Session One: CRISPR Biology

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Talk Title: Next Generation CRISPR tools to model and interrogate cancer

Abstract Title: Advancing the genetic engineering toolbox by combining AsCas12a knock-in mice with ultra-compact screening

Authors: Wei Jin1,2,3,4,#, Yexuan Deng1,2,3,4,5,#, John E. La Marca1,2,3,4,#, Emily J. Lelliott1,2,3,4, Sarah T. Diepstraten3,4, Christina König1,2,3, Lin Tai1, Valentina Snetkova6, Kristel M. Dorighi6, Luke Hoberecht7, Millicent G. Hedditch3, Lauren Whelan3, Geraldine Healey1, Dan Fayle1, Kieran Lau1, Margaret A. Potts1,2,3,4, Moore Z. Chen8, Angus P. R. Johnston8, Yang Liao1,2, Wei Shi1,2, Andrew J. Kueh1,2,3,4, Benjamin Haley6,9,*, Jean-Philippe Fortin7,*, Marco J. Herold1,2,3,4,*,^

Affiliations: 1Olivia Newton-John Cancer Research Institute, Heidelberg, Melbourne, Australia 2School of Cancer Medicine, La Trobe University, Bundoora, Melbourne, Australia 3The Walter and Eliza Hall Institute of Medical Research, Parkville, Melbourne, Australia 4Department of Medical Biology, University of Melbourne, Parkville, Melbourne, Australia 5The State Key Laboratory of Pharmaceutical Biotechnology, School of Life Sciences, Nanjing University, Nanjing, China 6Department of Molecular Biology, Genentech, Inc., South San Francisco, California, USA 7Computational Sciences, Genentech, Inc., South San Francisco, California, USA 8Drug Delivery, Disposition and Dynamics, Monash Institute of Pharmaceutical Sciences, Monash University, Parkville, Melbourne, Australia 9current address: Université de Montréal, Centre de recherche de l'Hôpital Maisonneuve Rosemont #share first authorship *share senior authorship ^corresponding author

Full Abstract: CRISPR-Cas9 technology has revolutionized genetic research, particularly in the discovery of novel tumour drivers and resistance factors through CRISPR screens. Cas12a (Cpf1) distinguishes itself from Cas9 by short crRNAs and a unique PAM site . Additionally, intrinsic RNase activity simplify multiplexed gene targeting via the processing of individual crRNAs from a pre-crRNA-encoding RNA. Here, we present a mouse model that constitutively expresses enhanced Acidaminococcus sp.Cas12a (enAsCas12a) linked to an mCherry fluorescent reporter. We validated robust expression of Cas12a in our mouse model across multiple tissues, with a particular focus on haematopoietic organs, a key facet of our research. We demonstrate efficient single and multiplexed gene-editing in vitro, using primary and transformed cells from enAsCas12a mice. We further demonstrate successful in vivo gene-editing, using normal and cancer-prone enAsCas12a stem cells to reconstitute the haematopoietic system of wild-type mice. We next generated compact, genome-wide Cas12a knockout libraries targeting each gene with four crRNAs encoded across one (Scherzo) or two (Menuetto) vectors, and demonstrated the utility of these libraries across multiple screens: in vitro enrichment screening in lymphoma cells, in vitro drop-out screening in immortalised MDFs, and in vivo enrichment screening in the haematopoietic lineage of mice to identify lymphoma-driving events. The consistency and robustness of the data extracted from each of our screens underscore the high effectiveness and broad potential of these new tools. Finally, we demonstrate CRISPR multiplexing via simultaneous gene knockout (via Cas12a) and activation (via dCas-SAM) using primary T cells and mouse dermal fibroblasts (MDFs). This highlights the compatibility of our enAsCas12a mouse model with other CRISPR technologies. Collectively, our enAsCas12a mouse model and accompanying crRNA expression libraries enhance genome engineering capabilities and complement current CRISPR technologies.

