other RNA-targeting systems (e.g., RADAR, CellREADR), is\nthe requirement for high levels of target RNA. The authors should test a range of endogenous target genes to evaluate\nDAMAGE efficiency across physiologically relevant RNA levels.\n• Fig. 6 claims LNP delivery of DAMAGE mRNA, but only Figs. 6h and 6i seem to include actual mRNA delivery. Since\nthese experiments are performed in HEK293 cells, likely overexpressing the targets, they don’t seem to add much value\nbeyond the plasmid transfection experiments.\n• The term “tgRNA” for target RNA feels unnecessary and complicates readability.\n• Some schematics, such as those in Fig. 1a and Fig. 4a, are too small. A larger, clearer depiction of a representative\nconstruct (such as crRNA-23 in the KRAS experiment) would be preferable.\n• Line 89: truncation analysis of Csx30 has already been performed in previous studies and should not be claimed as novel.\n• Certain panels, such as those in Fig. 1f and 1g, should directly indicate which DAMAGE constructs are being tested to\nimprove clarity.\n• Some western blot results are unclear. In Fig. 1b and 2b, the full-length and cleaved products of D-X are visible, but in Fig.\n3b, the cleaved product is absent. The separate panel showing D-X-N raises questions; why is the cleaved product not\nobserved in the FLAG blot, and what does D-X-N represent?\n• In Fig. 3f and Extended Fig. 13c, why does Cas7-11-HA expression depend on crRNA? Were these experiments\nperformed without crRNA rather than using a non-targeting control?", "reviewer_2": "Type III CRISPR-Cas systems provide adaptive immunity against foreign genetic elements. In the type III-E system, the\nCas7-11-crRNA-Csx29 complex recognizes and cleaves RNA targets that are complementary to the crRNA guide. In\naddition, the binding of target RNA activates the protease activity of Csx29, leading to the cleavage of the Csx30 protein and\nresulting in abortive infection. In this study, He et al. integrated gasdermin proteins with the type III-E CRISPR system to\ndevelop the DAMAGE (death manipulation gene) system, which triggers pyroptosis in human cells expressing specific target\nRNA. Furthermore, the authors demonstrated that DAMAGE can selectively eliminate virus-infected, cancerous, and\nsenescent cells, which exhibit altered RNA transcriptomes. The DAMAGE system has the potential to serve as a new\ntechnology for the programmable induction of pyroptosis. However, the quality of the manuscript, particularly the figures, is\ntoo poor to adequately understand the significance of the work. Therefore, I cannot support its publication in Nature\nCommunications.\nNumerous points need to be corrected, including the following:\nP4: “Cas7-11 specifically recognizes target RNA (tgRNA) that is complementary to CRISPR RNA (crRNA; CR).”\n- The abbreviation CR is defined for crRNA, but it is not mentioned afterward.\nP7: “Both forms exhibited significantly increased in the ON+ groups (Fig. 1b).”\n- This sentence is grammatically incorrect.\nP7: “Meanwhile, the ON+ groups showed decreased green fluorescence intensity (Fig. 1c and Extended Data Fig. 4d),\nincreased PI-labeled dead cells (Fig. 1c)…”\n- The abbreviation PI (propidium iodide) should be defined here.\nUnfortunately, the resolutions of all the figure panels and font sizes are too low to be understood. As stated in the journal\nguideline, resolutions should be ~300 dpi and font sizes should be at least 5 pt (typically 7 pt). In most graphs, replicates are\nnot provided in the legends. All data points should be plotted in the graphs. Bars should not overlap and should be\npresented separately (e.g., Fig. 1e).", "reviewer_3": "Pyroptosis is a highly inflammatory form of programmed cell death (PCD) that stands apart from other types of cell death due\nto its dramatic mechanism. Unlike conventional cell death processes, pyroptosis leads to the rupture of the cell membrane,\nreleasing cellular contents and triggering a robust immune response. This process is predominantly regulated by Gasdermin\nproteins, with Gasdermin D (GSDMD) playing a key role. Upon detection of pathogens or danger signals by intracellular\nimmune sensors, such as the inflammasome, enzymes like caspase-1 are activated. These enzymes cleave Gasdermin\nproteins, unleashing their pore-forming activity. The cleaved Gasdermin proteins then form pores in the cell membrane,\nallowing water to rush in. This influx causes the cell to swell and ultimately burst, releasing its contents—including\ninflammatory cytokines—that signal the immune system and help defend the body against infections. While pyroptosis is\nessential for immune defense, its excessive or inappropriate activation can be detrimental. It has been implicated in a range\nof diseases, including inflammatory bowel disease (IBD), sepsis, and various cancers.\nIn the submitted manuscript by Mingbin He et al., the authors introduce an innovative synthetic biology platform, DAMAGE,\nwhich creatively integrates CRISPR Type III-E systems with Gasdermins to induce pyroptosis. Based on their findings, I\nbelieve this represents a significant advancement in the field of programmable cell death technology. I have the following\nquestions/comments:\nMajor points:\nThe cell lines used are relatively limited, seemingly only HEK293T and HeLa cells, which does not sufficiently demonstrate\nthat the method works in other cell types. Therefore, it cannot be considered a universally applicable approach. The authors\nshould validate their technology in additional cell lines and/or primary mouse cell lines. It is not necessary to repeat all\nsimilar experiments; for example, performing PI staining or LDH release in different cell lines or even mouse primary cells\nwould be sufficient for broader validation.\n1.Figure 2: Since RSV primarily infects respiratory epithelial cells, it would strengthen the physiological relevance of your\nfindings to demonstrate the results using a respiratory epithelial cell line.\n2.Figure 4: To support the generalizability of your findings, please consider including additional cancer cell lines. It may not\nbe necessary to repeat all experiments—perhaps just the PI and LDH assays would suffice.\n3.Figure 5: Can the DAMAGE selectivity also target other types of senescent cells, such as those induced by doxorubicin or\nH₂O₂?\n4.Figure 6: Can this technology be used on other cell lines or in mouse primary cells? Expanding the validation would\nenhance the robustness of the findings.\nMinor points\n1.Figure 1c: Was the PI assay performed with three independent replicates? If not, please repeat the experiment to ensure\nstatistical validity. Also, please clarify the meaning of “N-” and “N+” in the figure legend.\n2.Figure 1: It appears that D-FL and D-SL did not work. Please explain or discuss these observations.\n3.Figure 1d: Was this experiment performed with three independent replicates? If not, please do so to support the reliability\nof the data.\n4.Figure 1e: Was this also a three-independent assay? Additionally, there is no N- control group shown, and the overlapping\ncolumns make it difficult to distinguish between groups. Consider displaying the data as in Figure 1c for better clarity.\n5.Figures 1b, 2b, 2f: Please specify which antibodies were used for the Western blots detecting GSDMs-Csx30, GSDMs-\nCsx30-N, D-X, and D-X-N. Indicate these details in the figure legends.\n6.Figure 1f: The zoomed-in view does not show pyroptotic bubbles. Please consider using a higher-resolution microscope to\nbetter visualize these features. In the absence of clear pyroptotic bubbles, it is inappropriate to classify the cells as pyroptotic\nbased solely on phase-contrast images.\n7.Figure 1g: Please clarify what FLAG label is and include this information in the figure legend.\n8.Figure 2b: What does “d” refer to in the figure? Please define it in the legend for clarity.\n9.Figure 2c: Please indicate how the immunoprecipitation (IP) was performed, including which antibody was used. This\ninformation should be included in the figure or figure legend.\n10.In general, please ensure that all experiments are performed with at least three independent replicates to support\nreproducibility.\n11.If possible, please include p-values in all relevant figures to indicate statistical significance.\n12.Figures 4g and 4h: Please indicate the statistical method used to calculate the p-values. Also, review other figures to\nensure this information is consistently provided.\n13.The Methods section requires significant revision to improve clarity and reproducibility. More detailed descriptions will\nallow other researchers to replicate your findings."}, "author_response": "We greatly appreciate the reviewer’s favorable summary and review and have addressed all\nthe points raised, which definitely help us to improve the manuscript substantially.\nDespite the groundbreaking discovery that the Cas7-11-Csx29 complex can specifically\ncleaves Csx301, substantial barriers persist in leveraging this activity for clinically viable\nRNA-targeted therapeutics. While current applications primarily exploit Cas7-11's nuclease\nactivity for targeted RNA editing, our study pioneers the integration of this system with gasdermin\n(GSDM) family proteins. This novel strategy not only circumvents the complex activation\nmechanisms of the GSDM family under natural conditions, but also converts CRISPR-detected\nRNA signals into protease-mediated cascades, enabling precise pyroptotic cell death for treating\nRNA-associated pathologies.\nThanks for your suggestions regarding the application of CRISPR systems in human cells. In\nthe final section of our study, we demonstrated that the system can be in vitro transcribed into\nRNA and successfully transfected into cells while retaining functionality. Furthermore,\nmRNA-LNP-mediated delivery achieved efficient transduction in human cell lines, resulting in\neffective clearance of target RNA-harboring cells. Please see the response, Fig. R1.\nFig. R1 The LDH release of mRNA transfection (a, b) or mRNA-LNP delivery (c), triggered\nby endogenous HPV18-E7 in HeLa cells\nIn response to queries on the efficacy of the DAMAGE system, we highlighted that\ntransduction-limited cells expressing high-potency effectors, notably GSDMB-Csx30-FL and\nGSDMC-Csx30-FL exhibit near-universal pyroptotic death even in cases of endogenous triggering,\nvalidating the functional potency despite constrained delivery. Please see the revised manuscript,\nFig. 1c and Fig. 3g.\nThe DAMAGE system exhibits single-base resolution in recognition of targeted\nKRAS-G12C mutations, as validated by allele-specific pyroptosis induction. Critical to this\nprecision, spacer positioning of the discriminatory nucleotide was optimized through\ncombinatorial screening. Results identified positions 16 and 23 (from the 3' spacer end) as optimal\nsites for G12C discrimination. Please see the revised manuscript, Figure. 5b. Additionally, we\ntransfected cells with either mCherry or EGFP combined with the DAMAGE system, but\nintroduced only crRNA targeting EGFP. Upon mixing these two cell populations, we observed\na significant reduction in green fluorescence compared to the control group, while red\nfluorescence remained unchanged. This result confirms the DAMAGE with strong selective\nspecificity. Please see the response, Fig. R2.\nFig. R2 The co-culture experiment to verify the specificity of DAMAGE in killing target cells\nWe appreciate your inquiry regarding endogenous triggering capability. To address this\nconcern, we conducted comprehensive validation at the following endogenously expressed genes:\n1) Endogenous triggering of HPV18 E6/E7 oncoproteins in HeLa cells. Please see the revised\nmanuscript, Fig. 3b and g.\n2) Endogenous activation in KRAS-G12C engineered stable HEK293T and HeLa cell lines.\nPlease see the revised manuscript, Fig. 4f and Supplementary Fig. 9h.\n3) NCI-H23 cells with endogenous G12C mutations and A549 cells with endogenous G12S\nmutation to trigger DAMAGE-KRAS. Please see the revised manuscript, Fig. 4j and k.\n4) Endogenous elevated p16/p21-mediated senescent cell death. Please see the revised\nmanuscript, Fig. 5g and Supplementary Fig. 11g.