Amplification editing enables efficient and precise duplication of DNA from short sequence to megabase and chromosomal scale.

Duplication is a foundation of molecular evolution and a driver of genomic and complex diseases. Here, we develop a genome editing tool named Amplification Editing (AE) that enables programmable DNA duplication with precision at chromosomal scale. AE can duplicate human genomes ranging from 20 bp to 100 Mb, a size comparable to human chromosomes. AE exhibits activity across various cell types, encompassing diploid, haploid, and primary cells. AE exhibited up to 73.0% efficiency for 1 Mb and 3.4% for 100 Mb duplications, respectively. Whole-genome sequencing and deep sequencing of the junctions of edited sequences confirm the precision of duplication. AE can create chromosomal microduplications within disease-relevant regions in embryonic stem cells, indicating its potential for generating cellular and animal models. AE is a precise and efficient tool for chromosomal engineering and DNA duplication, broadening the landscape of precision genome editing from an individual genetic locus to the chromosomal scale.

Coupling of N7-methyltransferase and 3'-5' exoribonuclease with SARS-CoV-2 polymerase reveals mechanisms for capping and proofreading.

The capping of mRNA and the proofreading play essential roles in SARS-CoV-2 replication and transcription. Here, we present the cryo-EM structure of the SARS-CoV-2 replication-transcription complex (RTC) in a form identified as Cap(0)-RTC, which couples a co-transcriptional capping complex (CCC) composed of nsp12 NiRAN, nsp9, the bifunctional nsp14 possessing an N-terminal exoribonuclease (ExoN) and a C-terminal N7-methyltransferase (N7-MTase), and nsp10 as a cofactor of nsp14. Nsp9 and nsp12 NiRAN recruit nsp10/nsp14 into the Cap(0)-RTC, forming the N7-CCC to yield cap(0) (7MeGpppA) at 5' end of pre-mRNA. A dimeric form of Cap(0)-RTC observed by cryo-EM suggests an in trans backtracking mechanism for nsp14 ExoN to facilitate proofreading of the RNA in concert with polymerase nsp12. These results not only provide a structural basis for understanding co-transcriptional modification of SARS-CoV-2 mRNA but also shed light on how replication fidelity in SARS-CoV-2 is maintained.

Cryo-EM Structure of an Extended SARS-CoV-2 Replication and Transcription Complex Reveals an Intermediate State in Cap Synthesis.

Transcription of SARS-CoV-2 mRNA requires sequential reactions facilitated by the replication and transcription complex (RTC). Here, we present a structural snapshot of SARS-CoV-2 RTC as it transitions toward cap structure synthesis. We determine the atomic cryo-EM structure of an extended RTC assembled by nsp7-nsp82-nsp12-nsp132-RNA and a single RNA-binding protein, nsp9. Nsp9 binds tightly to nsp12 (RdRp) NiRAN, allowing nsp9 N terminus inserting into the catalytic center of nsp12 NiRAN, which then inhibits activity. We also show that nsp12 NiRAN possesses guanylyltransferase activity, catalyzing the formation of cap core structure (GpppA). The orientation of nsp13 that anchors the 5' extension of template RNA shows a remarkable conformational shift, resulting in zinc finger 3 of its ZBD inserting into a minor groove of paired template-primer RNA. These results reason an intermediate state of RTC toward mRNA synthesis, pave a way to understand the RTC architecture, and provide a target for antiviral development.

Proteomic and Metabolomic Characterization of COVID-19 Patient Sera.

Early detection and effective treatment of severe COVID-19 patients remain major challenges. Here, we performed proteomic and metabolomic profiling of sera from 46 COVID-19 and 53 control individuals. We then trained a machine learning model using proteomic and metabolomic measurements from a training cohort of 18 non-severe and 13 severe patients. The model was validated using 10 independent patients, 7 of which were correctly classified. Targeted proteomics and metabolomics assays were employed to further validate this molecular classifier in a second test cohort of 19 COVID-19 patients, leading to 16 correct assignments. We identified molecular changes in the sera of COVID-19 patients compared to other groups implicating dysregulation of macrophage, platelet degranulation, complement system pathways, and massive metabolic suppression. This study revealed characteristic protein and metabolite changes in the sera of severe COVID-19 patients, which might be used in selection of potential blood biomarkers for severity evaluation.

Mutational Landscape of Secondary Glioblastoma Guides MET-Targeted Trial in Brain Tumor.