Abstract Title: Using a spatial CRISPR screen to identify regulators of the immune microenvironment in non-small cell lung cancer

Authors: Marina Leiwe1,2, Claire Marceaux1,2, Daniel Batey1, Velimir Gayevskiy1, Tracy Leong3, Marie-Liesse Asselin-Labat1,2

Affiliations: 1Walter and Eliza Hall Institute of Medical Research, 1G Royal Parade, Parkville Victoria 3052, Australia 2Department of Medical Biology, The University of Melbourne, Parkville, Victoria 3010, Australia 3Olivia Newton John Cancer Research Institute and Austin Health, Heidelberg, Victoria, 3084

Full Abstract: Although immunotherapy has shown major success in the clinic, most lung cancer patients fail to respond long-term to the current treatment options. Immune cell composition and spatial organisation of the tumour microenvironment (TME) determine patient response to immunotherapy. However, how cancer cells modulate cellular arrangements within the TME remains poorly understood. Here, we combine a spatial in vivo CRISPR screen, called Perturb-map, with spatial omic technologies to decipher the mechanisms tumour cells employ to reshape the immune microenvironment in non-small cell lung cancer.

Perturb-map adds spatial resolution to pooled CRISPR screens, hence allowing the identification of extracellular gene functions within the native tissue environment. 

As this approach is limited in the number of genes that can be studied in parallel, we curated a candidate gene library based on two RNA-seq datasets of lung adenocarcinoma patient biopsies: 1) comparing tumours sensitive or resistant to anti-PD-L1 therapy and 2) comparing tumours with high or low immune infiltration. Correlating these two transcriptomic datasets identified genes associated with both, response to immune checkpoint blockade and immune infiltration, suggesting these gene products may be involved in immunotherapy resistance via modulation of the tumour immune microenvironment.

We have completed a pilot screen depleting 12 candidate genes in murine lung carcinoma cells before injecting pooled knock-out cells into immunocompetent mice to grow orthotopic tumours. We identified two genes, which when depleted, decreased the number of tumour lesions as well as the percentage of total tumour burden compared to control lesions. Our ongoing work uses multiplex imaging techniques to investigate how the individual gene depletions influence composition of the immune microenvironment.

Genes identified in our study may suggest novel drug targets aiming to recruit immune cells to the tumour site and may improve patient outcome in combination with conventional immunotherapies like immune checkpoint blockade.

Abstract Title: Recursive CRISPR screening identifies a functional link between P300 and SF3B3 in transcriptional elongation control

Authors: Dane Vassiliadis 1,2, Jesse J. Balic1,2, Andrea Gillespie1,2, Olivia Braniff1,2, William Rothnie1,2, Oliver Sinclair1, Kelsy Prest1,2, Andrew Das1,2, Ching-Seng Ang3, and Mark A. Dawson1,2,4,^.Dane Vassiliadis1,2,*,^, Jesse J. Balic 1,2, Andrea Gillespie 1,2, Olivia Braniff 1,2, William Rothnie 1,2, Oliver Sinclair 1, Kelsy Prest 1,2, Andrew Das 1,2, Ching-Seng Ang 3, and Mark A. Dawson 1,2,4.

Affiliations: 1 Peter MacCallum Cancer Centre, Melbourne, Victoria, Australia; 2 Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Victoria, Australia; 3 Bio21 Mass Spectrometry and Proteomics Facility, The University of Melbourne, Parkville, Victoria 3052, Australia; 4 The University of Melbourne Centre for Cancer Research, The University of Melbourne, Melbourne, Victoria, Australia; 2 Sir Peter MacCallum Department of Oncology, The University of Melbourne, Melbourne, Victoria, Australia; 3 Bio21 Mass Spectrometry and Proteomics Facility, The University of Melbourne, Parkville, Victoria 3052, Australia; 4 The University of Melbourne Centre for Cancer Research, The University of Melbourne, Melbourne, Victoria, Australia.

Full Abstract: The expression of eukaryotic protein-coding genes is the product of transcriptional (e.g. initiation, elongation and termination) and co-transcriptional processes (e.g. splicing, modification and export). How diverse input signals are relayed to, and interpreted by transcription factors and associated co-factors to govern RNA Pol II activity remains unclear. Here we developed a gene expression reporter system traceable in real-time by flow cytometry and combined this with CRISPR/Cas9 screens to identify epistatic relationships underpinning transcriptional co-activator inhibition in human cells. We find that loss of the U2 spliceosome factor SF3B3 broadly desensitises cells to transcriptional co-activator inhibition, with short, exon-dense genes displaying the most prominent desensitisation. We show that splicing remains functional despite SF3B3 loss and that the desensitisation effect is mediated by the physical presence, but not the catalytic activity of the acetyltransferase and transcriptional co-activator P300. Mechanistically, SF3B3 depletion drives the redistribution of chromatinised P300 to newly licensed cis regulatory elements enriched for JUN/AP1; and perturbs the pause release and elongation dynamics of RNA Pol II. Moreover, the association of key transcriptional complexes, elongation factors and cyclin dependent kinases with RNA Pol II is reduced following SF3B3 depletion. Finally, using a CRISPR base editor screening approach, we performed tiled mutagenesis of P300 and discovered key residues in the histone acetyltransferase domain that either promote or prevent desensitisation to transcriptional coactivator inhibition. Together these data suggest a splicing-independent functional link between SF3B3, P300 and RNA Pol II required for the coordination and licensing of transcriptional elongation.

