Biology subjects

Trapani, J. A.

Publications and source records attributed to Trapani, J. A..

12 recordsLinked to original sources

bioRxiv2026

Addressing viral genomic variability towards developing a Cas13b-based therapy

Viral genome diversity may limit the effectiveness of antiviral RNA-editing tools such as CRISPR-Cas13 that can be used to destroy specific mRNA targets, by introducing mismatches between viral RNA targets and CRISPR guide RNAs (crRNAs). These mismatches can reduce target recognition and cleavage efficiency, diminishing antiviral activity and increasing the risk of viral escape. The extent to which natural viral genomic variability limits CRISPR-Cas13 efficacy remains unclear. Here, we used hepatitis B virus (HBV), which has substantial genetic diversity, as a model to assess the impact of viral genome variation on Cas13b activity in vitro. The efficacy of PspCas13b was examined across six HBV genotypes and sub-genotypes using five crRNAs that had up to five mismatches to the target region. We showed that crRNAs with one mismatch to the target strongly suppressed viral antigen expression for all genotypes tested, while some crRNAs with three or more mismatches were less effective. Restoring complementarity using spacer-target mutagenesis improved the level of knockdown for some but not all HBV genotypes, suggesting that sequence specificity alone did not control PspCas13b efficacy. Our findings show that a "one size fits all" approach for PspCas13b-mediated treatment of HBV is unlikely to be effective, but the impact of sequence variability on PspCas13b efficacy can be readily addressed through appropriate design of crRNAs. This approach will likely be necessary for all viral pathogens with highly variant genomes. IMPORTANCECRISPR-Cas13 is being explored as a novel antiviral for several viral infections. Viral sequence divergence can compromise CRISPR-Cas13 efficacy by introducing mismatches between therapeutic guide RNAs and viral targets. However, the impact of naturally occurring viral genomic variation on CRISPR-Cas13 efficacy remains poorly understood. Using hepatitis B virus (HBV) as a model, we showed that the effect of mismatches on Cas13b efficacy was context-dependent and varied for different crRNAs, HBV genotypes and target sites. Restoring complementarity improved the efficacy for some, but not all crRNAs, suggesting that Cas13b efficacy was not solely influenced by the number of mismatches. As the target sequence may differ between viral variants, this study advances our understanding of the impact of mismatches on Cas13b efficacy and provides further insights into using Cas13b as a novel antiviral.

microbiology↗

bioRxiv2026

Target RNA abundance controls the collateral activity of RfxCas13d in human cells and zebrafish embryos

Collateral RNA cleavage by CRISPR-Cas13 effectors presents a critical obstacle to their application in biological research and therapeutics, yet the molecular determinants of this activity remain poorly understood. Here, we systematically investigate the collateral activity of various Cas13 variants in human cells in vitro and zebrafish embryos in vivo. Among these nucleases, RfxCas13d displays robust collateral activity that is highly dependent on target abundance. Targeting moderately expressed RNAs activates only a limited subset of RfxCas13d molecules, resulting in selective degradation of ectopically expressed transcripts, while endogenous RNAs remain largely protected. This selectivity indicates higher accessibility to exogenous RNAs through spatial proximity and temporal colocalization with activated RfxCas13d nuclease domains. In contrast, the recognition of highly abundant RNA targets drives simultaneous activation of a large fraction of cellular RfxCas13d, leading to widespread collateral cleavage of cellular RNAs, disruption of proteome homeostasis, and consequent cell toxicity and developmental defects in zebrafish embryos. Notably, transgenic zebrafish with target RNA expression restricted to endothelial or neuronal cell lineages exhibit localized collateral activity, leading to tissue-specific developmental abnormalities and motility deficits. These findings reveal that RfxCas13ds collateral activity is threshold-dependent, with abundant target RNA acting as a molecular switch for widespread collateral RNA degradation. This work underscores the need for careful consideration of target abundance when deploying RfxCas13d, and highlights PspCas13b as an alternative for RNA silencing free of collateral activity.