\nThese experiments collectively substantiate the system's robust performance in response to\nendogenously expressed genes.\nI have two primary concerns with this work.\nThe first is an issue with the presentation and clarity of the paper. I found the manuscript difficult\nto follow, and I believe that too much unnecessary data is presented, which obscures the main\nfindings. This is a case where “less is more,” and the authors could have selected the best\nDAMAGE construct from Fig. 1 and presented it more clearly and rigorously for the rest of the\npaper. To me it would have been more useful to optimize one construct rather than testing all the\nreporters in parallel with multiple target RNAs. In my opinion, Figs. 2-6 are not that useful, as we\nknow the system is programmable and can be applied to different targets.\nWe sincerely regret the issues with manuscript formatting and image clarity in our initial\nsubmission. Due to journal file size restrictions, necessary image compression inadvertently\nimpeded viewing clarity, hindering your review process. In this revised manuscript, we have\nprovided uncompressed, high-resolution versions of all figures to ensure optimal viewing quality.\nWe fully concur with your view that \"less is more.\" However, at the outset of our research, it\nwas unclear which gasdermin family protein fused with the Csx30 effector possessed the strongest\npore-forming activity. Therefore, we investigated all GSDMs-Csx30 effectors and discovered that\ndifferent effectors exhibit varying pyroptosis-inducing capacities. In our subsequent experiments,\nwe primarily focused on the two most potent GSDMs, GSDMB-Csx30 or GSDMC-Csx30\n(containing B-FL, B-SL, C-FL and C-SL) for further study.\nIn this study, we primarily aimed to investigate whether DAMAGE could effectively\nrecognize target cell RNA and achieve the transduction of nucleic acid signals to protease signals,\nand whether this capability can be applied to disease treatment. Therefore, after constructing this\nsystem, we conducted investigations in different disease models (exogenous viral infection (RSV),\ncancers with integrated exogenous genomes (HPV), KRAS-mutant cancers (KRAS-G12C/\nKRAS-G12S) and senescent cells (p16 and p21), Fig. 2 to 5), demonstrating that the system\npossesses the capability to eliminate target cells. In Figure 6, we demonstrated that the system\ncould be delivered into cells in the form of mRNA-LNP, opening possible avenues for the\ntreatment of certain diseases associated with abnormal mRNA.\nNevertheless, we fully recognize the limitations of our study. Although the DAMAGE\nsystem has demonstrated a specific cytotoxic effect on target cells across multiple cell lines, its\nefficacy and functional behavior have not yet been evaluated in more complex biological systems,\nsuch as organoid or murine models. Therefore, the system is still in an early, exploratory phase of\ndevelopment. We greatly appreciate the suggestion that you gave, we can select the\nhighest-activity effector for optimization in subsequent experiments. Meanwhile, additional in\nvivo studies are necessary to assess its translational potential and feasibility for future clinical\napplications.\nI also found that the text and methods lacked sufficient detail, making some experiments unclear.\nIt is not always explicitly stated how the experiments were conducted, whether target genes were\ntransfected or integrated, which promoters were used, and whether targets were overexpressed.\nAdditionally, certain figure abbreviations do not seem to be explained in the text or legends.\nWe are deeply sorry for not giving the sufficient detail of the text and methods. We strongly\nagree that additional details in the methods and results sections would enhance the clarity of our\nexperimental procedures. In the revised manuscript, we have provided detailed descriptions of the\nfigure elements and defined all relevant abbreviations in the text or figure legends.\nThe second major concern is the practical utility of this system outside of overexpression\nexperiments in HEK293 cells. Many core ideas of this paper—using the CRISPR protease in\nhuman cells, truncating Csx30 as a linker to trigger biological activities, and engineering\ngasdermins for cleavage by other proteases—have already been explored. While this work extends\nthese concepts in an interesting direction, it does not fundamentally advance our understanding of\nCRISPR protease biology or inducible cell death circuits.\nThanks for reviewer’s attention on the experiments, outside the overexpression experiments\nin HEK293 cells, we also applied DAMAGE in some endogenous genes that mentioned above.\nThese experiments collectively substantiate the system's robust performance in response to\nendogenously expressed genes.\nThe Cas7-11-Csx29-Csx30 system, as the first RNA-guided protease system1, 2, has seen its\nmechanistic understanding advance from structural biology3 insights to dynamic regulatory\nnetworks, while its applications are centered on RNA diagnostics and synthetic biology tools\nwhich are primarily focused on the Cas7-11’s nuclease activity. On the other hand, engineering\nheterologous protease cleavage systems has enabled Gasdermin-based platforms to overcome the\ninherent limitation of caspase dependency. This breakthrough underscores the tremendous\ntherapeutic potential of Gasdermin proteins in treating tumors, fibrotic disorders, and\ninfections. Significantly, our study pioneers the use of Csx30 as a linker connecting the N-\nand C-terminal domains of GSDM proteins.This innovative design capitalizes on the unique\nattributes of the Cas7-11-Csx29-Csx30 complex as an RNA-targeted proteolytic system, enabling\nspecific detection of endogenous target RNAs and selective elimination of pathogenic cells,\nthereby providing a promising therapeutic strategy for RNA heterogeneous diseases.\nI therefore disagree with the claim that this work demonstrates “considerable promise” for\ntherapeutic applications. The PI-positive cell quantification in Fig. 1c shows an increase from 5%\nto 15-20%, and the ATP-based readout in Fig. 1e shows viability decreasing from 85-90% to\n60-70%. As such, it seems to fall into a “proof of principle” zone but I am not sure this is\nsufficient for publication. In my opinion the authors would need to improve the system and show\nmore compelling results beyond HEK293 cells.\nRegarding the concern you raised about the relatively modest increase in the PI-positive rate,\nwe propose two main explanations. Firstly, in our experiments, PI positive (PI+) rate (%) was\nquantified as the proportion of PI+ cells relative to the total cell population. Samples were\ncollected at approximately 36 hours post-transfection, by which time nearly all successfully\ntransfected cells had undergone death, consistent with the marked reduction in EGFP-positive\n(EGFP+) cells observed. However, due to inherent limitations in transfection efficiency and the\nrapid proliferation of HEK293 cells, un-transfected cells remained viable and expanded\nsubstantially. This led to a high total cell number at the time of sampling, thereby diluting the\nproportion of PI+ cells relative to the total population. Secondly, during the late stages of cell\ndeath, cellular disintegration generates fragmented debris. In Flow cytometry analysis, these\nfragments were gated out as cellular debris and excluded from the total cell count, further\ncontributing to the apparent low PI-positive rate. To address your concerns, we collected samples\nbefore the cells were completely lysed, and the PI staining level was significantly increased,\nplease see the response, Fig. R3. Additionally, due to the earlier sample collection time, the\nmaximum EGFP+ rate also decreased.\nFig. R3 Characterization of pyroptotic activity of GSDMs-Csx30 effectors\nthrough Flow Cytometry assay, using EGFP as tgRNA\nWith respect to the relatively low reduction in ATP levels, two factors may be relevant. First,\nour system directly exploits the pore-forming activity of GSDMs function as the downstream\nexecutors of pyroptosis, which does not interfere with ATP synthesis pathways via signal\ntransduction. Secondly, similar to the low PI-positive issue, the rapid proliferation of HEK293\ncells likely counteracts the ATP reduction, as the increased number of viable un-transfected cells\nmaintains overall ATP levels.\nMeanwhile, we also measured LDH release, which increased by approximately 40%, also\nsuggests the occurrence of intense pyroptosis.Please see revised manuscript, Fig. 1d. Western\nblot analysis of intracellular effector proteins revealed that full-length B-FL and C-FL were barely\ndetectable in ON groups. Please see B-FL and C-FL group in revised manuscript, Fig. 1b.\nThank you for your comment regarding the initial limitation of our experiments to HEK293\ncells. To address this concern and better demonstrate the applicability of DAMAGE system as you\nsuggested, we have extended our investigations to additional cell models. Specifically, we have\nperformed the core experiments and relevant supplementary assays in HeLa cells, 293T and HeLa\nstable cell lines, C33-A cells, A549 cells, and NCI-H23 cells harboring the KRAS-G12C mutation.\nWe hope these expanded data will meet the expectations regarding the system's applicability.\nOverall, while the study presents a potentially interesting application of previous work and\nattempts to tackle an important biological problem, to me it does not sufficiently address key\nchallenges or demonstrate a substantial advance.\nIn our study, although the DAMAGE system still has aspects requiring improvement, it also\ndemonstrates the capability to precisely recognize target RNA (including single-nucleotide\nmutations in KRAS), please see the revised manuscript, Fig. 4b and e; it can respond to\nextremely low levels of target RNA (responding to RNA transcribed from plasmid concentrations\nof 2-7 μg and RSV infection at an MOI of approximately 2-7), please see the revised manuscript,\nSupplementary Fig. 7a-e, and effectively trigger pyroptosis. Importantly, the DAMAGE system is\ncapable to detect HPV18 mRNA transcribed from the HeLa cell genome and inducing pyroptosis,\nsuggesting its promising potential for application in the treatment of cervical cancer. As\ndemonstrated in Fig. 6, the DAMAGE system targeting HPV18-E7 mRNA—whether delivered in\nplasmid (Fig. 6c, d), mRNA (Fig. 6e-h), or mRNA-LNP (Fig. 6i) form—exhibited the ability to\nspecifically induce pyroptosis. Please see the revised manuscript, Fig. 6. These characteristics of\nthe system providepromising therapeutic strategiesfor treating RNA-related diseases.\nSpecific Comments:\n• The figure legends and text lack clarity in explaining the experiments and different conditions\ntested. For instance, the abbreviations in Fig. 1 (N- and N+) are not clearly defined. Some of the\ndata presented feels a bit unnecessary and makes the paper “heavy” and could be moved to\nsupplementary materials or omitted for ease of reading.\nWe are sorry for the lack of clarity in explaining the experiments and different conditions\ntested. Based on the principle that Csx29 cleaves Csx30 at residues 427-429, a plasmid expressing\nGSDMs-Csx30-N, which mimics N-terminal product generated by effector cleavage, was\nconstructed as a positive control. For example, A-SL-N represents a fusion protein of GSDMA-N\nwith a fragment of amino acids 407 to 429 of Csx30. \"-\" represents the group transfected with\nnon-targeting crRNA (CR-NT), and \"+\" represents the group transfected with crRNA that can\nrecognize the target RNA, for example EGFP-crRNA-Mix in Figure 1c.\nIn the revised manuscript, we have elaborated on the relevant experiments and specified the\nexperimental conditions. The issues you raised regarding the abbreviations \"N-\" and \"N+\" (please\nsee the revised manuscript, Line 157-159) and other ambiguous descriptions have been addressed.\nFollowing your suggestion, we have defined all relevant abbreviations in the corresponding figure\nlegends. Thank you for your suggestions on the presentation of our paper. To better present our\nresearch, some redundant data have been deleted or moved to the supplementary materials (eg:\nSupplementary Fig. 6d in revised manuscript). Additionally, we have reorganized the figures to\nensure a logical progression throughout the manuscript.