Abstract Title: Expanding the molecular biology toolkit for paediatric cancer: generation of a comprehensive library of CRISPR/Cas cell lines

Authors: Alice Salib1, Teresa Sadras2,3, Fatimah Jalud2,3, Tasnia Ibnat2,3, Paul G Ekert1,2,4,5,6

Affiliations: 1. Translational Tumour Biology, Children’s Cancer Institute, Australia. 2. Peter MacCallum Cancer Centre, Australia 3. The Sir Peter MacCallum Department of Oncology, University of Melbourne, Australia 4. School of Clinical Medicine, University of New South Wales, Australia 5. University of New South Wales Centre for Childhood Cancer Research, University of New South Wales, Australia 6. Cancer Immunology Program, Peter MacCallum Cancer Centre, Australia

Full Abstract: Personalised medicine initiatives like the Zero Childhood Cancer Program have highlighted the genomic heterogeneity of paediatric cancers, and the value of molecular characterisation to deliver more effective treatments. As novel variants are identified, there is a growing need to characterise their functions and therapeutic targetability. To address this, we are developing a comprehensive library of CRISPR/Cas-engineered isogenic paediatric cancer cell lines, enabling manipulation of gene expression through knockout (CRISPR/Cas9), knockdown (CRISPR/Cas13), or overexpression (CRISPRa) strategies. A nuclease-dead Cas13d variant (dCas13d) serves as a control. Leveraging these tools, we aim to functionally characterise candidate driver genes and novel variants in paediatric malignancies.

To date, we have generated 22 CRISPR/Cas cell lines, encompassing a wide range of paediatric cancers including neuroblastoma, medulloblastoma, rhabdomyosarcoma, osteosarcoma, and Ewing's sarcoma. Across the CRISPR/Cas13d lines generated, we observe variable doxycycline (dox)-induced Cas13d expression. Further, our findings suggest guide expression levels may limit gene silencing efficiency. Clonogenic assays revealed a dose-dependent reduction in colony-forming capacity upon Cas13d induction, independent of guide expression. Our results indicate that careful control of both guide and Cas13d expression is needed and strategies to mitigate Cas13d toxicity while enhancing guide efficacy are in progress.

In parallel, we are using CRISPR/Cas9 cell lines we have generated in combination with a unique pipeline we have developed to generate PDGFRA structural variants by targeting intronic regions adjacent to the deleted exons. This approach enables us to recreate these PDGFRA variants endogenously to further characterise their function. 

In summary, the cell lines developed offer a valuable resource for investigating the molecular mechanisms driving paediatric cancers. Furthermore, they serve as a toolkit for identifying and exploring potential therapeutic vulnerabilities in paediatric cancer to select targeted and more effective treatments for patients.

Abstract Title: Reactivation of δ-globin via CRISPR in treating β-haemoglobinopathies

Authors: Wing Fuk Chan, James J. J. The, Bradon Rumler, Stephanie Anderson, Huimin Cai, Zane S. Kaplan, Andrew C. Perkins

Affiliations: 1Australian Centre for Blood Diseases, Monash University

Full Abstract: β-haemoglobinopathies are inherited blood disorders that affect haemoglobin in erythrocytes, with hundreds of thousands of affected new-borns each year. While sickle cell disease results from a single-nucleotide substitution, β-thalassaemia is caused by hundreds of different mutations, making the direct repair of mutations challenging. As such, a universal therapeutic approach is widely sought.

