Revealing cancer glycome drivers using CRISPR activation screens.

Cells deficient in the DNA repair pathway homologous recombination (HR) show particular sensitivity to inhibitors of poly-ADP-ribose polymerases (PARPi). Based on this observation, several PARPi, such as olaparib, have been FDA approved for the treatment of BRCA2-mutated ovarian cancer. Indeed, clinical trials have demonstrated that use of these agents significantly improved progression-free survival (PFS), for example, from 5.5 months to 19 months in one cohort of ovarian cancer patients harboring BRCA1/2 mutations (SOLO2 trial). However, even in this trial, only 65% of patients who were predicted to be genetically susceptible to this treatment attained 12 months PFS. This indicates that sensitivity to PARPi is mediated by more than simply BRCA2 status. Moreover, investigations into mechanisms governing sensitivity and resistance to PARPi continue to further our understanding of basic DNA repair and replication pathways. To this end, we conducted dual CRISPR screens in BRCA2-deficient HeLa cells as an unbiased approach for identifying proteins whose loss or overexpression confers resistance to PARPi. First, over 19,000 genes were individually knocked out in BRCA2-deficient cells. Then, these cells were treated or not with olaparib. Sequencing and computational analysis were used to identify which genes were lost most often in cells surviving olaparib treatment, yielding hundreds of potential hits. Hits were tested in multiple cell lines using cellular viability, apoptosis, and clonogenic survival assays. Several of these hits were successfully validated, including the histone acetyltransferase TIP60 (KAT5) and the ubiquitin ligase HUWE1. Mechanistically, we show that the rescue of PARPi sensitivity caused by TIP60 depletion is dependent on the downstream proteins 53BP1 and REV7, proteins known to inhibit end resection, thus promoting NHEJ; this indicates that the role of TIP60 as a modulator of double-strand break repair pathway choice underlies this resistance. The finding that increased 53BP1 binding contributes to PARPi resistance in BRCA2-deficient cells is particularly interesting given that loss of 53BP1 has been shown to cause resistance to PARPi in BRCA1-deficient cells. This indicates that pathways that govern sensitivity to PARPi in BRCA1-deficient cells may have opposite effects on this phenotype in BRCA2-deficient cells. To further understand the networks governing PARPi resistance in BRCA2-deficient cells, we also completed a CRISPR activation screen using the same experimental design as the knockout screen. By combining the knockout and activation screens for the first time in this context, we are able to better construct overarching networks regulating PARPi sensitivity in BRCA2-deficient cells. We have identified many potential mediators of PARPi resistance in BRCA2-deficient cells by combining CRISPR-knockout and CRISPR-activation screens. Our work identifies TIP60 as a novel potential biomarker for PARPi response in BRCA2-deficient cells. Citation Format: Kristen E. Clements, Anastasia Hale, Nathanial J. Tolman, George-Lucian Moldovan. Understanding poly-ADP-ribose polymerase (PARP) inhibitor resistance in BRCA2-deficient cells through dual CRISPR knockout and activation screens [abstract]. In: Proceedings of the AACR Special Conference on Advances in Ovarian Cancer Research; 2019 Sep 13-16, 2019; Atlanta, GA. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(13_Suppl):Abstract nr A39.

Functional genomic screens identify positive and negative regulators of venetoclax resistance

The hostile tumor microenvironment (TME) remains a major challenge for cancer immunotherapy. Here, we performed TME-targeted in vivo CRISPR activation (CRISPRa) screen to identify factors that promote anti-tumor immunity, culminating in rationally designed immune gene therapy combinations. Multiplexed activation of genes encoding antigen presentation, T cell proliferation, co-stimulation, and migration (APCM) leads to enhanced anti-tumor responses. An APCM-focused CRISPRa screen in metastatic tumors identified CD80, TNFSF14, CXCL10, TNFSF18, TNFSF9, and IFNG as top immunostimulatory candidates. Further optimization pinpointed TNFSF9 (4-1BBL) + IFNG + IL12B (4II) as a potent therapeutic combination. AAV-4II enhanced antigen presentation, T cell activation, proliferation, cytotoxicity, and tumor infiltration. Preconditioning the TME with AAV-4II synergized with CAR-T and TCR-T cell therapies to suppress primary and metastatic solid tumors in vivo. These findings establish TME-targeted CRISPRa screening as a rapid route to develop immune gene therapy combinations against solid tumors.

Dystrophic epidermolysis bullosa (DEB) is a skin-blistering disease caused by mutations in COL7A1, which encodes type VII collagen (C7). There is no cure for DEB, but previous work has shown potential therapeutic benefit of increased production of even partially functional C7. Genome-wide screens using CRISPR-Cas9 have enabled the identification of genes involved in cancer development, drug resistance and other genetic diseases, suggesting that they could be used to identify drivers of C7 production. A keratinocyte C7 reporter cell line was created and used in a genome-wide CRISPR activation (CRISPRa) screen to identify genes and pathways that increase C7 expression. The CRISPRa screen results were used to develop a targeted drug screen to identify compounds that upregulate C7 expression. The C7_tdTomato cell line was validated as an effective reporter for detection of C7 upregulation. The CRISPRa screen identified DENND4B and TYROBP as top gene hits plus pathways related to calcium uptake and immune signalling in C7 regulation. The targeted drug screen identified several compounds that increase C7 expression in keratinocytes, of which kaempferol, a plant flavonoid, also significantly increased C7mRNA and protein in DEB patient cells.