Low-grade gliomas almost invariably progress into secondary glioblastoma (sGBM) with limited therapeutic option and poorly understood mechanism. By studying the mutational landscape of 188 sGBMs, we find significant enrichment of TP53 mutations, somatic hypermutation, MET-exon-14-skipping (METex14), PTPRZ1-MET (ZM) fusions, and MET amplification. Strikingly, METex14 frequently co-occurs with ZM fusion and is present in ∼14% of cases with significantly worse prognosis. Subsequent studies show that METex14 promotes glioma progression by prolonging MET activity. Furthermore, we describe a MET kinase inhibitor, PLB-1001, that demonstrates remarkable potency in selectively inhibiting MET-altered tumor cells in preclinical models. Importantly, this compound also shows blood-brain barrier permeability and is subsequently applied in a phase I clinical trial that enrolls MET-altered chemo-resistant glioma patients. Encouragingly, PLB-1001 achieves partial response in at least two advanced sGBM patients with rarely significant side effects, underscoring the clinical potential for precisely treating gliomas using this therapy.

FDA-approved disulfiram inhibits pyroptosis by blocking gasdermin D pore formation.

Cytosolic sensing of pathogens and damage by myeloid and barrier epithelial cells assembles large complexes called inflammasomes, which activate inflammatory caspases to process cytokines (IL-1ß) and gasdermin D (GSDMD). Cleaved GSDMD forms membrane pores, leading to cytokine release and inflammatory cell death (pyroptosis). Inhibiting GSDMD is an attractive strategy to curb inflammation. Here we identify disulfiram, a drug for treating alcohol addiction, as an inhibitor of pore formation by GSDMD but not other members of the GSDM family. Disulfiram blocks pyroptosis and cytokine release in cells and lipopolysaccharide-induced septic death in mice. At nanomolar concentration, disulfiram covalently modifies human/mouse Cys191/Cys192 in GSDMD to block pore formation. Disulfiram still allows IL-1ß and GSDMD processing, but abrogates pore formation, thereby preventing IL-1ß release and pyroptosis. The role of disulfiram in inhibiting GSDMD provides new therapeutic indications for repurposing this safe drug to counteract inflammation, which contributes to many human diseases.

SPARCLE, a p53-induced lncRNA, controls apoptosis after genotoxic stress by promoting PARP-1 cleavage.

p53, master transcriptional regulator of the genotoxic stress response, controls cell-cycle arrest and apoptosis following DNA damage. Here, we identify a p53-induced lncRNA suicidal PARP-1 cleavage enhancer (SPARCLE) adjacent to miR-34b/c required for p53-mediated apoptosis. SPARCLE is a ∼770-nt, nuclear lncRNA induced 1 day after DNA damage. Despite low expression (<16 copies/cell), SPARCLE deletion increases DNA repair and reduces DNA-damage-induced apoptosis as much as p53 deficiency, while its overexpression restores apoptosis in p53-deficient cells. SPARCLE does not alter gene expression. SPARCLE binds to PARP-1 with nanomolar affinity and causes apoptosis by acting as a caspase-3 cofactor for PARP-1 cleavage, which separates PARP-1's N-terminal (NT) DNA-binding domain from its catalytic domains. NT-PARP-1 inhibits DNA repair. Expressing NT-PARP-1 in SPARCLE-deficient cells increases unrepaired DNA damage and restores apoptosis after DNA damage. Thus, SPARCLE enhances p53-induced apoptosis by promoting PARP-1 cleavage, which interferes with DNA-damage repair.

DNA topology regulates PAM-Cas9 interaction and DNA unwinding to enable near-PAMless cleavage by thermophilic Cas9.

CRISPR-Cas9-mediated genome editing depends on PAM recognition to initiate DNA unwinding. PAM mutations can abolish Cas9 binding and prohibit editing. Here, we identified a Cas9 from the thermophile Alicyclobacillus tengchongensis for which the PAM interaction can be robustly regulated by DNA topology. AtCas9 has a relaxed PAM of N4CNNN and N4RNNA (R = A/G) and is able to bind but not cleave targets with mutated PAMs. When PAM-mutated DNA was in underwound topology, AtCas9 exhibited enhanced binding affinity and high cleavage activity. Mechanistically, AtCas9 has a unique loop motif, which docked into the DNA major groove, and this interaction can be regulated by DNA topology. More importantly, AtCas9 showed near-PAMless editing of supercoiled plasmid in E. coli. In mammalian cells, AtCas9 exhibited broad PAM preference to edit plasmid with up to 72% efficiency and effective base editing at four endogenous loci, representing a potentially powerful tool for near-PAMless editing.