Much of the effort has been put into reactivating the foetal γ-globin by various means in the adult cells. As an alternative, δ-globin is expressed in adult red blood cells and shares a much higher sequence homology to β-globin. As such, δ-globin, and the resultant haemoglobin A2 (HbA2), is biochemically more similar and fully functional as the conventional adult haemoglobin HbA. However, δ-globin is expressed at a low level and HbA2 only constitutes 2-3% of the total adult haemoglobin. The low expression is believed to be due to mutations at the promoter that disrupt transcription factor binding. Nevertheless, overexpression of δ-globin transgene is able to rescue the sickling phenotype of red cell in a mouse model. As such, we propose reactivating the δ-globin by editing the promoter as effective alternative therapeutics of SCD and β-thalassaemia.

In this project we employed CRISPR gene editing techniques to install an KLF1 motif at the δ-globin promoter in the immortalised HUDEP-2 cell line. Through RNP transfection along with an HDR repair template, we were able to drive the expression of δ-globin to ~160 fold in the HUDEP-2 cells compared to the control. The upregulated δ-globin transcript level is around 60% of that of β-globin. Upon differentiation, we observed the HbA2 level is upregulated to 20% by HPLC. Using NGS, we demonstrated the editing is highly efficient with over 50% HDR events. Lastly, we showed that this upregulation of δ-globin is highly dependent on the insertion site at the promoter.

Abstract Title: Genome-wide CRISPR Screen Identify Menin as a Mediator of Encorafenib Plus Cetuximab Resistance in BRAF V600E Mutant Colorectal Cancer

Authors: Akash Srivaths (1), Tirta Mario Djajawi (1), Kristen Needham (1), Chloe Gerak (2), Liam Neil (1), Sarahi Mendoza Rivera (2), Bhupinder Pal (1), Stephin Vervoort (2), John Mariadason (1), Conor Kearney (1)

Affiliations: 1. Olivia Newton-John Cancer Research Institute, 2. Walter and Eliza Hall Institute of Medical Research

Full Abstract: Colorectal cancer is the second most commonly diagnosed cancer and one of the leading causes of cancer-related deaths in Australia. Mutations in the BRAF gene are frequently detected in several cancers including colorectal cancer. Up to one in five patients with metastatic colorectal cancer have a BRAF mutation, with the V600E substitution being the most prevalent. These patients generally have a poor prognosis. A combination of encorafenib plus cetuximab has significantly improved the overall survival of such patients, compared to standard chemotherapy regimens in clinical trials, establishing it as the new standard of care for colorectal cancer with BRAF V600E mutation. While this treatment strategy is promising, it does not eradicate the tumor completely as tumor-intrinsic resistance poses a challenge. We performed whole-genome CRISPR screens to identify such resistance-causing genes in response to encorafenib plus cetuximab. We identified that MEN1 (Menin) confers resistance to encorafenib plus cetuximab treatment in colorectal cancer with BRAF V600E mutation. Indeed, MEN1 knockout, or treatment with revumenib, a small-molecule inhibitor of the Menin-MLL interaction, augmented the cytostatic effect of encorafenib plus cetuximab in BRAF V600E mutant colorectal cancer cells, whereas re-expression of MEN1 into MEN1-null cells reversed this phenotype. We further demonstrated that the protective role of menin is dependent on its interaction with MLL1, a histone methyltransferase. Taken together, we identify Menin as a key factor in driving encorafenib plus cetuximab resistance, providing rational for targeting of Menin in combination with encorafenib plus cetuximab for improved treatment of colorectal cancers with BRAF V600E mutation.

Abstract Title: High depth broad scale CRISPR screening to identify fundamental synthetic lethal targets to P300/CBP inhibition

Authors: Oliver Sinclair, Jesse Balic, Tim Somervaille, Mark Dawson

Affiliations: Peter MacCallum Cancer Centre, Cancer Research UK Manchester Institute

Full Abstract: Multiple myeloma (MM) and acute myeloid leukaemia (AML) are distinct haematopoietic malignancies which together provide a large unmet clinical need. While both are discrete they progress stepwise through premalignant states, and share dependency on aberrant transcriptional programmes that block differentiation. This dependency renders both diseases susceptible to inhibition of core transcriptional machinery, including the transcriptional co-activators p300 and CBP. These highly homologous acetyltransferases occupy (super)enhancer and promoter cis-regulatory elements to critically govern cell-type and disease-specific gene regulatory networks. In MM, p300/CBP activity sustains elevated IRF4 levels, driving proliferation and survival. In AML, p300/CBP supports MYC expression, promoting rapid growth.