molecular biology↗

bioRxiv2025

A TAK1Cytokine Toxicity Checkpoint Controls Anti-Cancer Immunity

The success of cancer immunotherapies is currently limited to a subset of patients, which underscores the urgent need to identify the processes by which tumours evade immunity. Through screening a kinome-wide CRISPR/Cas9 sgRNA library we identified MAP3K7 (TAK1) as a suppressor of CD8+ T cell mediated killing. We demonstrate that TAK1 acts as a cancer-intrinsic checkpoint by integrating signals from T cell-secreted TNF and IFNy effector cytokines to elicit a cytoprotective response. This cytoprotective response profoundly limits the anti-cancer activity these key effector molecules and completely abrogates bystander killing by perforin deficient T cells. Inhibition of the TAK1 checkpoint effectively redirects the combined TNF/IFNy pathway activation to promote inflammatory cell death via RIPK1 and Caspase-8 and simultaneously amplifies the output of the IFNy pathway, thereby priming cells for cytokine-induced cell death. Mechanistically, TAK1 deficiency led to proteasomal degradation of cFLIP, enhancing the formation of Complex II and subversion of other cytoprotective responses. Targeting the TAK1 checkpoint led to profound attenuation of tumour growth in immune competent mice, with minimal impact in immune deficient counterparts. Adoptive cell therapy led to preferential elimination of TAK1 deficient clones. Collectively, our study uncovers a cancer-intrinsic checkpoint controlled by TAK1 activity that switches TNF and IFNy responses from cytoprotective to apoptosis. Cancer cells exploit this to limit cell death in the presence of the cytotoxic lymphoctye cytokines TNF and IFN{gamma} and therapeutic intervention can fully unleash the impact of these effector molecules both on the direct target and bystander cells. These findings highlight the clinical development of TAK1 biologics as a potential strategy to improve cancer immunotherapies through harnessing and enhancing the cytotoxic potential of CTL-derived cytokines. In BriefDjajawi et al. identify TAK1 as a cancer-intrinsic cytokine toxicity checkpoint that limits the efficacy of anti-cancer immune responses. Cancer cells exploit TAK1 activity to confers resistance to CD8+ T cell-derived TNF and IFN{gamma}-induced apoptosis, limiting both direct and bystander killing. Mechanistically, TAK1 loss acts through 1. cFLIP and RIPK1 to promote cell death, 2. The inactivation of pro-survival signals and 3. Via amplification of the IFNy response. Targeting of this TAK1 checkpoint enhanced anti-tumour immunity in vivo and improved adoptive cellular therapy. These findings identify a strategy employed by transformed cells to avoid destruction by inflammatory cytokines and provide new therapeutic vulnerabilities for enhancing immunotherapies. HighlightsO_LICRISPR screens identify TAK1 as a tumour-intrinsic survival checkpoint limiting destruction by CD8+ T cells. C_LIO_LITAK1 protects tumour cells from combined TNF and IFN{gamma}-induced apoptosis. C_LIO_LITAK1 loss destabilizes cFLIP, priming cells for STAT1 and RIPK1-dependent cell death. C_LIO_LITAK1 loss promotes tumour control in immune competent animals and enhances the efficacy of adoptive cell therapy. C_LI

cancer biology↗

bioRxiv2025

Systematic silencing of oncogenic fusion transcripts with ultra-precise CRISPR-Cas13b

Oncogenic gene fusions are key drivers of cancer, yet most remain untargetable by current therapies. Here, we establish CRISPR-PspCas13b as a personalizable platform for systematic silencing of various fusion transcripts. We reveal that recognition and cleavage of the breakpoint sequence by PspCas13b disrupts the fusion transcript, resulting in unexpected RNA nicking and ligation near the cleavage site, which generates out-of-frame, translation-incompetent transcripts. This approach efficiently degrades canonical and drug-resistant BCR::ABL1 mutants (e.g., T315I), a primary cause of resistance to tyrosine kinase inhibitors (TKIs) and relapse in chronic myeloid leukemia (CML). Silencing T315I BCR::ABL1 mRNA in drug-resistant CML cells triggers extensive transcriptomic, proteomic, and phosphoproteomic remodelling, causing erythroid differentiation and apoptosis. Beyond BCR-ABL1 mutants, personalized design of PspCas13b effectively silences other undruggable fusions, including RUNX1::RUNX1T1 and EWSR1::FLI1, key drivers in acute myeloid leukemia and in Ewing sarcoma, respectively. Collectively, this study establishes a framework for systematic, precise, and personalizable targeting of otherwise undruggable or drug-resistant oncogenic transcripts.