\n• Line 134: “All GSDMs-Csx30 effectors can respond to tgRNA and induce pyroptosis, but they\ndisplay a distinct control over pyroptotic activity.” However, D-SL and D-FL clearly do not\nrespond to target RNA. Why do the authors make this claim and continue to test these constructs\nthat do not seem promising for this application?\nWe acknowledge that D-SL and D-FL do not perform as effectively as other GSDMs-Csx30\neffectors. However, both D-SL and D-FL are capable to recognize and respond to target RNA. As\nshown in Fig. 1c and 1d, although significant cell death occurred in both D-SL and D-FL groups,\nthe PI-positive cell rate and LDH levels in the untriggered condition (OFF) were statistically\nsignificantly lower than those in the triggered condition (ON). Please see the response, Fig. R3\nand revised manuscript, Fig. 1c, d. (P values were calculated by two way ANOWA with Sidak's\nmultiple comparisons test. And the p-values between OFF and ON of D-SL and D-FL were all\nless than 0.0001. (****p < 0.0001)).\nWe continued to investigate unmodified GSDMD constructs (D-FL and D-SL) for two\nprimary reasons. On the one hand, the pronounced cytotoxicity of both D-FL and D-SL induces\novert pyroptosis in transfected cells. This provides a visually distinguishable positive control that\nallows us to objectively evaluate experimental reliability and transfection efficiency. More\nimportantly, D-SL and D-FL, as well as D-X and D-Y, represent two particularly intriguing\ncontrasting combinations. In comparison with D-SL and D-FL, the D-X and D-Y constructs\nincorporated an additional short NES sequence at the N-terminus. Please see the revised\nmanuscript, Fig. 1a. Notably, this modification was found to effectively reduce the non-specific\npyroptotic activity of the GSDMD-Csx30 effectors, particularly in the case of D-X. Furthermore,\nas we mentioned in the manuscript, the pyroptosis-inducing capability of GSDMs-Csx30-SL was\nattenuated, while non-specific pyroptotic activity was significantly diminished, especially between\nB-SL and B-FL. These findings suggest that a moderate attenuation of the pyroptosis-inducing\ncapacity in cleaved GSDMs-Csx30 may effectively reduce nonspecific pyroptotic events. We\ndiscussed this phenomenon in the Discussion section, please see the revised manuscript, Line\n575-592. We believe that this information is important for readers who want to apply and further\nimprove the DAMAGE system, and that they can gain valuable experience and inspiration.\n• There appears to be selective presentation of different constructs across figures, which\nintroduces confusion. For example, Fig. 1f shows C-FL, Fig. 1g shows A-FL, and Fig. 2b switches\nto D-X. A more consistent focus on the best reporter construct would improve the manuscript.\nThank you for reaching out the question on the presentation of the figures. During the initial\nphase of the experiment, we primarily used D-X to assess the pyroptosis-inducing capacity of the\nfusion proteins incorporating the Csx30 linker between GSDMs N-terminus and C-terminus. The\nresults showed that the GSDMX-Csx30 effector can still be successfully cleaved by activated\nCsx29, please see the revised manuscript, Supplementary Fig. 2b and c. Subsequently, to\ncomprehensively evaluate the potential of different GSDM family proteins, we tested\neffectors GSDMA to GSDME with Csx30 as linker, and identified GSDMB and GSDMC\neffectors exhibited superior activity. Therefore, to best demonstrate DAMAGE's efficacy, these\neffectors were predominantly used in subsequent experiments. However, the rapid cell death\nkinetics induced by B-FL and C-FL impeded timely capture of membrane ballooning during\nconfocal microscopy. Consequently, for the Fig. 1g confocal analysis, we employed the less\ncytotoxic A-FL effector, which enabled timely visualization of pyroptotic ballooning dynamics.\n• The positive control in Fig. 1e at ~48% is unclear. Additionally, where are the error bars indicating standard deviation\nbetween replicates?\nWe sincerely apologize for omitting details regarding the positive control. The positive\ncontrol consisted of cells only transfected with the GSDMD-N-terminal fragment (D-N). This\ninformation has been added to the revised manuscript, please see the revised manuscript, Line\n166-167. Additionally, we have added error bars to the figures and specified the number of\nbiological replicates in the figure legends.\n• A critical issue with CRISPR protease systems, as seen with other RNA-targeting systems (e.g.,\nRADAR, CellREADR), is the requirement for high levels of target RNA. The authors should test\na range of endogenous target genes to evaluate DAMAGE efficiency across physiologically\nrelevant RNA levels.\nWe appreciate your suggestion to include endogenous activation experiments. We performed\nthe following endogenous triggering assays:\n1) Targeting endogenous HPV18 E6/E7 mRNA in HeLa cells，please see the revised manuscript,\nFig. 3b-g.\n2) Endogenous activation in NCI-H23 cells carrying the KRAS G12C mutation, please see the\nrevised manuscript, Fig. 4j. Endogenous activation in A549 cells harboring the KRAS G12S\nmutation, please see the revised manuscript, Fig. 4k.\n3) Endogenous triggering assays targeting p16/p21 in both 293T and HeLa cell lines, please see\nthe revised manuscript, Fig. 5g.\nUnlike other RNA-targeting systems, we propose that the Cas7-11-Csx29-Csx30 complex in\nDAMAGE functions as a reaction cascade with signal amplification. When even trace amounts of\ntgRNA are present in cells, they can be recognized by crRNA, which mediates the activated\nCas7-11 to potently activate abundant Csx29. Once activated, Csx29 continuously cleaves the\nGSDMX-Csx30 effector, thereby constantly releasing its N-terminal fragment. The released\nN-terminal fragment possesses pore-forming functionality, ultimately mediating the occurrence of\npyroptosis. Our titration experiments in RSV demonstrate the remarkable sensitivity of the\nDAMAGE system. We observed DAMAGE activation even at minimal stimulation levels:\ntransfection of only 2–7 μg RSV-N plasmid or infection with trace amounts of RSV virus. Please\nsee the revised manuscript, Supplementary Fig.7d, e. Endogenous assays successfully conducted\nacross multiple cell lines as mentioned above further corroborate this conclusion. This strongly\nsuggests that DAMAGE can detect target RNAs with high sensitivity in intact cells.\n• Fig. 6 claims LNP delivery of DAMAGE mRNA, but only Figs. 6h and 6i seem to include\nactual mRNA delivery. Since these experiments are performed in HEK293 cells, likely\noverexpressing the targets, they don’t seem to add much value beyond the plasmid transfection\nexperiments.\nWe are grateful for your valuable suggestions regarding the limitations of the cell line\nselection and experimental design in mRNA delivery. We have supplemented the manuscript with\nmRNA transfection and mRNA-LNP delivery experiments in HEK293T cells, demonstrating that\nthe DAMAGE system enables RNA-based therapeutic delivery. Please see the response, Fig. R4.\nFig. R4 The PI-positive rate and LDH release level of 293T when treated with mRNA (a) and\nmRNA-LNP (b), using HPV18 E7 as tgRNA\nBeyond targeting exogenous RNA, we supplemented the study with RNA transfection\nexperiments in HeLa cells. The result confirmed that endogenous HPV18 E7 mRNA can activate\nthe DAMAGE system introduced via RNA and induce pyroptosis. Moreover, we conducted\nmRNA transfection and mRNA-LNP delivery experiments in HeLa cells and demonstrated that it\ncould successfully respond to endogenous mRNA. Please see the response, Fig. R5.\nFig. R5 mRNA transfection (a) and mRNA-LNP delivery experiments (b) in HeLa cells,\nusing endogenous HPV18 E7 as tgRNA\n• The term “tgRNA” for target RNA feels unnecessary and complicates readability.\nIn accordance with the abbreviation \"tgRNA\" (target RNA) used by Kazuki Kato et al1, we\nhave adopted this terminology. Additionally, we also found that spelling out \"target RNA\" in\nfigures caused uneven edges that compromised layout integrity and visual coherence.\n• Some schematics, such as those in Fig. 1a and Fig. 4a, are too small. A larger, clearer depiction\nof a representative construct (such as crRNA-23 in the KRAS experiment) would be preferable.\nWe are sorry for the small size of some schematics. For the sake of image layout, we have\nmade slight adjustments to the image sizes (including Fig. 1a and Fig. 4a). Meanwhile, we have\nuploaded zoomable, clear images for viewing details and have highlighted key steps or\nrepresentative construct to improvedepiction clarity in the revised manuscript.\n• Line 89: truncation analysis of Csx30 has already been performed in previous studies and\nshould not be claimed as novel.\nWe sincerely appreciate your identification of the problem in our Csx30 truncation analysis\nexperiment. During our investigation, we referenced previous studies reporting Csx30 truncation\nexperiments and did not present this as a novel discovery. However, to determine the optimal\nlength of Csx30 serving as the linker between GSDM-N and GSDM-C, we conducted similar\ntruncation experiments to ensure the specificity these effectors. Our results identified Csx30\n407-565aa as the optimal linker among the screened mutants. Please see the revised manuscript,\nLine 97-105.\n• Certain panels, such as those in Fig. 1f and 1g, should directly indicate which DAMAGE\nconstructs are being tested to improve clarity.\nThank you for your suggestion. We have provided clarifications in the figure legends. Please\nsee the revised manuscript, Line 167 and 174. For Fig. 1f, we employed C-FL effector for\nexperiment and it was a fluorescence microscopy image that does not involve antibody staining.\nAnd for Fig. 1g, we employed anti-Flag antibody to detect Flag-tagged A-FL effector. For Fig. 1f\nand 1g, we have labeled the tested DAMAGE construct in the figure legend.\n• Some western blot results are unclear. In Fig. 1b and 2b, the full-length and cleaved products of\nD-X are visible, but in Fig. 3b, the cleaved product is absent. The separate panel showing D-X-N\nraises questions; why is the cleaved product not observed in the FLAG blot, and what does D-X-N\nrepresent?\nWe regret the insufficient clarity of some western blot results in the original submission. In\nthe revised manuscript, we have provided additional clarification in the text and figure legends to\nfacilitate interpretation of our findings.We also sincerely appreciate the questions you have raised.\nD-X-N represents the N-terminal fragment released after the GSDMD-X-Csx30 effector cleaved.\nThe transfection experiments in Fig. 1b and 2b were conducted in 293T cells via exogenous\nmRNA, whereas Fig. 3b shows endogenous triggering in HeLa cells. Relative to 293T cells, HeLa\ncells display both lower transfection efficiency and weaker endogenous signal induction compared\nto the robust exogenous overexpression achievable in 293T cells. Consequently, no cleaved\nproduct was detected in Fig. 3b under the same exposure conditions. The D-X-N fragment shown\nin Fig. 3b was visualized through extended exposure time. Regarding the undetectable cleaved\nN-terminal products in FLAG blots, this occurs for the FLAG tag is fused to the effector's\nC-terminus. Thus, only the cleaved N-terminal fragment can be detected using specific\nendogenous antibodies (GSDMD, 39754S，CST) which binds to the N-terminal region of\nGSDMD.\n• In Fig. 3f and Extended Fig. 13c, why does Cas7-11-HA expression depend on crRNA? Were\nthese experiments performed without crRNA rather than using a non-targeting control?\nThank you for your questions and we sincerely apologize for the misunderstanding we may\nhave caused. The decrease in the expression level of Cas7-11-HA is due to cell death but not\ndepends on crRNA. After the cells undergo pyroptosis, the cell membrane ruptures and the\ncellular contents are released into the supernatant. In the ON group, the presence of Cas7-11 or\nother released proteins can be detected in the immunoprecipitated supernatant, please see the\nresponse, Fig. R6. We added an explanation this phenomenon in lines 122-131 of the revised\nmanuscript. The experiment of OFF group was performed using a non-targeting crRNA, which\nwas described in lines 119-120 of the revised manuscript.