This work investigates CCS1477, a novel p300/CBP bromodomain inhibitor in clinical evaluation. While early trials report responses in 67% of MM patients, relapse and resistance remain significant barriers. To address these challenges, we performed unbiased CRISPR screening across AML and MM cell lines to identify synthetic lethal targets and resistance mechanisms.

CRISPR screens were conducted in five cell lines across two doses and timepoints, with the hope of identifying fundamental sensitising hits to p300/CBP inhibition. Hits were ranked by average RRA scores, revealing the metazoan INO80 chromatin remodelling complex as a key sensitising target. Validation in both disease states and in screen-naïve cell lines confirmed these hits sensitize cells to CCS1477 and to alternative p300/CBP inhibitors targeting histone acetyltransferase activity. 

Our findings establish the INO80 complex as a critical regulator of p300/CBP-driven transcription and a disease, cell line and inhibitor agnostic synthetic lethal target to enhance efficacy. These hits would be overlooked in standard individual screening approaches. Therefore, this study presents a robust CRISPR screening approach to prioritise synthetic lethal targets which outperforms standard single cell line approaches.

Abstract Title: Identifying New Roles For Ubiquitination Machinery In Regulating Dendritic Cell Function

Authors: Huw Morgan1, Haiyin Liu1, Laura E Edgington-Mitchell1, Justine D Mintern1

Affiliations: 1Department of Biochemistry and Pharmacology, The Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne, Parkville, Australia

Full Abstract: Ubiquitination is a post-translational modification that regulates protein degradation and activity, such as in regulating inflammatory signalling or protein surface expression. A cascade of enzymes carries out ubiquitination of proteins. One key player is the E3 ubiquitin (Ub) ligases, which are involved in the addition of ubiquitin. This project aims to unveil novel roles for these enzymes in immunity, focusing on dendritic cells (DCs). DCs are antigen-presenting cells specialised in regulating T cells during the adaptive immune response, maintaining the balance between health and disease. Of the over 600 known and putative E3 Ub ligases, only a handful have a described role in DC function. This project aims to identify other E3 Ub ligases that participate in the regulation of DCs.

To identify E3 Ub ligases that participate in DC function, we employed an arrayed CRISPR/Cas9 screen of all known and putative E3 Ub ligases and scaffold proteins in the mutuDC line. The expression of surface markers involved in DC function were analysed by spectral flow cytometry to assess the impact of gene knockout on DCs. This screen identified Cul3 as a new regulator of DC function. The screen results were confirmed in bone marrow-derived dendritic cells (BMDCs) differentiated from gene-edited Cas9 transgenic haematopoietic progenitors. Knockout of Cul3 upregulated the expression of co-stimulatory molecules CD80 and CD86 in unstimulated BMDCs and upregulated the expression of the inhibitory molecule PD-L2 following TLR3 and TLR9 stimulation, relative to wild-type BMDCs. 

Using a functional genomics approach, we have identified new roles for ubiquitination machinery in regulating the function of DCs. We identified a new role for Cul3, a gene with no known role in DC function. Further exploration of these pathways will gain insight into the complex regulatory network of DCs and highlight new ways to manipulate DC function in health and disease.

Abstract Title: Tumour-site directed expression of adenosine receptor subtype 1 (A1R) enhances CAR T cell function and improves efficacy against solid tumours

Authors: Kevin Sek (1,2), Amanda Chen (1,2), Thomas Cole (1,2), Philip K. Darcy (1,2,6), Paul A. Beavis (1,2,6)

Affiliations: (1) Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, 3000, Victoria, Australia, (2) Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, 3010, Australia, (3) Department of Pathology, University of Melbourne, Parkville.

Full Abstract: The efficacy of Chimeric Antigen Receptor (CAR) T cells against solid tumours is limited by immunosuppressive factors including adenosine, which suppresses CAR T cells through activation of the A2A receptor (A2AR). Deletion of the A2AR with CRISPR/Cas9 significantly enhances the in vivo efficacy of both mouse and human CAR T cells. Alternatively, CAR T cells were engineered to express A1R, a receptor that signals inversely to A2AR. Using murine and human CAR T cells, constitutive A1R overexpression was demonstrated to significantly enhanced CAR T cell effector function but at the expense of CAR T cell persistence. Through a novel CRISPR/Cas9 “knock-in” approach CAR T cells were engineered to express A1R upon CAR T cell activation, which led to enhanced anti-tumour efficacy concomitant with improved long-term persistence and was dependent on the transcription factor IRF8. These data provide a novel approach for enhancing CAR T cell efficacy in solid tumours and provides proof of principle for site-directed expression of factors that promote effector T cell differentiation.