molecular biology↗

bioRxiv2024

Acidic pH attenuates immune killing through inactivation of perforin

Cytotoxic lymphocytes are pivotal effectors in our immune system. Their most potent way to delete virus infected or cancerous target cells is by perforin/granzyme dependent killing. Perforin is secreted into the immunological synapse and forms transmembrane pores in the target cell plasma membrane, allowing granzymes to enter the target cell cytosol and trigger apoptosis. The prowess of cytotoxic lymphocytes to efficiently eradicate target cells has been widely harnessed in immunotherapies against haematological cancers. Despite efforts to achieve a similar outcome against solid tumours, the immunosuppressive and acidic tumour microenvironment poses a persistent obstacle. Using different types of effector cells, including therapeutically relevant anti-CD19 CAR T cells, we demonstrate that the acidic pH typically found in solid tumours hinders the efficacy of immune therapies by impeding perforin pore formation within the immunological synapse. Nanometre-scale study of purified recombinant perforin undergoing oligomerization reveals that pore formation is inhibited specifically by preventing the formation of a transmembrane {beta}-barrel. The absence of perforin pore formation directly prevents target cell death. This finding uncovers a novel layer of immune effector inhibition that must be considered in the development of effective immunotherapies for solid tumours. One Sentence SummaryOur study reveals that an acidic environment hinders the efficacy of immune therapies by impeding the action of perforin, a critical protein in immune-mediated cell killing, directly within the immunological synapse.

immunology↗

bioRxiv2023

Molecular principles of CRISPR-Cas13 mismatch intolerance enable selective silencing of point-mutated oncogenic RNA with single-base precision

Single nucleotide variants (SNVs) are extremely prevalent in human cancers. For instance, KRAS mutations occur in over 90% of pancreatic cancers and [~]40% of colorectal cancers. Virtually all KRAS mutations are SNVs, most of which remain clinically unactionable. The programmable RNA nuclease CRISPR-Cas13 has been deployed to specifically target RNAs such as overexpressed oncogenes and fusion transcripts. However, silencing oncogenic SNVs with single-base precision remains extremely challenging due to the intrinsic mismatch tolerance of Cas13. Here, we developed a comprehensive mutagenesis analysis of target-spacer interactions at single-nucleotide resolution, which revealed key spacer nucleotide positions intolerant to mismatches. We show that introducing synthetic mismatches at these precise positions enables de nova design of CRISPR RNA (crRNA) with strong preferential silencing of SNV transcripts. We demonstrate that our top-performing crRNAs possess prominent SNV-selectivity with dose-dependent silencing activity against all KRAS G12 variants at both the RNA and protein levels with minimal off-target silencing of wildtype KRAS. We applied these design principles to effectively silence oncogenic NRAS G12D and BRAF V600E transcripts, underscoring the adaptability of this platform to silence various SNVs. These findings demonstrate that the CRISPR Cas13 system can be reprogrammed to target mutant transcripts with single-base precision, showcasing the tremendous potential of this tool in personalized transcriptome editing. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=176 SRC="FIGDIR/small/557083v1_ufig1.gif" ALT="Figure 1"> View larger version (58K): org.highwire.dtl.DTLVardef@20a7b3org.highwire.dtl.DTLVardef@1e1f79corg.highwire.dtl.DTLVardef@4724c1org.highwire.dtl.DTLVardef@be3063_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2022

A novel transcriptional signature identifies T-cell infiltration in high-risk paediatric cancer

Molecular profiling of the tumour immune microenvironment (TIME) has enabled the rational choice of immunotherapies in some adult cancers. In contrast, the TIME of paediatric cancers is relatively unexplored. We speculated that a more refined appreciation of the TIME in childhood cancers, rather than a reliance on commonly used biomarkers such as tumour mutation burden (TMB), neoantigen load and PD-L1 expression, is an essential prerequisite for improved immunotherapies in childhood solid cancers. We combined immunohistochemistry (IHC) and molecular profiling to develop an alternative, expression-based signature associated with CD8+ T-cell infiltration of the TIME in high-risk paediatric tumours. Using this novel 15-gene immune signature, Immune Paediatric Signature Score (IPASS), we estimate up to 31% of high-risk cancers harbour infiltrating T-cells. Our data provides new insights into the variable immune-suppressive mechanisms dampening responses in paediatric solid cancers. Effective immune-based interventions in high-risk paediatric cancer will require individualised analysis of the TIME.

cancer biology↗

bioRxiv2022

Single-base precision design of CRISPR-Cas13b enables systematic silencing of oncogenic fusions

Precision oncology programs can rapidly identify oncogenic gene fusions in individual patients1-3. However, despite their established oncogenic status, the vast majority of gene fusions remain undruggable due to the lack of specific inhibitory molecules4, 5. Here, we establish PspCas13b, a poorly characterized programmable RNA nuclease, as a versatile tool to silence various oncogenic fusion transcripts. Our Single-Base Tiled crRNA screens (SiBTil), unbiased computational analysis, and comprehensive spacer-target mutagenesis revealed key determinants of PspCas13b activity. De novo design of crRNAs harbouring basepaired or mismatched guanosine bases at key spacer positions greatly enhances the silencing efficacy of otherwise inefficient crRNAs, expanding the targeting spectrum of this enzyme. We also reveal the interface between mismatch tolerance and intolerance, which unlocks an unexpected single-base precision targeting capability of this RNA nuclease. Notably, our de novo design principles enable potent and selective silencing of various gene fusion transcripts and their downstream oncogenic networks, without off-targeting of non-translocated variants that share extensive sequence homology. We demonstrate that PspCas13b targeting the breakpoint of fusion transcripts enables efficient suppression of ancestral and single-nucleotide mutants (e.g. BCR-ABL1 T315I) that often drive clinical cancer relapse. Collectively, this study provides new design principles for PspCas13b programming to specifically recognise and degrade any undruggable fusion oncogenic transcript, thus providing a new conceptual framework for personalized oncology.