\nFig. R6 Immunoblotting assay of the cell lysates and supernatants-IP for A-FL effectors,\nusing RSN-N as tgRNA\nReviewer #2 (Remarks to the Author):\nType III CRISPR-Cas systems provide adaptive immunity against foreign genetic elements. In the\ntype III-E system, the Cas7-11-crRNA-Csx29 complex recognizes and cleaves RNA targets that\nare complementary to the crRNA guide. In addition, the binding of target RNA activates the\nprotease activity of Csx29, leading to the cleavage of the Csx30 protein and resulting in abortive\ninfection. In this study, He et al. integrated gasdermin proteins with the type III-E CRISPR system\nto develop the DAMAGE (death manipulation gene) system, which triggers pyroptosis in human\ncells expressing specific target RNA. Furthermore, the authors demonstrated that DAMAGE can\nselectively eliminate virus-infected, cancerous, and senescent cells, which exhibit altered RNA\ntranscriptomes. The DAMAGE system has the potential to serve as a new technology for the\nprogrammable induction of pyroptosis. However, the quality of the manuscript, particularly the\nfigures, is too poor to adequately understand the significance of the work. Therefore, I cannot\nsupport its publication in Nature Communications.\nWe sincerely appreciate your recognition of the research content presented in our manuscript.\nWe would like to offer our earnest apologies for any difficulties encountered in reading the initial\ndraft due to issues with clarity. In the revised manuscript, we have included high-resolution\nimages, with the aim of addressing any inconveniences this may have caused. We kindly hope that\nour research will meet your approval.\nNumerous points need to be corrected, including the following:\nP4: “Cas7-11 specifically recognizes target RNA (tgRNA) that is complementary to CRISPR\nRNA (crRNA; CR).”\n- The abbreviation CR is defined for crRNA, but it is not mentioned afterward.\nTo optimize the layout of the figures, we primarily employed the abbreviation CR during the\nfigure preparation process and figure legend, such as illustrated in Fig. 4h and Fig. 5b, etc. Please\nsee the revised manuscript, Fig. 4h and 5b.\nP7: “Both forms exhibited significantly increased in the ON+ groups (Fig. 1b).”\n- This sentence is grammatically incorrect.\nBoth forms exhibited significantly increase in the ON+ groups (Fig. 1b)\nWe are sorry for the mistakes, and have modified according to grammatical rules. Please see\nthe revised manuscript, Line 122-131.\nP7: “Meanwhile, the ON+ groups showed decreased green fluorescence intensity (Fig. 1c and\nExtended Data Fig. 4d), increased PI-labeled dead cells (Fig. 1c)…”\n- The abbreviation PI (propidium iodide) should be defined here.\nWe sincerely apologize for our omission in specifying the full name of PI. We have rectified\nthis by adding PI (propidium iodide) in the revised manuscript (Line 133).\nUnfortunately, the resolutions of all the figure panels and font sizes are too low to be understood.\nAs stated in the journal guideline, resolutions should be ~300 dpi and font sizes should be at least\n5 pt (typically 7 pt). In most graphs, replicates are not provided in the legends. All data points\nshould be plotted in the graphs. Bars should not overlap and should be presented separately (e.g.,\nFig. 1e).\nWe sincerely apologize for the difficulties you encountered while reviewing the initial draft,\nwhich arose from poor image clarity and excessively small annotation font sizes. During the initial\nsubmission, the images were compressed due to file size restrictions for upload. However, in the\nrevised manuscript, we have therefore provided high-resolution versions. Thanks for your\nsuggestions regarding image clarity and font size, we have revised all the relevant figures to\nrequested resolutions, and increased all undersized font size to ≥5 pt as requested in the revised\nmanuscript. With respect to data reproducibility, we have defined all the biological replicates\n(n-values) in the figure legends, and incorporated all data points in the figure as possible. For Fig.\n1e, given the large number of experimental groups, we employed a composite layout to present the\ndata, with group names clearly labeled in the figure legend to optimize overall arrangement.\nPlease see the revised manuscript, Line 179-180. We have also included the error bar for all data\nas per your recommendations.\nReviewer #3 (Remarks to the Author):\nPyroptosis is a highly inflammatory form of programmed cell death (PCD) that stands apart from\nother types of cell death due to its dramatic mechanism. Unlike conventional cell death processes,\npyroptosis leads to the rupture of the cell membrane, releasing cellular contents and triggering a\nrobust immune response. This process is predominantly regulated by Gasdermin proteins, with\nGasdermin D (GSDMD) playing a key role. Upon detection of pathogens or danger signals by\nintracellular immune sensors, such as the inflammasome, enzymes like caspase-1 are activated.\nThese enzymes cleave Gasdermin proteins, unleashing their pore-forming activity. The cleaved\nGasdermin proteins then form pores in the cell membrane, allowing water to rush in. This influx\ncauses the cell to swell and ultimately burst, releasing its contents—including inflammatory\ncytokines—that signal the immune system and help defend the body against infections. While\npyroptosis is essential for immune defense, its excessive or inappropriate activation can be\ndetrimental. It has been implicated in a range of diseases, including inflammatory bowel disease\n(IBD), sepsis, and various cancers.\nIn the submitted manuscript by Mingbin He et al., the authors introduce an innovative synthetic\nbiology platform, DAMAGE, which creatively integrates CRISPR Type III-E systems with\nGasdermins to induce pyroptosis. Based on their findings, I believe this represents a significant\nadvancement in the field of programmable cell death technology. I have the following\nquestions/comments:\nWe sincerely appreciate your meticulous review and valuable suggestions. Your insightful\ncomments on the content of the manuscript have provided us with significant guidance for\nimprovement. In response, we have added relevant experimental contents and made major\nrevisions to the manuscript as recommended.\nMajor points:\nThe cell lines used are relatively limited, seemingly only HEK293T and HeLa cells, which does\nnot sufficiently demonstrate that the method works in other cell types. Therefore, it cannot be\nconsidered a universally applicable approach. The authors should validate their technology in\nadditional cell lines and/or primary mouse cell lines. It is not necessary to repeat all\nsimilar experiments; for example, performing PI staining or LDH release in different cell lines or even\nmouse primary cells would be sufficient for broader validation.\nWe sincerely appreciate your suggestion on validating our technology in additional cell lines. In\naccordance with your advice, we have supplemented relevant experiments in cell lines such as\nA549, C33-A, and NCI-H23. The details are provided in the following response or the referenced\nsection of the revised manuscript.\n1. Figure 2: Since RSV primarily infects respiratory epithelial cells, it would strengthen the\nphysiological relevance of your findings to demonstrate the results using a respiratory\nepithelial cell line.\nThank you for your suggestion on employment of the respiratory epithelial cell line to\nstrengthen our findings. We performed infection experiments using RSV-susceptible A549 cells,\nwith the DAMAGE system transfection after infection with RSV, please see the response, Fig.\nR7. It was observed that morphological apoptosis occurs in A549 cells upon normal RSV\ninfection. However, following transfection of the DAMAGE system, it can recognize the mRNA\ngenerated post-RSV infection and trigger pyroptosis in the infected cells, thereby eliminating\nthem.\nFig. R7 Cell imaging (a) and LDH release (b) of A549 after infected with RSV and\ntransfected with DAMAGE-RSV\n2. Figure 4: To support the generalizability of your findings, please consider including\nadditional cancer cell lines. It may not be necessary to repeat all experiments—perhaps\njust the PI and LDH assays would suffice.\nThank you for your suggestion on adding cancer cell lines. We have included the following\nadditional cell lines. In the DAMAGE-RSV section, we supplemented the transfection and\ninfection experiments in A549 cell line, please see the revised manuscript, Fig. 2i, j; In the\nDAMAGE-HPV section, we employed HPV18-negative C33-A cell line as a control, please see\nrevised manuscript, Supplementary Fig. 8h; In the DAMAGE-KRAS section, we initially\nconducted a crRNA screening targeting six common KRAS G12 mutations (G12A, G12C, G12D,\nG12R, G12S, G12V) in the HEK293T cell line. We selected the most effective crRNA and\nsupplemented endogenous triggering experiments using the A549 cell line harboring the\nKRAS-G12S mutation and the NCI-H23 cell line carrying the KRAS G12C mutation, please see\nthe revised manuscript Fig. 4j, k. PI staining, LDH release assays, or cell imaging were\nperformed on the referenced cell lines.\n3. Figure 5: Can the DAMAGE selectivity also target other types of senescent cells, such as\nthose induced by doxorubicin or H₂O₂?\nThank you for your question on the DAMAGE selectivity. In this study, we targeted the\nelevated transcriptional levels of p16 and p21 characteristic of senescent cells. In principle, any\nintervention capable of upregulating p16 or p21 transcription could potentially activate the\nDAMAGE system. Following your recommendation, we treated HeLa cells with Etoposide (2 μM)\nand Doxorubicin (100 nM) which act as the transcriptional activator of p16 and p21, please see\nthe revised manuscript, Supplementary Fig. 11g, and h. Following the treatment, cells exhibited\ncellular enlargement and mitotic arrest. Notably, both drug-treated groups displayed significant\nenhanced pyroptosis compared to untreated group (DMSO), with the Doxorubicin (100 nM) group\nshowing particularly pronounced effects. This was evidenced by distinct pyroptotic pore formation\nand increased LDH release, please see the response, Fig. R8.\nFig. R8 LDH release of HeLa with the treatment of Etoposide or Doxorubicin (a) and Cell\nimaging of HeLa with the treatment ofDoxorubicin (b)\n4. Figure 6: Can this technology be used on other cell lines or in mouse primary cells?\nExpanding the validation would enhance the robustness of the findings.\nThank you for your suggestion. Validation of the DAMAGE system in A549 cells and other cell\nlines mentioned above confirmed its robust functionality. Regrettably, due to the inherent\ntechnical challenges of transfecting primary cells, we were unable to perform corresponding\nexperiments. However, the successful cellular delivery of the DAMAGE system via mRNA-LNP\nin our findings has given us partial confidence to advance DAMAGE toward application. In future\nstudies, we aim to achieve efficient delivery in murine primary cells and relevant disease models.\nMinor points\n1. Figure 1c: Was the PI assay performed with three independent replicates? If not, please\nrepeat the experiment to ensure statistical validity. Also, please clarify the meaning of\n“N-” and “N+” in the figure legend.\nWe sincerely regret the omission of experimental details. The PI assay was performed with\nthree independent replicates in Fig. 1c. Following the suggestion from Reviewer 1, we have\nrepeated the relevant experiment and presented the PI assay data as Mean with SD in the revised\nmanuscript.