Abstract Title: Improving neuroblastoma response to BH3-mimetic drugs with CRISPRi

Authors: Ziyan Liu, Shuai Huang, Grant Dewson

Affiliations: WEHI

Full Abstract: High-risk neuroblastoma patients face a poor prognosis and limited response to chemotherapy, with 50% of children relapse following aggressive chemotherapy regimens, and median overall survival after relapse is 11 months. Hence there is critical need to identify more effective therapeutic targets with limited toxicities. Targeted BH3 mimetic drugs such as VENCLEXTA®/Venetoclax/ABT-199 are changing the treatment landscape for certain blood cancers, but their use in neuroblastoma has been hindered by resistance. To address this challenge, this study leverages CRISPRi screening technology to identify targets that can sensitize neuroblastoma to venetoclax treatment and overcome resistance. 

Investigating these targets and utilizing the ubiquitin system could uncover synergistic pathways, enhancing venetoclax efficacy in neuroblastoma. By elucidating the underlying molecular mechanisms, this project aims to advance targeted therapeutic strategies and improve outcomes for neuroblastoma patients.

 Talk Title: CRISPR engineering of armored CAR T cells enables tumor restricted payload delivery with enhanced efficacy and safety 

Abstract Title: TAK1 protects tumour cells from combined CTL-derived TNF and IFN-y

Authors: Tirta M. Djajawi, Anne Huber, Akash Srivaths, Oliver Ozaydin, Sarahi Mendoza Rivera, Stephin J. Vervoort and Conor J. Kearney

Affiliations: Olivia Newton-John Cancer Research Institute, Melbourne, VIC

Full Abstract: Cancer immunotherapies, particularly immune checkpoint blockade, have demonstrated remarkable efficacy in some cancer types including melanoma. However, regardless of the clinical success of checkpoint inhibitor therapies, many patients do not respond or develop resistance, leading to relapse. Whereas some mechanisms of tumour immune escape are understood, the full spectrum of immune evasion routes are not well understood. Given the issue of high non-response rates and patient relapse, there is an urgent need to molecularly investigate tumour immune evasion tactics in order to develop improved immunotherapy approaches.

Here, we conducted a kinome-wide CRISPR-Cas9 screen in melanoma cells to identify factors that limit CD8+ T cell-mediated anti-tumour immunity. We identified that TAK1 suppresses combined CTL-derived IFNy- and TNF-induced cell death, thus protecting from CD8+ T cell-mediated killing. Indeed, TAK1 knockout or pharmacological targeting of TAK1 with the clinical inhibitor Takinib enhances CD8+ T cell-mediated cancer cell killing in a TNF and IFNy-dependent manner, while re-introduction of TAK1 in TAK1-deficient cells reverses this effect. Furthermore, through genome-wide CRISPR screening in a TAK1 deficient background, we identified STAT1, RIPK1 and Caspase-8 as key effectors of the combined TNF and IFNy-induced cell death. Taken together, we identify TAK1 as a negative regulator of anti-tumour immunity, unveiling TAK1 targeting as a novel immunotherapeutic approach or as an adjunct to existing immunotherapies.

Abstract Title: Generating CRISPR/CAS9 armoured TCR-T cells for the treatment of solid tumours

Authors: Phoebe Dunbar (1, 2), Amanda Chen (1, 2), Kah Min Yap (1, 2), Phil Darcy (1, 2), Paul Beavis (1, 2)

Affiliations: 1. Cancer Immunology Program, Peter MacCallum Cancer Centre, Melbourne, 3000, Victoria, Australia. 2. Sir Peter MacCallum Department of Oncology, The University of Melbourne, Parkville, 3010, Australia