Absence of near-ambient superconductivity in LuH2±xNy.

A recent study demonstrated near-ambient superconductivity in nitrogen-doped lutetium hydride1. This stimulated a worldwide interest in exploring room-temperature superconductivity at low pressures. Here, by using a high-pressure and high-temperature synthesis technique, we have obtained nitrogen-doped lutetium hydride (LuH2±xNy), which has a dark-blue colour and a structure with the space group [Formula: see text] as evidenced by X-ray diffraction. This structure is the same as that reported in ref. 1, with a slight difference in lattice constant. Raman spectroscopy of our samples also showed patterns similar to those observed in ref. 1. Energy-dispersive X-ray spectroscopy confirmed the presence of nitrogen in the samples. We observed a metallic behaviour from 350 K to 2 K at ambient pressure. On applying pressures from 2.1 GPa to 41 GPa, we observed a gradual colour change from dark blue to violet to pink-red. By measuring the resistance at pressures ranging from 0.4 GPa to 40.1 GPa, we observed a progressively improved metallic behaviour; however, superconductivity was not observed above 2 K. Temperature dependence of magnetization at high pressure shows a very weak positive signal between 100 K and 320 K, and the magnetization increases with an increase in magnetic field at 100 K. All of these are not expected for superconductivity above 100 K. Thus, we conclude the absence of near-ambient superconductivity in this nitrogen-doped lutetium hydride at pressures below 40.1 GPa.

The NK cell receptor NKp46 recognizes ecto-calreticulin on ER-stressed cells.

Natural killer (NK) cell kill infected, transformed and stressed cells when an activating NK cell receptor is triggered1. Most NK cells and some innate lymphoid cells express the activating receptor NKp46, encoded by NCR1, the most evolutionarily ancient NK cell receptor2,3. Blockage of NKp46 inhibits NK killing of many cancer targets4. Although a few infectious NKp46 ligands have been identified, the endogenous NKp46 cell surface ligand is unknown. Here we show that NKp46 recognizes externalized calreticulin (ecto-CRT), which translocates from the endoplasmic reticulum (ER) to the cell membrane during ER stress. ER stress and ecto-CRT are hallmarks of chemotherapy-induced immunogenic cell death5,6, flavivirus infection and senescence. NKp46 recognition of the P domain of ecto-CRT triggers NK cell signalling and NKp46 caps with ecto-CRT in NK immune synapses. NKp46-mediated killing is inhibited by knockout or knockdown of CALR, the gene encoding CRT, or CRT antibodies, and is enhanced by ectopic expression of glycosylphosphatidylinositol-anchored CRT. NCR1)-deficient human (and Nrc1-deficient mouse) NK cells are impaired in the killing of ZIKV-infected, ER-stressed and senescent cells and ecto-CRT-expressing cancer cells. Importantly, NKp46 recognition of ecto-CRT controls mouse B16 melanoma and RAS-driven lung cancers and enhances tumour-infiltrating NK cell degranulation and cytokine secretion. Thus, NKp46 recognition of ecto-CRT as a danger-associated molecular pattern eliminates ER-stressed cells.

Proteome plasticity in response to persistent environmental change.

Temperature is a variable component of the environment, and all organisms must deal with or adapt to temperature change. Acute temperature change activates cellular stress responses, resulting in refolding or removal of damaged proteins. However, how organisms adapt to long-term temperature change remains largely unexplored. Here we report that budding yeast responds to long-term high temperature challenge by switching from chaperone induction to reduction of temperature-sensitive proteins and re-localizing a portion of its proteome. Surprisingly, we also find that many proteins adopt an alternative conformation. Using Fet3p as an example, we find that the temperature-dependent conformational difference is accompanied by distinct thermostability, subcellular localization, and, importantly, cellular functions. We postulate that, in addition to the known mechanisms of adaptation, conformational plasticity allows some polypeptides to acquire new biophysical properties and functions when environmental change endures.

Targeting thalamic circuits rescues motor and mood deficits in PD mice.