\nBased on the principle that Csx29 cleaves Csx30 at residues 427-429, a plasmid expressing\nGSDMs-Csx30-N, which mimics N-terminal product generated by effector cleavage, was\nconstructed as a positive control. For example, A-SL-N represents a fusion protein of GSDMA-N\nwith a fragment of amino acids 407 to 429 of Csx30. \"-\" represents the group transfected with\nnon-targeting crRNA (CR-NT), and \"+\" represents the group transfected with crRNA that can\nrecognize the target RNA, for example EGFP-crRNA-Mix in Figure 1c. Additionally, the\ndefinitions of N- and N+ have been explicitly defined in the figure legend, please see revised\nmanuscript Line 157-159.\n2. Figure 1: It appears that D-FL and D-SL did not work. Please explain or discuss these\nobservations.\nWe acknowledge that D-SL and D-FL do not perform as effectively as other GSDMs-Csx30\neffectors. However, both D-SL and D-FL are capable to recognize and respond to target RNA. As\nshown in Fig. 1c and 1d, although significant cell death occurred in both D-SL and D-FL groups,\nthe PI-positive cell rate and LDH levels in the untriggered condition (OFF) were statistically\nsignificantly lower than those in the triggered condition (ON). Please see the response Fig. R9\nand revised manuscript, Fig. 1c, d. (P values were calculated by two way ANOWA with Sidak's\nmultiple comparisons test (****p < 0.0001)).\nFig. R9 The PI-positive and EGFP-positive rate of D-SL and D-FL, using EGFP as tgRNA.\nWe continue to investigate unmodified GSDMD constructs (D-FL and D-SL) for two\nprimary reasons. On the one hand, the pronounced cytotoxicity of both D-FL and D-SL induces\novert pyroptosis in transfected cells. This provides a visually distinguishable positive control that\nallows us to objectively evaluate experimental reliability and transfection efficiency. More\nimportantly, D-SL and D-FL, as well as D-X and D-Y, represent two particularly intriguing\ncontrasting combinations. In comparison with D-SL and D-FL, the D-X and D-Y constructs\nincorporated an additional short NES sequence at the N-terminus. Please see the revised\nmanuscript, Fig. 1a. Notably, this modification was found to effectively reduce the non-specific\npyroptotic activity of the GSDMD-Csx30 effectors, particularly in the case of D-X. Furthermore,\nas we mentioned in the manuscript, the pyroptosis-inducing capability of GSDMs-Csx30-SL was\nattenuated, while non-specific pyroptotic activity was significantly diminished, especially between\nB-SL and B-FL. These findings suggest that a moderate attenuation of the pyroptosis-inducing\ncapacity in cleaved GSDMs-Csx30 may effectively reduce nonspecific pyroptotic events. We\ndiscussed this phenomenon in the Discussion section, please see the revised manuscript, Line\n575-592. We believe that this information is important for readers who want to apply and further\nimprove the DAMAGE system, and that they can gain valuable experience and inspiration.\n3. Figure 1d: Was this experiment performed with three independent replicates? If not,\nplease do so to support the reliability of the data.\nWe are sorry that not explained clearly about the details of the experiment. The experiment in\nFigure 1d was performed with six independent replicates. Following your recommendation, we\nhave now specified the number of experimental replicates in the figure legends. Please see the\nrevised manuscript, Line 176-178.\n4. Figure 1e: Was this also a three-independent assay? Additionally, there is no N- control\ngroup shown, and the overlapping columns make it difficult to distinguish between\ngroups. Consider displaying the data as in Figure 1c for better clarity.\nThe experiment in Figure 1e was conducted with three biological replicates. Whether crRNA\nrecognizes the target sequence, both GSDMs-N-Csx30 407-429aa can directly induce pyroptosis\nby permeabilizing the cell membrane. Given that N- and N+ constructs exhibit comparable\ncytotoxic effects (Fig. 1c, d), we only presented data for the N+ group in Fig. 1e.\nThank you for your suggestion on the presentation of Fig. 1e. However, for better optimize\nthe layout of the figures and given the multiplicity of experimental groups, we employed a\nchimeric display strategy for data presentation. To enhance clarity, we provided detailed labeling\nof distinct groups in both the figure legend and superscripts. Please see the revised manuscript,\nLine179-180. We have also included the error bar for the data as recommended.\n5. Figures 1b, 2b, 2f: Please specify which antibodies were used for the Western blots\ndetecting GSDMs-Csx30, GSDMs-Csx30-N, D-X, and D-X-N. Indicate these details in\nthe figure legends.\nThe C-terminus of GSDMs-Csx30 is tagged with FLAG allowing detection using an\nanti-Flag antibody. However, since the tag is located at the C-terminus, detection of the cleaved\nN-terminus requires the use of endogenous antibodies. All endogenous antibodies against GSDM\nproteins employed in this study target epitopes located at the N-terminus. In Figure 1b, to\nsimultaneously detect all the effectors and their cleaved N-terminal fragments, we diluted the\nendogenous antibody anti-GSDMA/B/C/D/E by the recommended dilution ratio according to the\ninstructions, and followed by detection of GSDMs-Csx30 and GSDMs-Csx30-N. Similarly, D-X\nand D-X-N in Figure 2b were detected by the endogenous antibody anti-GSDMD (GSDMD,\n39754S, CST) with its antigenic epitope located at the N-terminus. Thanks for your suggestion to\nindicate antibody details in the figure legends. However, considering that including all antibodies\nin the figure legends would make the article appear cumbersome, we indicated these details in the\nfigure and made an explanation in the Methods section, please see the revised manuscript Line\n831-853.\n6. Figure 1f: The zoomed-in view does not show pyroptotic bubbles. Please consider using a\nhigher-resolution microscope to better visualize these features. In the absence of clear pyroptotic\nbubbles, it is inappropriate to classify the cells as pyroptotic based solely on phase-contrast\nimages.\nWe sincerely apologize for the failure to clearly display the bubbling of pyroptotic cells due to\ninadequate image resolution. In the revised manuscript, we have uploaded high-definition images,\nwith arrows specifically indicating the bubbling phenomenon of the cells.\n7. Figure 1g: Please clarify what FLAG label is and include this information in the figure legend.\nWe are sorry for the lack of detailed clarification. Given that the C-terminus of\nGSDMx-Csx30 is fused with a FLAG tag, so the effector can be detected using anti-FLAG\nantibody. In Figure 1g, FLAG label denotes the A-FL effector incubated with anti-Flag antibody.\nFollowing your recommendation, we have supplemented this information in the figure legend,\nplease see revised manuscript, Line 169-174.\n8. Figure 2b: What does “d” refer to in the figure? Please define it in the legend for clarity.\nWe sincerely apologize for our oversight in failing to provide this clarification in the figure\nlegend. The notation \"d\" denotes catalytically inactive mutants of a nuclease-inactive Cas7-11\nvariant (dCas7-11) or a protease-inactive Csx29 variant (dCsx29), please see the revised\nmanuscript Line 82-84. Thank you for your suggestion, we have now explicitly stated this\ndefinition in the figure legend, please see the revised manuscript, Line 235.\n9. Figure 2c: Please indicate how the immunoprecipitation (IP) was performed, including which\nantibody was used. This information should be included in the figure or figure legend.\nGiven that the effector protein harbors a FLAG tag at its C-terminus, we performed\nimmunoprecipitation (IP) of supernatant using Flag-conjugated agarose beads. HA-tagged\ndCas-7-11 was immunoprecipitated using HA-conjugated magnetic beads, while Myc-tagged\nCsx29 and RSV-N were immunoprecipitated with Myc-conjugated magnetic beads. The detailed\nprocedure was described in Methods section, please see the revised manuscript, Line 831-853.\nFor the detection of proteins obtained via IP, we can use tag-specific antibodies (anti-FLAG,\nanti-HA, anti-Myc) for the corresponding tagged proteins. Following your recommendation, we\nhave supplemented this information in the figure.\n10. In general, please ensure that all experiments are performed with at least three independent\nreplicates to support reproducibility.\nAll experiments were performed with three biological replicates. Thank you for your suggestion,\nwe have indicated the replicate values (n-values) for all experiments in the figure legends of the\nrevised manuscript.\n11. If possible, please include p-values in all relevant figures to indicate statistical significance.\nThank you for your suggestion. In the revised manuscript, we have made every effort to\npresent all p-values and the calculation methods in the figure legends.\n12. Figures 4g and 4h: Please indicate the statistical method used to calculate the p-values. Also,\nreview other figures to ensure this information is consistently provided.\nThank you for your suggestion on the details of the statistical method. P values in Figure 4g and\n4h were calculated by one way ANOVA with Sidak's multiple comparisons test. We have checked\nall the images and provided the explanation of all p-values calculation method in the figure\nlegends.\n13. The Methods section requires significant revision to improve clarity and reproducibility.\nMore detailed descriptions will allow other researchers to replicate your findings.\nThank you for your valuable suggestion on the Methods section. We have uploaded\nhigh-definition images and supplemented detailed descriptions of the relevant experiments in the\nfigure legends and Methods section which may assist other researchers to understand or replicate\nour findings.\nReferences:\n1. Strecker J, Demircioglu FE, Li D, et al. RNA-activated protein cleavage with a\nCRISPR-associated endopeptidase. Science. 378 (6622), 874-881 (2022).\nhttps://doi.org/10.1126/science.add7450\n2. Kazuki Katoet al. A-triggered protein cleavage and cell growth arrest by the type III-E CRISPR\nnuclease-protease. Science378,882-889 (2022). https://doi.org/10.1126/science.add7347\n3. Cui N, Zhang JT, Li Z, et al. Structural basis for the non-self RNA-activated protease activity of\nthe type III-E CRISPR nuclease-protease Craspase. Nat Commun. 13 (1), 7549.\nhttps://doi.org/10.1038/s41467-022-35275-5"}, {"round": 2, "reviewer_comments": {"reviewer_1": "I commend the authors for their additional work in revising their manuscript. In particular, I like the addition of the\nGFP/mCherry co-culture experiment, which is a nice demonstration of the DAMAGE system; I think this could have been\nmoved to a main figure. Unfortunately, despite the point-by-point rebuttal and additional data provided, the main figures are\nessentially unchanged from the initial submission and do not meet the standard required for publication. This is not an issue\nof image resolution, but rather of what data are shown and how much is included in the figures.\nSome issues are technical, with panels still being too small to be readable or useful (e.g., Figs. 1a and 4a), but the problems\nare broader than that. The paper takes a simple and clever idea and makes it complicated and difficult to get through.\nWhile the rebuttal appears to address the concerns raised by me and the other reviewers, the manuscript does not. One of\nmy primary issues was the selective use of different DAMAGE constructs in different assays and the lack of focus on the\nbest-performing version across Figs. 2–6. The authors write, “In our subsequent experiments, we primarily focused on the\ntwo most potent GSDMs, GSDMB-Csx30 or GSDMC-Csx30 (containing B-FL, B-SL, C-FL, and C-SL) for further study.”\nHowever, the figures are mostly unchanged and still contain a variety of constructs without explanation.\nFig. 2c uses A-FL; Fig. 2d uses a large panel; Fig. 3b uses D-X; Fig. 3i uses a large panel; Fig. 5b uses D-X; Fig. 5c uses a\nlarge panel; Fig. 6b uses a large panel.