molecular biology↗

bioRxiv2021

The pore conformation of lymphocyte perforin

Perforin is a pore-forming protein that facilitates rapid killing of pathogen-infected or cancerous cells by the immune system. Perforin is released from cytotoxic lymphocytes, together with pro-apoptotic granzymes, to bind to the plasma membrane of the target cell where it oligomerises and forms pores. The pores allow granzyme entry, which rapidly triggers the apoptotic death of the target cell. Here we present a 4 [A] resolution cryo-EM structure of the perforin pore, revealing new inter- and intra-molecular interactions stabilising the pore. During assembly and pore formation, the helix-turn-helix motif at the bend in the central {beta}-sheet moves away from the bend to form an intermolecular contact. Cryo-electron tomography shows that prepores form on the membrane surface with minimal conformational changes. Our findings suggest the sequence of conformational changes underlying oligomerisation and membrane insertion, and explain how several pathogenic mutations affect function.

biochemistry↗

bioRxiv2020

Reprogrammed CRISPR-Cas13b suppresses SARS-CoV-2 replication and circumvents its mutational escape through mismatch tolerance

Mutation-driven evolution of SARS coronavirus-2 (SARS-CoV-2) highlights the need for innovative approaches that simultaneously suppress viral replication and circumvent viral escape routes from host immunity and antiviral therapeutics. Here, we employed genome-wide computational prediction and singlenucleotide resolution screening to reprogram CRISPR-Cas13b against SARS-CoV-2 genomic and subgenomic RNAs. Reprogrammed Cas13b effectors targeting accessible regions of Spike and Nucleocapsid transcripts achieved >98% silencing efficiency in virus free-models. Further, optimized and multiplexed gRNAs suppressed viral replication by up to 90% in mammalian cells infected with replication-competent SARS-CoV-2. Unexpectedly, the comprehensive mutagenesis of guide-target interaction demonstrated that single-nucleotide mismatches do not impair the capacity of a potent single gRNA to simultaneously suppress ancestral and mutated SARS-CoV-2 in infected mammalian cells, including the highly infectious and globally disseminated Spike D614G mutant. The specificity, efficiency and rapid deployment properties of reprogrammed Cas13b described here provide a molecular blueprint of antiviral therapeutics to simultaneously suppress a wide range of SARS-CoV-2 mutants, and is readily adaptable to other emerging pathogenic viruses.

microbiology↗

bioRxiv2020

Lipid specificity of the immune effector perforin

Perforin is a pore forming protein used by cytotoxic T lymphocytes to remove cancerous or virus-infected cells during immune response. During the response, the lymphocyte membrane becomes refractory to perforin function by accumulating densely ordered lipid rafts and externalizing negatively charged lipid species. The dense membrane packing lowers the capacity of perforin to bind, and negatively charged lipids scavenge any residual protein before pore formation. Using atomic force microscopy on model membrane systems, we here provide insight into the molecular basis of perforin lipid specificity.

biophysics↗

bioRxiv2019

Efficient CRISPR/Cas9 gene ablation in uncultured naive mouse T cells for in vivo studies

CRISPR/Cas9 technologies have revolutionised our understanding of gene function in complex biological settings, including T cell immunology. Current CRISPR-mediated gene deletion strategies in T cells require in vitro stimulation or culture that can both preclude studies of gene function within unmanipulated naive T cells and can alter subsequent differentiation. Here we demonstrate highly efficient gene deletion within uncultured primary naive murine CD8+ T cells by electroporation of recombinant Cas9/sgRNA ribonucleoprotein immediately prior to in vivo adoptive transfer. Using this approach, we generated single and double gene knock-out cells within multiple mouse infection models. Strikingly, gene deletion occurred even when the transferred cells were left in a naive state, suggesting that gene deletion occurs independent of T cell activation. This protocol thus expands CRISPR-based probing of gene function beyond models of robust T cell activation, to encompass both naive T cell homeostasis and models of weak activation, such as tolerance and tumour models.

immunology↗