Although bradykinesia, tremor and rigidity are the hallmark motor defects in patients with Parkinson's disease (PD), patients also experience motor learning impairments and non-motor symptoms such as depression1. The neural circuit basis for these different symptoms of PD are not well understood. Although current treatments are effective for locomotion deficits in PD2,3, therapeutic strategies targeting motor learning deficits and non-motor symptoms are lacking4-6. Here we found that distinct parafascicular (PF) thalamic subpopulations project to caudate putamen (CPu), subthalamic nucleus (STN) and nucleus accumbens (NAc). Whereas PF→CPu and PF→STN circuits are critical for locomotion and motor learning, respectively, inhibition of the PF→NAc circuit induced a depression-like state. Whereas chemogenetically manipulating CPu-projecting PF neurons led to a long-term restoration of locomotion, optogenetic long-term potentiation (LTP) at PF→STN synapses restored motor learning behaviour in an acute mouse model of PD. Furthermore, activation of NAc-projecting PF neurons rescued depression-like phenotypes. Further, we identified nicotinic acetylcholine receptors capable of modulating PF circuits to rescue different PD phenotypes. Thus, targeting PF thalamic circuits may be an effective strategy for treating motor and non-motor deficits in PD.

A six-amino-acid motif is a major determinant in functional evolution of HOX1 proteins.

Gene duplication and divergence is a major driver in the emergence of evolutionary novelties. How variations in amino acid sequences lead to loss of ancestral activity and functional diversification of proteins is poorly understood. We used cross-species functional analysis of Drosophila Labial and its mouse HOX1 orthologs (HOXA1, HOXB1, and HOXD1) as a paradigm to address this issue. Mouse HOX1 proteins display low (30%) sequence similarity with Drosophila Labial. However, substituting endogenous Labial with the mouse proteins revealed that HOXA1 has retained essential ancestral functions of Labial, while HOXB1 and HOXD1 have diverged. Genome-wide analysis demonstrated similar DNA-binding patterns of HOXA1 and Labial in mouse cells, while HOXB1 binds to distinct targets. Compared with HOXB1, HOXA1 shows an enrichment in co-occupancy with PBX proteins on target sites and exists in the same complex with PBX on chromatin. Functional analysis of HOXA1-HOXB1 chimeric proteins uncovered a novel six-amino-acid C-terminal motif (CTM) flanking the homeodomain that serves as a major determinant of ancestral activity. In vitro DNA-binding experiments and structural prediction show that CTM provides an important domain for interaction of HOXA1 proteins with PBX. Our findings show that small changes outside of highly conserved DNA-binding regions can lead to profound changes in protein function.

UVRAG cooperates with cargo receptors to assemble the ER-phagy site.

ER-phagy is a selective autophagy process that targets specific regions of the endoplasmic reticulum (ER) for removal via lysosomal degradation. During cellular stress induced by starvation, cargo receptors concentrate at distinct ER-phagy sites (ERPHS) to recruit core autophagy proteins and initiate ER-phagy. However, the molecular mechanism responsible for ERPHS formation remains unclear. In our study, we discovered that the autophagy regulator UV radiation Resistance-Associated Gene (UVRAG) plays a crucial role in orchestrating the assembly of ERPHS. Upon starvation, UVRAG localizes to ERPHS and interacts with specific ER-phagy cargo receptors, such as FAM134B, ATL3, and RTN3L. UVRAG regulates the oligomerization of cargo receptors and facilitates the recruitment of Atg8 family proteins. Consequently, UVRAG promotes efficient ERPHS assembly and turnover of both ER sheets and tubules. Importantly, UVRAG-mediated ER-phagy contributes to the clearance of pathogenic proinsulin aggregates. Remarkably, the involvement of UVRAG in ER-phagy initiation is independent of its canonical function as a subunit of class III phosphatidylinositol 3-kinase complex II.

T Cell Receptor-Regulated TGF-ß Type I Receptor Expression Determines T Cell Quiescence and Activation.

It is unclear how quiescence is enforced in naive T cells, but activation by foreign antigens and self-antigens is allowed, despite the presence of inhibitory signals. We showed that active transforming growth factor ß (TGF-ß) signaling was present in naive T cells, and T cell receptor (TCR) engagement reduced TGF-ß signaling during T cell activation by downregulating TGF-ß type 1 receptor (TßRI) through activation of caspase recruitment domain-containing protein 11 (CARD11) and nuclear factor κB (NF-κB). TGF-ß prevented TCR-mediated TßRI downregulation, but this was abrogated by interleukin-6 (IL-6). Mitigation of TCR-mediated TßRI downregulation through overexpression of TßRI in naive and activated T cells rendered T cells less responsive and suppressed autoimmunity. Naive T cells in autoimmune patients exhibited reduced TßRI expression and increased TCR-driven proliferation compared to healthy subjects. Thus, TCR-mediated regulation of TßRI-TGF-ß signaling acts as a crucial criterion to determine T cell quiescence and activation.