\nI think the authors are on to something with their approach, and I generally like the idea of this work; however, I do not\nsupport publication of this work in its current form.", "reviewer_2": "Although the authors have improved most figures in the revised manuscript, some remain difficult to interpret. For instance,\nFig. 1a, 3g, 4f, 5g, h, EDF2f, and EDF5 contain very small labels, which hampers readability. Further enhancement of these\nfigures is recommended to improve clarity. Based on the comments from Reviewer 1, there appear to be several concerns\nregarding the cellular experiments. As my expertise lies in biochemistry rather than cell biology, I defer to the other\nreviewers' judgment on whether all issues related to the cellular experiments are sufficiently addressed.", "reviewer_3": "The authors have answered my questions. Another important point is to further refine the figures, their legends, and the\nmethods, ensuring that readers can clearly and readily understand the content and significance of each figure and its\nassociated experiments. I don't have other questions.", "reviewer_4": "I have only seen the revised manuscript and I am a substitute for reviewer 1.\nFirst, on the clarity of the manuscript. R1 mentioned that the manuscript was difficult to follow and that too much unnecessary\ndata was included, which obscures the main findings. R2 raised similar points. I don’t think this has been addressed\nproperly, including the remark that unclarity may be due to image compression issues, which is besides the point. It feels like\nthe authors misunderstood the comment. The manuscript still reads as cluttered, and the key results don’t stand out clearly.\nSecond, I’m not convinced by their response about the PI-positive cells and ATP-based readout. They explained it by talking\nabout how to interpret PI staining and the rapid proliferation of HeLa cells. But if you look at Fig R4b, the difference between\nLDH release and PI staining is not that big. This makes the argument weak. To me, it looks like the cell death efficiency is\nsimply low for this construct, which is not surprising considering it involves bulky proteins, target RNA recognition, and\ngasdermin activation. This is still an important step forward, but it feels very much like proof of principle, just as R1 pointed\nout.\nWhat’s missing is any discussion about how to move past proof of concept. If you think about this as a therapeutic, you’d\nprobably need cell death efficiencies of 50 percent or more. Since these constructs would likely be delivered intratumorally,\nyou can’t assume multiple dosing like chemotherapy. You basically have one shot to kill as many cells as possible. It would\nbe useful if the authors talked about possible ways to improve efficiency, even if just conceptually, so that the paper\nbecomes a resource for others working in this area.\nI also find the figures hard to interpret without reading the text. Even something simple, like explaining abbreviations in the\nfigure captions, would help. Right now, it’s difficult to figure out what things like N-, N+, OFF, and ON mean without digging\nthrough the main text.\nOverall, I agree with the authors that this is a new concept. They’ve shown that you can kill cells based on an RNA\nsignature, which they demonstrated using persistent viral infections and oncogenic mutations. Even though the efficiencies\nare low, it’s still a meaningful step forward. That said, some of the claims are overstated. For example, line 75 says, “Overall,\nour study established a new method for the treatment of RNA-heterogeneous diseases.” This just is incorrect with the data\nthey have now. There are similar overstatements throughout the manuscript, and I think these need to be toned down before\npublication.\nI do think this is a publishable piece of work, but I’m not fully sure it’s at Nature Communications level yet without further\nrefinement in the text. The data is there, and the authors should keep it more factual."}, "author_response": "PointbypointResponse\nREVIEWERCOMMENTS\nReviewer #1(Remarks to theAuthor):\nI commend the authors for their additional work in revising their manuscript. In\nparticular, I like the addition of the GFP/mCherry co-culture experiment, which is a\nnice demonstration of the DAMAGE system; I think this could have been moved to a\nmain figure. Unfortunately, despite the point-by-point rebuttal and additional data\nprovided, the main figures are essentially unchanged from the initial submission and\ndo not meet the standard required for publication. This is not an issue of image\nresolution, butrather ofwhat data are shownand how much is included in thefigures.\nWe are grateful for your positive assessment on our additional data and for your\nfurther constructive suggestions. Following your advice, we have now moved the\nEGFP/mCherry cell co-culture experiment (Before: Extended Data Fig. 5c) to Fig. 1g.\nPleasesee theresponse, Fig.R1.\nFig. R1 Co-cultureexperiment of EGFP-expressing and mCherry-expressing cells.\nEGFP or mCherry was transiently transfected into 293T cells, with simultaneous\ntransfection of DAMAGE targeting EGFP mRNA. And then EGFP-expressing and\nmCherry-expressing cells were mixed at a 1:1 ratio and co-cultured. In the ON group,\na significant reduction in green fluorescence from EGFP was observed, whereas the\nred fluorescence from mCherry remained largely unchanged. Scalebar,200μm.\nBesides, in the first round of review comments, you mentioned “The figure\nlegends and text lack clarity in explaining the experiments and different conditions\ntested. For instance, the abbreviations in Fig. 1 (N- and N+) are not clearly defined.\nSome of the data presented feels a bit unnecessary and makes the paper ‘heavy’ and\ncould be moved to supplementary materials or omitted for ease of reading”, this is\nessential for further improving the manuscript. Accordingly, we have removed the\ndata of \"N-\" and only retain \"N+\" in the new revised manuscript. The N+ groups,\nwhich employed GSDMs-Csx30-N (simulate the N-terminus of GSDMs-Csx30,\nwhich is cleaved by activated Csx29) to replace the GSDMs-Csx30 in the ON group,\nwere used as the positive control group. The specific changes include: Fig. 1b, 1c, 2b\nand Extended Data Fig. 6a. For details, please see the response, Fig. R2 and Table\nR1.\nFig. R2 Characterization of pyroptotic activity of GSDMs-Csx30 effectors through\nFlowCytometry analysis.\nThe experimental treatments of the OFF group, ON group and N+ group are shown in\nthefollowing table(crRNA×：non-target crRNA, CR-NT):\nTableR1\nWesincerely apologize that our previous revisions did not fundamentally address\nyour concerns and have now made further adjustments on the data for better\npresentation. This included removing, relocating, and supplementing data as\nrecommended. We hope these changes could significantly improve the clarity and the\noverall readability of the manuscript, which might help this work to meet the standard\nrequired for publication.\nSome issues are technical, with panels still being too small to be readable or useful\n(e.g., Figs. 1a and 4a), but the problems are broader than that. The paper takes a\nsimpleand clever idea and makes itcomplicated and difficulttoget through.\nWe sincerely thank you for your constructive suggestions and your positive\nfeedback on our idea. Following your advice, we have adjusted Fig. 1a and Fig. 4a by\nhighlighting the most essential information and enlarging the font size in accordance\nwiththe journal's formatting guidelines.\nFor Fig 1a, we redesigned the schematic diagrams of the 12 GSDMs-Csx30\neffector proteins to emphasize the distinct sequence architecture of each component,\nincluding GSDMs-N, Csx30, and GSDMs-C. Taking D-X as an example, the\nsequence from the N-terminal to the C-terminal is organized as follows: a short NES\nsequence, GSDMD-N 1→241, Csx30 407→565,and GSDMD-C 287→484. Different\ncolors were used to represent the different components. Through these modifications,\nwe consider that readers can clearly understand the sequence composition of these 12\nGSDMs-Csx30 effectors. Pleasesee theresponse,Fig. R3.\nFig. R3 Theschematic diagram oftheDAMAGE system.\nFor Fig. 4a, we highlighted the important sequences through boxes of different\ncolors, including KRAS-WT (blue), KRAS-G12C (red), as well as crRNA-16\n(green), crRNA-23 (purple), and crRNA-24 (orange) that exhibited the most the\noutstanding performance. For other crRNAs with poor performance, we marked them\nin gray font, and employed the lowercase red letters a/t to mark the mutant bases of\nKRAS-G12C. Pleasesee theresponse, Fig.R4.\nBy adopting this approach, all screening sequences can be incorporated without\ninterfering the presentation of key information. Additionally, we have included the\ncomplete set of screening sequences in the supplementary materials in the revised\nmanuscript. Please see the revised manuscript, Extended Table 1. Through these\nmodifications, we consider that readers can fully obtain the detailed sequence\ninformation ofthetarget RNAandcrRNAweused in Fig. 4.\nFig. R4 CRISPRRNAscreening ofthe DAMAGE-KRAS system.\nWhile the rebuttal appears to address the concerns raised by me and the other\nreviewers, the manuscript does not. One of my primary issues was the selective use of\ndifferent DAMAGE constructs in different assays and the lack of focus on the\nbest-performing version across Figs. 2–6. The authors write, “In our subsequent\nexperiments, we primarily focused on the two most potent GSDMs, GSDMB-Csx30\nor GSDMC-Csx30 (containing B-FL, B-SL, C-FL, and C-SL) for further study.”\nHowever, the figures are mostly unchanged and still contain a variety of constructs\nwithoutexplanation.\nWesincerely apologize that theprevious revised manuscript did not fully address\nthe concerns raised by you and the other reviewers, and we also greatly appreciate\nyour critical suggestions. In this revised manuscript, we have moved some data to the\nsupplementary materials or removed them entirely, aiming to present the findings in a\nmorestraightforward, clear,and logically coherent manner.\nRegarding your concern on the selective use of different DAMAGE constructs,\nwe first need to point out that the 12 GSDMs-Csx30 effectors designed in this study\nare all execution proteins of the DAMAGE system. While there are differences line in\nthe capability to induce pyroptosis, they all share a common underlying mechanism:\nthey respond totarget RNAand trigger pyroptosisintarget cells.\nIn this work, it was challenging to choose one \"best-performing\" effector, while\nhigher lethality often comes with increased non-specific pyroptotic effects. This\ndistinction is particularly evident between B-FL and B-SL. Therefore, we believe that\nB-FL and B-SL, C-FL and C-SL, as well as A-FL, A-SL, and D-X are all strong\ncandidates among the GSDMs-Csx30 effectors. They can be utilized for different\nexperimental purposes to yield more robust and clearer outcomes, thereby enabling a\nmore comprehensive characterization of the DAMAGE system's functionality and its\nspecificity in identifying target cells.Their selection was based solely on experimental\nrequirements, not onany intentional bias.\nScreening under different target conditions revealed that GSDMB-Csx30 or\nGSDMC-Csx30 (including B-FL, B-SL, C-FL, and C-SL) demonstrated potent\ncytotoxicity. The ranking of their lethality is B-FL > C-FL > C-SL > B-SL, but the\nstronger lethality also comes with higher non-specificity. We further discuss the\ndifferences in performance among these effectors in the Discussion section. Please\nseetherevised manuscript, Line635-638for moredetails.\nTherefore, in Figures 2-6, we primarily investigated the stronger GSDMB-Csx30\nor GSDMC-Csx30. Following your advice, to enhance experimental coherence and\nnarrative flow of the manuscript, we consistently employed the same type of\nGSDMs-Csx30 effector for each data figure in the revised manuscript. For instance,\nFig. 2presents data derived from B-SL, Fig. 3,Fig. 4, and Fig. 6were based on B-FL,\nand Fig. 5 utilized C-SL. Regarding the insufficient explanations in the manuscript,\nwe have made efforts in this revised manuscript to clarify the choice of the effectors\nineach relevant section as suggested.\nFig. 2c uses A-FL; Fig. 2d uses a large panel; Fig. 3b uses D-X; Fig. 3i uses a large\npanel; Fig. 5buses D-X; Fig. 5cuses a largepanel; Fig. 6buses alarge panel.