Full Abstract: CAR T cell therapy has exhibited remarkable clinical success in the treatment of haematological malignancies, however, their efficacy in solid tumours is limited by antigen heterogeneity and immunosuppression imposed by the tumour microenvironment. To overcome these barriers, ‘armoured’ CAR T cells which secrete pro-inflammatory cytokines have been developed. However, toxicities related to the unrestricted expression of the armouring transgene has limited the application of these CAR T cells in the clinic. Our lab has developed a novel CRISPR/Cas9-mediated homology directed repair (HDR) strategy whereby we aimed to engage endogenous gene regulatory mechanisms drive transgene expression in a tumour-localised manner (in review, Nature). Genome-wide RNA-sequencing was used to identify genes in CAR T cells with tumour-specific expression. The promotor NR4A2 was identified as a key candidate due to its inhibitory role in T cells. This novel CRISPR HDR strategy was employed to knock in proinflammatory cytokine, IL-12 into NR4A2 promoter which supports the delivery of cytokines directly to the tumour site, leading to enhanced anti-tumour efficacy and long-term survival of mice in both syngeneic and xenogeneic models. 

Although we have developed a novel technology that tightly restricts expression of pro-inflammatory cytokines to T cell activation, the application of this technology in a CAR T setting is limited by on-target toxicity in healthy cells where tumour antigen is expressed. I therefore hypothesise that the application of tumour neoantigen specific TCR-T cells would provide an even greater safety profile for this technology. I have generated T cells expressing an engineered TCR targeting the tumour neoantigen KRAS G12D and subsequently verified that the NR4A2 promotor effectively turns on within this neoantigen specific TCR-T cell setting. I have further engineered TCR-T cells to express an NR4A2/IL-12 knock in, which elicits improved anti-tumour efficacy and an improved safety profile in xenogeneic mouse models.

Abstract Title: Robust Genomewide Application of Deactivated CRISPR-Cas9 Systems for Expression Regulation

Authors: Emily Anderson1, Clarence Mills1, Joanna Gawden-Bone2, Jera Law1, Abhijit Patil2, Matthew Brockman3, Brian Ziemba1, Andrew Riching1, Kevin Hemphill1, Simon Scrace2, Zaklina Strezoska1, and Josien Levenga1

Affiliations: 1. Revvity, Lafayette, Colorado, United States 2. Revvity, Cambridge, United Kingdom 3. Revvity, Mulgrave, Australia

Full Abstract: CRISPR-Cas9 has been engineered for modulating transcription and epigenetics; CRISPR activation (CRISPRa) and CRISPR interference (CRISPRi) systems act via a nuclease-deactivated Cas9 (dCas9) fused to modifiers and targeted to promoters by gene-specific synthetic guide RNAs (gRNAs). We have optimized CRISPR-based modulation for use in primary and difficult cells (requiring chemically synthesized gRNAs transiently delivered with dCas9 mRNA or ribonucleoprotein (RNP)). Synthetic gRNA delivery was also tested for streamlined high-throughput arrayed screening using stably-expressing Cas9 cell lines. Finally, high-titer All-in-one dCas9-effector lentiviral systems were produced allowing up to full genome pooled screens.

We demonstrate a simple, robust CRISPRa approach compatible with dCas9 fused to the VP64-p65-Rta tripartite activator (dCas9-VPR). Endogenous activation can be tuned/enhanced using multiple chemically modified synthetic gRNAs. Using the All-in-one dCas9-VPR lentiviral system, we have conducted a whole-genome CRISPRa screen, which faithfully recapitulates data from prior drug-resistance screening using the complex, three-vector Synergistic Activation Mediator (SAM).

The Dharmacon CRISPRi system is more efficient than first-generation CRISPRi. This novel dCas9 fusion uses domains from transcriptional repressors Sal-like protein 1 (SALL1)/Sin3 histone deacetylase corepressor complex component (SDS3). An unbiased protein interaction experiment demonstrates dCas9-SALL1-SDS3 interacts with key members of the histone deacetylase and Swi-independent 3 complexes (endogenous functional effectors of SALL1 and SDS3). dCas9-SALL1-SDS3 with chemically synthesized gRNAs can be used orthogonally to siRNA to verify downstream phenotypes. Finally, we performed the first genome-wide pooled screen with single lentiviral vector (All-in-one) dCas9-SALL1-SDS3 CRISPRi and observed enhanced essential gene dropout compared to first-generation single-vector dCas9-KRAB CRISPRi.

These CRISPR modulation systems were optimized for short-term, non-viral delivery or for pooled screening with high-titer lentiviral libraries. These formats enable simple, robust, and rapid gain- or loss-of-function experiments. Complex end-point assays are now accessible, and scale up to high throughput arrayed or pooled screening platforms is possible for multiple compounds, hypotheses, or cell types.