The electrochemistry of stable sulfur isotopes versus lithium.

Sulfur in nature consists of two abundant stable isotopes, with two more neutrons in the heavy one (34S) than in the light one (32S). The two isotopes show similar physicochemical properties and are usually considered an integral system for chemical research in various fields. In this work, a model study based on a Li-S battery was performed to reveal the variation between the electrochemical properties of the two S isotopes. Provided with the same octatomic ring structure, the cyclo-34S8 molecules form stronger S-S bonds than cyclo-32S8 and are more prone to react with Li. The soluble Li polysulfides generated by the Li-34S conversion reaction show a stronger cation-solvent interaction yet a weaker cation-anion interaction than the 32S-based counterparts, which facilitates quick solvation of polysulfides yet hinders their migration from the cathode to the anode. Consequently, the Li-34S cell shows improved cathode reaction kinetics at the solid-liquid interface and inhibited shuttle of polysulfides through the electrolyte so that it demonstrates better cycling performance than the Li-32S cell. Based on the varied shuttle kinetics of the isotopic-S-based polysulfides, an electrochemical separation method for 34S/32S isotope is proposed, which enables a notably higher separation factor than the conventional separation methods via chemical exchange or distillation and brings opportunities to low-cost manufacture, utilization, and research of heavy chalcogen isotopes.

Bivalent Omicron BA.1-Adapted BNT162b2 Booster in Adults Older than 55 Years.

BACKGROUND: The emergence of immune-escape variants of severe acute respiratory syndrome coronavirus 2 warrants the use of sequence-adapted vaccines to provide protection against coronavirus disease 2019. METHODS: In an ongoing phase 3 trial, adults older than 55 years who had previously received three 30-µg doses of the BNT162b2 vaccine were randomly assigned to receive 30 µg or 60 µg of BNT162b2, 30 µg or 60 µg of monovalent B.1.1.529 (omicron) BA.1-adapted BNT162b2 (monovalent BA.1), or 30 µg (15 µg of BNT162b2 + 15 µg of monovalent BA.1) or 60 µg (30 µg of BNT162b2 + 30 µg of monovalent BA.1) of BA.1-adapted BNT162b2 (bivalent BA.1). Primary objectives were to determine superiority (with respect to 50% neutralizing titer [NT50] against BA.1) and noninferiority (with respect to seroresponse) of the BA.1-adapted vaccines to BNT162b2 (30 µg). A secondary objective was to determine noninferiority of bivalent BA.1 to BNT162b2 (30 µg) with respect to neutralizing activity against the ancestral strain. Exploratory analyses assessed immune responses against omicron BA.4, BA.5, and BA.2.75 subvariants. RESULTS: A total of 1846 participants underwent randomization. At 1 month after vaccination, bivalent BA.1 (30 µg and 60 µg) and monovalent BA.1 (60 µg) showed neutralizing activity against BA.1 superior to that of BNT162b2 (30 µg), with NT50 geometric mean ratios (GMRs) of 1.56 (95% confidence interval [CI], 1.17 to 2.08), 1.97 (95% CI, 1.45 to 2.68), and 3.15 (95% CI, 2.38 to 4.16), respectively. Bivalent BA.1 (both doses) and monovalent BA.1 (60 µg) were also noninferior to BNT162b2 (30 µg) with respect to seroresponse against BA.1; between-group differences ranged from 10.9 to 29.1 percentage points. Bivalent BA.1 (either dose) was noninferior to BNT162b2 (30 µg) with respect to neutralizing activity against the ancestral strain, with NT50 GMRs of 0.99 (95% CI, 0.82 to 1.20) and 1.30 (95% CI, 1.07 to 1.58), respectively. BA.4-BA.5 and BA.2.75 neutralizing titers were numerically higher with 30-µg bivalent BA.1 than with 30-µg BNT162b2. The safety profile of either dose of monovalent or bivalent BA.1 was similar to that of BNT162b2 (30 µg). Adverse events were more common in the 30-µg monovalent-BA.1 (8.5%) and 60-µg bivalent-BA.1 (10.4%) groups than in the other groups (3.6 to 6.6%). CONCLUSIONS: The candidate monovalent or bivalent omicron BA.1-adapted vaccines had a safety profile similar to that of BNT162b2 (30 µg), induced substantial neutralizing responses against ancestral and omicron BA.1 strains, and, to a lesser extent, neutralized BA.4, BA.5, and BA.2.75 strains. (Funded by BioNTech and Pfizer; ClinicalTrials.gov number, NCT04955626.).