\nThank you for pointing out this issue. In this revised manuscript, we have\nrepositioned the figures you mentioned and made every effort provided corresponding\nexplanations. To answer your question, we first make a simple categorization of these\nfigures:\n1. “Fig. 2c usesA-FL”;\n2. “Fig. 3buses D-X; Fig. 5buses D-X”;\n3. \"Fig. 2d uses a large panel; Fig. 3i uses a large panel; Fig. 5c uses a large\npanel; Fig. 6buses alarge panel”.\n1. “Fig. 2c usesA-FL”;\nFor Fig. 2c, the main purpose of conducting this experiment is to enrich the\ncomponents of the DAMAGE system in the cell culture medium supernatant through\nIP, for further demonstrating the release of cellular contents resulting from pyroptosis.\nIn the Supernatant-IPcomponent, HA-tagged Cas7-11, Myc-tagged Csx29,full-length\nGSDMs-Csx30 with Flag tag and its cut form GSDMs-Csx30-C can be detected by\nWestern Blotanalysis.\nWe presented the results of A-FL in Fig. 2c because it is highly representative.\nDue to its weak lethality, the pyroptosis induced by A-FL seems to be a slow and\nprogressive process. Therefore, when samples were collected at the same time point\nafter transfection for Western blot assay, A-FL and its cleavage form A-FL-C were\nmore easily detectable in both cell lysates and culture supernatants compared to other\nstronger effectors. Following your suggestion, to maintain consistency in the use of\neffectors, we repeated the experiment using B-SL and obtained consistent results and\npresented as the new Fig. 2c. Meanwhile, we also conducted the same experiment\nusing A-SL (Please see the revised manuscript, Extended Data Fig. 6d), and the\nresults remained consistent. Please seetheresponse, Fig.R5.\nFig. R5 Western blot and immunoprecipitation (IP) of supernatants assay to validate\nthepyroptoticactivity ofDAMAGE-RSV, using A-SL (a), A-FL (b), B-SL (c).\n2. “Fig. 3buses D-X; Fig. 5buses D-X”;\nFor Fig. 3b and Fig. 5b. The purpose of these two experiments is to\npreliminarily screen crRNA for tgRNA. Fig. 3b that screening the crRNA for\nendogenous transcription of HPV18-E6/E7 mRNA by HeLa cell genome, was done\nwith the aim of preliminarily confirming that the endogenous mRNAin Hela cells can\nindeed activate the system, and of laying the groundwork for the subsequent\nconstruction of CR-Mix. And, the previous Fig. 5b is for screening the crRNA of\np16/p21 mRNA, and it has been presented in the supplementary data, please see the\nrevised manuscript, Extended Data Fig. 3a (Please see the response, Fig. R6a). As\nan exemplar effector, D-X is widely used in these preliminary screenings. However,\nan effector even if GSDMs-Csx30 effectors other than D-X are used, there would\nexpose no significant impact on the screening results. To confirm, we repeated the\nexperiments on all targets using B-SL (Extended Data Fig. 3b) and the results was\nconsistent withthe D-Xscreening data. Please seetheresponse, Fig. R6b.\nFig. R6 CRISPRRNAscreening ofall target RNAs, using D-X (a), B-SL (b).\nFollowing your advice, the previous Fig. 3b, which shows the endogenous\ntriggering using D-X, was replaced by the experiment employing B-FL that obtaining\nconsistent results. Please see the response, Fig. R7.We have also added a description\ninthe main text as suggested (Pleasesee therevised manuscript, Line295-299).\nFig. R7 Screening of crRNA complementary to HPV18-E6/E7 mRNA transcribed by\nHeLa genome, using B-FL.\n3. “Fig. 2d uses a large panel; Fig. 3i uses a large panel; Fig. 5c uses a large\npanel; Fig. 6buses alarge panel”.\nTo answer this question, it is necessary to provide the following explanation of\ntheselarge panel figures.\nFirstly, from the research perspective, although EGFP was employed for initial\nvalidation, we cannot conclusively confirm whether the DAMAGE system exhibits\nidentical sensing effects on different target RNAs. Therefore, in the experimental\ndesign, we planned to screen all GSDMs-Csx30 effectors for each individual target\nRNA. In the new revised manuscript, to reduce reading barriers, we have relocated all\nthese large panel data prior to the main data figures and provided relevant\nexplanationswithin thearticle toenhance overall coherence.\nSecondly, the experimental designs of these large panel data figures are not\nentirely uniform. Based on the activation of the DAMAGE system requires five\ncomponents and the diversity of target RNA properties, we have designed distinct\nOFF groups for different targets. The results indicated that effective activation of the\nDAMAGE system occurs only when all five components are simultaneously present\nwithin a single cell. This suggests that the DAMAGE system is a controllable\nsynthetic biology framework capable of responding to target RNAs.Wehave provided\nthe detailed experimental design in the corresponding figure legends, with specific\ndetails outlined intheTableR2 (Please see theresponse, TableR2).\nTable R2 The experimental design of the OFF group and the ON group of the\nDAMAGE System\nBeyond the two reasons mentioned above, since RSV infection itself causes a\ncertain degree of cell damage, the nonspecific lethality of strong effectors combined\nwith virus-induced damage under infection conditions, is not an outcome we intended\nto observe. Therefore, to comprehensively evaluate the capabilities of each effector,\nwe included all of them in the gradient experiments (The previous Fig. 2f-h which has\nbeen moved to the Supplementary Fig. 6g, h). Based on these findings, we\nsubsequently selected B-SL, which exhibits weaker lethality compared to B-FL, for\nthe follow-up experiments. To directly present the key findings, only the results of\ngradient experiment using B-SL are shown in the main figures (Fig. 2f, 2g in the\nrevised manuscript). Please seetheresponse, Fig. R8).\nFig. R8 RSV-N plasmid gradient analysis (a) and RSV infection gradient analysis (b)\nofDAMAGE-RSV, using B-SL.\nFig. R9 Immunofluorescence analysis for DAMAGE-RSV in RSV-infected HeLa\ncells\nFurthermore, to better present the findings, we have removed the original Fig. 2e\ntothe supplementary dataand replaced it with aconfocal microscopy assay performed\nin Hela cells under infection conditions. This addition data provides direct visual\nevidence of pyroptosis induced by DAMAGE activation upon RSV infection. Please\nseetheresponse, Fig. R9.\nFor the previous Fig. 3h and 3i, since most of the previous experiments were\nconducted in HEK293T cells, an initial screening was performed to evaluate the\nlethality of various effectors under endogenous triggering conditions in HeLa cells.A\nstatement has been added in the manuscript, please see the revised manuscript, Line\n301-303. For better presentation of the data, they have been relocated to Fig. 3c and\n3d. The results turned out that B-FL and C-FL exhibited the strongest triggering\ncapabilities under endogenous conditions which was consistent with the previous\nresults in 293T. So, B-FL and C-FL were selected as the primary effectors for further\ninvestigation. Following your suggestion, we also have added relevant explanations in\nthemanuscript, please see therevised manuscript, Line305-308.\nMeanwhile, we have also adjusted other sections of Fig. 5 to improve the\npresentation of the data. Such as the Extend Data Fig.10e, was adjusted and moved to\nFig.5din revised manuscript, please seetheresponse, Fig. R10.\nFig. R10 Cell imaging of the activation of DAMAGE-Aging by endogenous p16/p21\nmRNAin 293Tcell line\nThe original Fig. 5c and 5d have been adjusted to Fig. 5b and 5c as the\npreliminary screening GSDMs-Csx30 effectors with p16 and p21 as targets.\nFurthermore, to better illustrate the impact of p16 and p21 mRNAlevels on induction\nof DAMAGE, the drug (Etoposide and Doxorubicin function as the activator of\np16/p21) treatment experiments in 293T cells were added, please see the response,\nFig.R11.\nFig. R11 Pro-aging drugs (Etoposide and Doxorubicin) further exacerbate\nDAMAGE-Aging-induced pyroptosis\nFor Fig. 6b, after constructing DAMAGE-Plus, we re-conducted a preliminary\ninvestigation into the lethality of all effectors. The resulted showed that B-FL\npossessed the highest lethality based on the screening and was chosen for the\nsubsequent experiments. A relevant explanation has been provided in the main text,\npleasesee revised manuscript,Line528-532.\nI think the authors are on to something with their approach, and I generally like the\nidea of this work; however, I do not support publication of this work in its current\nform.\nWe truly appreciate your recognition of our approach and idea. We are also very\ngrateful for your constructive comments. We have carefully addressed each point in\nthe revised manuscript and hope that these revisions could fully resolved your\nconcerns, bringing thepaper to apublishable standard.\nReviewer #2(Remarks to theAuthor):\nAlthough the authors have improved most figures in the revised manuscript, some\nremain difficult to interpret. For instance, Fig. 1a, 3g, 4f, 5g, h, EDF2f, and EDF5\ncontain very small labels, which hampers readability. Further enhancement of these\nfigures is recommended to improve clarity. Based on the comments from Reviewer 1,\nthere appear to be several concerns regarding the cellular experiments. As my\nexpertise lies in biochemistry rather than cell biology, I defer to the other reviewers'\njudgment on whether all issues related to the cellular experiments are sufficiently\naddressed.\nWe sincerely thank you for your suggestion. Following your advice, we have\nmaximized the font size of the scale bars in Fig. 1a, 3g, 4f, 5g, h (main figure)\naccording to the journal's formatting guidelines. And for EDF2f, and EDF5 that\ncontains numerous small images, we removed the labels and retained only the bars,\nand the specific scale values are clearly described in the figure legends. Furthermore,\nwe have fully addressed the issues raised by the other reviewers to the best of our\nability.We hope that the comprehensive revisions have now satisfactorily resolved all\nconcerns and themanuscript meets the journal's standard for publication.\nReviewer #3(Remarks to theAuthor):\nThe authors have answered my questions.Another important point is to further refine\nthe figures, their legends, and the methods, ensuring that readers can clearly and\nreadily understand the content and significance of each figure and its associated\nexperiments.I don't have other questions.\nThank you for your valuable suggestions which has greatly helped us in\nenhancing this work. Following your and other reviewers' suggestions, we have\nfurther refined the data presentation in the figures and optimized several labels that\nwere too small. We have also taken care to provide thorough descriptions of the\nexperimental procedures in both the figure legends and the Methods section, ensuring\nthat readers can more easily and accurately grasp the content and significance of each\nfigure and its associated experiments. Finally, we sincerely appreciate your positive\nassessment ofour work.\nReviewer #4(Remarks to theAuthor):\nIhave only seen the revised manuscript and Iam a substitutefor reviewer 1.\nThank you for reviewing our manuscript and providing the valuable suggestions.\nWe have revised the manuscript accordingly and hope that these changes could fully\naddress all your concerns, thereby meeting thejournal's requirements forpublication.\nFirst,ontheclarity ofthemanuscript. R1 mentioned thatthe manuscript was difficult\ntofollow andthat too muchunnecessary datawas included,which obscures the main\nfindings. R2 raised similar points.I don’t thinkthis has been addressed properly,\nincludingthe remark that unclarity may be dueto image compression issues,which is\nbesides thepoint. It feels liketheauthors misunderstood thecomment.The\nmanuscript stillreads as cluttered, and the key results don’t stand outclearly.\nWe are grateful for your feedback and sincerely regret that our initial revisions\ndid not adequately resolve the concerns of Reviewers 1 and 2. In response to the\nvaluable comments from you and the other reviewers, we have refined the data\npresentation in the revised manuscript avoiding obscuring the main findings. These\nchanges include:\n• The removal of redundant datasets (Please see revised manuscript, Fig. 1b,\n1c, 2fand2getc.).\n• The relocation of less critical data to the supplementary figures (Please see\nrevised manuscript,Extend Data Fig.6b,6f, 6gand6h etc.).\n• Adjustments to specific panels in the main figures (Please see revised\nmanuscript, Fig.2etc.).\n• The addition of new data to strengthen our conclusions (Please see revised\nmanuscript, Fig.2c, 2dand 2eetc.).\nFurthermore, the manuscript has been extensively refined to enhance the\nreadabilityand stand out thekey results clearly.\nSecond, I’m not convinced by their response about the PI-positive cells and\nATP-based readout. They explained it by talking about how to interpret PI staining\nand the rapid proliferation of HeLa cells. But if you look at Fig R4b, the difference\nbetween LDH release and PI staining is not that big. This makes the argument weak.\nTo me, it looks like the cell death efficiency is simply low for this construct, which is\nnot surprising considering it involves bulky proteins, target RNA recognition, and\ngasdermin activation. This is still an important step forward, but it feels very much\nlikeproofof principle,just as R1 pointed out.\nThank you very much for your positive feedback on our methodological design.\nWefully agree with your perspective that factors such as the large-scale protein, target\nRNA recognition, and the activation of gasdermin family proteins can indeed\nsignificantly influence the overall system performance. Thus, the system still faces\nconsiderable challenges and requires significant improvements before its practical\napplication. We fully acknowledge your and Reviewer 1's perspective that our work\nrepresents a proof of principle since data in Figure 6 provided only preliminary\nevidence that DAMAGE holds thepotentialof delivering via LNPs.\nHowever, under the transfection conditions which ensures adequate protein\nexpression, we are inclined to think that the pyroptosis induced by DAMAGE\nresembles a cascading amplification reaction. The upstream recognition of tgRNA by\ncrRNA mediates the activation of Cas7-11—Csx29, subsequently triggering the\ncleavage of downstream effectors. But, the varying potency of different effectors is\nindeed a key factor limiting the overall lethality of the system. While, all full-length\neffectors (GSDMs-Csx30-FL: A-FL to E-FL) exhibited greater lethality than their\nshorter counterparts (GSDMs-Csx30-FL: A-SL to E-SL) (Discuss in Discussion\nSection, please see revised manuscript, Line 629-649). The experiments in this study\nhave confirmed that transfection efficiency is a major reason for the relatively low PI\nstaining. Even under conditions where GSDMD-Csx30-N was directly transfected to\nperforate cell membranes, flow cytometry detected only approximately 45%\nPI-positive cells. Therefore, whether in transfection experiments or the practical\napplications, delivery efficiency poses asignificant challenge for DAMAGE.\nWhat’s missing is any discussion about how to move past proof of concept. If you\nthink about this as a therapeutic, you’d probably need compared to the separate\ncomponents.. Since these constructs would likely be delivered intratumorally, you\ncan’t assumemultipledosing likechemotherapy.You basically have one shotto killas\nmany cells as possible. It would be useful if the authors talked about possible ways to\nimprove efficiency, even if just conceptually, so that the paper becomes a resource for\nothers working in this area.\nWesincerely appreciate you highlighting this limitation. Our study was primarily\nfocused on the initial proof-of-concept for the delivery system, and we regret not\nhaving discussed its practical efficiency orpotential strategies for improvement.\nIndeed, we have actively explored strategies to enhance delivery efficiency. As\nillustrated in Figure 6, we developed the DAMAGE-Plus system by effectively\nlinking the three protein components of the DAMAGE—Cas7-11, Csx29, and\nGSDMs-Csx30—using T2A and P2A self-cleaving peptides. This modification not\nonly streamlined the DAMAGE system but also partially enhance efficient cellular\ndelivery of the components compared to the separate components. For LNP delivery,\nwe attempted to fuse Csx29, GSDMB-Csx30-FL into a single plasmid. This approach\nachieved successful delivery and subsequently induced pyroptosis (Fig. 6g-i).\nNevertheless, these studies constitute only initial explorations and additional work is\nwarranted toenhance thesystem's efficacy.\nFollowing your insightful suggestions, we have discussed potential strategies on\nthe possible ways to improve efficiency, please see revised manuscript, Line 650-658\nin Discussion section. We hope that the proposed concepts will serve as a valuable\nresourcefor researchers and practitioners in thisfield.\nMeanwhile, it is noteworthy that pyroptosis is a form of inflammatory\nprogrammed cell death characterized byits abilitytorecruit andactivate immunecells,\nthereby enhancing the elimination of tumor tissues. The scenario depicted below is\nour most desired outcome; however, it currently remains an ambitious\nhypothesis that requires substantial experimental validation. Specifically, in the\ncontext of cancer therapy, the DAMAGE system is expected to function as a\nmolecular trigger that induces pyroptosis through specific recognition and targeting of\ntumor-associated mRNAs. This process may promote immune infiltration and\nactivation, potentially transforming immunologically \"cold\" tumors into \"hot\" tumors\n(please see the response, Reference 1 and 2 for details, and Fig. R12). The relevant\ncontent has also been mentioned in the Introduction section. Please see the revised\nmanuscript, Line 41-43. Consequently, we hypothesize that achieving a tumor cell\ndeath efficiency of 50% or higher may not be strictly necessary for effective\nsuppression of tumor progression. Nevertheless, it should be emphasized that current\nstudies remain at the cellular level, and in vivo validation has not yet been conducted.\nTherefore, whether DAMAGE system-mediated pyroptosis can elicit a robust\nanti-tumor immune response through immune recruitment and activation warrants\nfurther investigation.\n[Figure Redacted]\nFig. R12 Immunological outcomes of gasdermin pore formation and pyroptosis.\n(NatRev Immunol. 2020;20(3):143-157.)\nPyroptotic cells release numerous intracellular molecules that can activate the immune\nsystem by acting as alarmins and ‘find me’ signals.\nI also find the figures hard to interpret without reading the text. Even something\nsimple, like explaining abbreviations in the figure captions, would help. Right now,\nit’s difficult to figure out what things likeN-, N+, OFF, and ON mean without digging\nthrough themain text.\nThanks for your suggestions, and meanwhile, to better highlight the key findings,\nwe have removed the data of \"N-\" and only retain \"N+\" in the new revised manuscript.\nThe N+ groups, which employed GSDMs-Csx30-N (simulate the N-terminus of\nGSDMs-Csx30, which is cleaved by activated Csx29) to replace the GSDMs-Csx30\nin the ON group, were used as the positive control groups. The specific changes\ninclude: Fig. 1b, 1c, 2b and Extended Data Fig. 6a. For details, please see the\nresponse, Fig.R2 andTableR1.\nFollowing your suggestion, we have accordingly added explanations for all\nrelevant abbreviations and details of the experiments in each figure caption. This\ncould facilitateeasier interpretation foryou and otherreaders.\nFig. R2 Characterization of pyroptotic activity of GSDMs-Csx30 effectors through\nFlowCytometry analysis.\nThe experimental treatments of the OFF group, ON group and N+ group are shown in\nthefollowing table(crRNA×：non-target crRNA, CR-NT):\nTableR1\nOverall, I agree with the authors that this is a new concept. They’ve shown that you\ncan kill cells based on an RNA signature, which they demonstrated using persistent\nviral infections and oncogenic mutations. Even though the efficiencies are low, it’s\nstill a meaningful step forward. That said, some of the claims are overstated. For\nexample, line 75 says, “Overall, our study established a new method for the treatment\nof RNA-heterogeneous diseases.” This just is incorrect with the data they have now.\nThere are similar overstatements throughout the manuscript, and I think these need to\nbetoned down before publication.\nWe sincerely appreciate your encouraging feedback and insightful comments.\nWhile our study only represents a preliminary attempt to explore practical\napplications, we acknowledge and apologize for the inclusion of some overly\nassertive statements in the original manuscript. In the revised version, we have\ncarefully toned down these overstated claims to present a more scientific explanation\nof the findings, ensuring they meet the standards required for publication. Line 75 that\nyou mentioned has revised to “In summary, DAMAGE represents a sophisticated\ncircuit that transduces signals by converting RNA information to proteolytic activity,\nfacilitating the cascading amplification of these signals. We maintain high\nexpectations regarding the potential applications of DAMAGE, it may offer a new\ntherapeutic strategies for a wide range of RNA-heterogeneous diseases”. The other\nsimilar overstatements were also refined, please see revised manuscript, Line\n659-663.\nI do think this is a publishable piece of work, but I’m not fully sure it’s at Nature\nCommunications level yet without further refinement in the text. The data is there,\nand theauthors shouldkeep it morefactual.\nWe sincerely appreciate your positive assessment of our work and your critical\nsuggestions. In the revised manuscript, we have performed comprehensive refinement\nin the text and the data presentation to enhance the scientific rigor and accuracy,\nensuring that it meets thestandards for publicationat NatureCommunications.\nReferences:\n[1] Broz, P., Pelegrı´n, P., and Shao, F. The gasdermins, a protein family executing\ncell death and inflammation. Nat. Rev.Immunol 20,143–157(2020).\n[2] Liu, X., Xia, S., Zhang, Z., Wu, H., and Lieberman, J. Channelling inflammation:\ngasdermins inphysiology and disease. Nat. Rev.Drug Discov 20,384–405(2021)."}, {"round": 3, "reviewer_comments": {"reviewer_1": "I appreciate the authors’ detailed explanation of the different DAMAGE constructs and the corresponding revisions to the text to better justify their inclusion. These changes address my major concerns with the paper and provide appropriate context for readers and potential future users of this system.\nOverall, the revised manuscript is substantially improved relative to the previous version and, in my opinion, is suitable for publication. I would have liked to see further simplification of the figures, as they remain somewhat data-dense and additional material could potentially be moved to the Supplementary Information; however, I defer to the other reviewers and the editor on this point, as this is largely a matter of presentation style.", "reviewer_4": "The authors have addressed my comments sufficiently and made adjustments to the manuscript to improve it."}, "author_response": "We sincerely appreciate your positive feedback on our work and are truly delighted that we\nhave been able to address all your concerns. Thanks for the time you dedicated and the invaluable\nsuggestions you raised, which have played a significant and essential role in refining our manuscript.\nThank you for your positive assessment that our manuscript is suitable for publication. In light\nof the suggestions from you and other reviewers, and given the extensive scope of this work, we\nhave strived to include only the most essential data in the main figures, while placing supplementary\nor more detailed elements in the Supplementary Figures. Following your advice, we have further\nrefined the presentation of several figures in the revised manuscript (Please see Figure. 1d) to\nimprove the clarity and readability. Additionally, we have carefully revised and polished the\nmanuscript point-by-point according to the author checklist provided by the editorial office. We trust\nthese adjustments will support the successful publication of this work.\nWe are very pleased to have addressed all your concerns and are grateful for your positive\nfeedback on our work. Thank you once more for your time and the crucial advice provided during\nreview."}]

Decision letter