Fast and sensitive CRISPR detection by minimized interference of target amplification.

Despite the great potential of CRISPR-based detection, it has not been competitive with other market diagnostics for on-site and in-home testing. Here we dissect the rate-limiting factors that undermine the performance of Cas12b- and Cas13a-mediated detection. In one-pot testing, Cas12b interferes with loop-mediated isothermal amplification by binding to and cleaving the amplicon, while Cas13a directly degrades the viral RNA, reducing its amplification. We found that the protospacer-adjacent motif-interacting domain engineered Cas12b accelerated one-pot testing with 10-10,000-fold improved sensitivity, and detected 85 out of 85 SARS-CoV-2 clinical samples with a sensitivity of 0.5 cp µl-1, making it superior to wild-type Cas12b. In parallel, by diminishing the interference of Cas13a with viral RNA, the optimized Cas13a-based assay detected 86 out of 87 SARS-CoV-2 clinical samples at room temperature in 30 min with a sensitivity of 0.5 cp µl-1. The relaxed reaction conditions and improved performance of CRISPR-based assays make them competitive for widespread use in pathogen detection.

DDX5 inhibits inflammation by modulating m6A levels of TLR2/4 transcripts during bacterial infection.

DExD/H-box helicases are crucial regulators of RNA metabolism and antiviral innate immune responses; however, their role in bacteria-induced inflammation remains unclear. Here, we report that DDX5 interacts with METTL3 and METTL14 to form an m6A writing complex, which adds N6-methyladenosine to transcripts of toll-like receptor (TLR) 2 and TLR4, promoting their decay via YTHDF2-mediated RNA degradation, resulting in reduced expression of TLR2/4. Upon bacterial infection, DDX5 is recruited to Hrd1 at the endoplasmic reticulum in an MyD88-dependent manner and is degraded by the ubiquitin-proteasome pathway. This process disrupts the DDX5 m6A writing complex and halts m6A modification as well as degradation of TLR2/4 mRNAs, thereby promoting the expression of TLR2 and TLR4 and downstream NF-κB activation. The role of DDX5 in regulating inflammation is also validated in vivo, as DDX5- and METTL3-KO mice exhibit enhanced expression of inflammatory cytokines. Our findings show that DDX5 acts as a molecular switch to regulate inflammation during bacterial infection and shed light on mechanisms of quiescent inflammation during homeostasis.

Gasdermin E suppresses tumour growth by activating anti-tumour immunity.

Cleavage of the gasdermin proteins to produce pore-forming amino-terminal fragments causes inflammatory cell death (pyroptosis)1. Gasdermin E (GSDME, also known as DFNA5)-mutated in familial ageing-related hearing loss2-can be cleaved by caspase 3, thereby converting noninflammatory apoptosis to pyroptosis in GSDME-expressing cells3-5. GSDME expression is suppressed in many cancers, and reduced GSDME levels are associated with decreased survival as a result of breast cancer2,6, suggesting that GSDME might be a tumour suppressor. Here we show that 20 of 22 tested cancer-associated GSDME mutations reduce GSDME function. In mice, knocking out Gsdme in GSDME-expressing tumours enhances, whereas ectopic expression in Gsdme-repressed tumours inhibits, tumour growth. This tumour suppression is mediated by killer cytotoxic lymphocytes: it is abrogated in perforin-deficient mice or mice depleted of killer lymphocytes. GSDME expression enhances the phagocytosis of tumour cells by tumour-associated macrophages, as well as the number and functions of tumour-infiltrating natural-killer and CD8+ T lymphocytes. Killer-cell granzyme B also activates caspase-independent pyroptosis in target cells by directly cleaving GSDME at the same site as caspase 3. Uncleavable or pore-defective GSDME proteins are not tumour suppressive. Thus, tumour GSDME acts as a tumour suppressor by activating pyroptosis, enhancing anti-tumour immunity.