Ewing’s sarcoma is an aggressive pediatric bone and soft-tissue cancer with a pathognomonic chromosomal translocation t(11;22) resulting in expression of EWS-FLI1, an “undruggable” fusion protein acting as a transcriptional modulator. Identification and ranking of repressed EWS-FLI1 target genes essential for cancer cell survival will potentially provide much-needed insights to develop novel therapeutic strategies. We performed a CRISPR activation (CRISPRa) dropout screen in Ewing cells. We generated a clonal SKNMC cell line homogenously expressing the synergistic activation mediator (SAM) CRISPRa system to functionally interrogate repressed EWS-FLI1 target genes. The systems functionality was tested using CD44, a surface marker absent on the surface of Ewing cells. We found robust and stable expression of CD44 after introduction of promoter-targeting gRNAs. The library of repressed EWS-FLI1 target genes, named LIBerty, was constructed to target 872 genes bioinformatically selected from publicly available silenced EWS-FLI1 RNA-Seq datasets as well as genes identified as repressed signature genes in Ewing’s sarcoma via meta-analysis with 3,777 unique guideRNAs. LIBerty was lentivirally delivered into SAM SKNMC cells and data from four biologic replicates were gathered at three time points: three, ten, and 21 days after infection. Cells were harvested and samples were prepared for next-generation sequencing (NGS) via PCR. The NGS data were evaluated with the analysis tool PinAPL-Py. Preliminary analysis of the screen revealed efficient selection of the positive control genes BAD and BBC3. In addition, high-ranking hits included CDKN1A and CDKN1C, both cell cycle regulators and known tumor-suppressor genes. Furthermore, we identified PCDH7 and TGFBR2, two genes already known from the literature to play a role in Ewing’s sarcoma. The presence of validated genes amid highest-ranking candidates confirms robustness of the conducted CRISPRa screen. Further analysis and validation of previously unexplored targets and pathways is currently ongoing. Our CRISPRa screen revealed both known as well as previously unknown EWS-FLI1 repressed genes whose absence of function is essential for tumor cell survival. Citation Format: Vadim Saratov, Qui A. Ngo, Gloria Pedot, Felix K. Niggli, Beat W. Schaefer. Identification of physiologically relevant EWS-FLI1 target genes in Ewing’s sarcoma via CRISPRa screening [abstract]. In: Proceedings of the AACR Special Conference on the Advances in Pediatric Cancer Research; 2019 Sep 17-20; Montreal, QC, Canada. Philadelphia (PA): AACR; Cancer Res 2020;80(14 Suppl):Abstract nr B19.

Background & AimsHepatocellular carcinoma (HCC) frequently undergoes regional chromosomal amplification, resulting in elevated gene expression levels. We aimed to elucidate the role of these poorly understood genetic changes by using CRISPR activation (CRISPRa) screening in mouse livers to identify which genes within these amplified loci are cancer driver genes.MethodsWe used data from The Cancer Genome Atlas to identify that frequently copy number-amplified and up-regulated genes all reside on human chromosomes 1q and 8q. We generated CRISPRa screening transposons that contain oncogenic Myc to drive tumor formation. We conducted CRISPRa screens in vivo in the liver to identify tumor driver genes. We extensively validated the findings in separate mice and performed RNA sequencing analysis to explore mechanisms driving tumorigenesis.ResultsWe targeted genes that frequently undergo amplification in human HCC using an in vivo CRISPRa screening system in mice, which induced extensive liver tumorigenesis. Human chromosome 1q genes Zbtb7b, Vps72, Gba1, and Mrpl9 emerged as drivers of liver tumorigenesis. In human HCC there is a trend in correlation between levels of MRPL9, VPS72, or GBA1 and poor survival. In validation assays, activation of Vps72, Gba1, or Mrpl9 resulted in extensive liver tumorigenesis and decreased survival in mice. RNA sequencing revealed different mechanisms driving HCC, with Mrpl9 activation altering genes functionally related to mitochondrial function, Vps72 levels altering phospholipid metabolism, and Gba1 activation enhancing endosomal-lysosomal activity, all leading to promotion of cellular proliferation. Analysis of human tumor tissues with high levels of MRPL9, VPS72, or GBA1 revealed congruent results, indicating conserved mechanisms driving HCC.ConclusionsThis study reveals chromosome 1q genes Vps72, Gba1, and Mrpl9 as drivers of HCC. Future efforts to prevent or treat HCC can focus on these new driver genes.

Inhibitors of poly-ADP-ribose polymerase 1 (PARPi) are highly effective in killing cells deficient in homologous recombination (HR); thus, PARPi have been clinically utilized to successfully treat BRCA2-mutant tumors. However, positive response to PARPi is not universal, even among patients with HR-deficiency. Here, we present the results of genome-wide CRISPR knockout and activation screens which reveal genetic determinants of PARPi response in wildtype or BRCA2-knockout cells. Strikingly, we report that depletion of the ubiquitin ligase HUWE1, or the histone acetyltransferase KAT5, top hits from our screens, robustly reverses the PARPi sensitivity caused by BRCA2-deficiency. We identify distinct mechanisms of resistance, in which HUWE1 loss increases RAD51 levels to partially restore HR, whereas KAT5 depletion rewires double strand break repair by promoting 53BP1 binding to double-strand breaks. Our work provides a comprehensive set of putative biomarkers that advance understanding of PARPi response, and identifies novel pathways of PARPi resistance in BRCA2-deficient cells.

Regulation of cytokine production in stimulated T cells can be disrupted in autoimmunity, immunodeficiencies, and cancer. Systematic discovery of stimulation-dependent cytokine regulators requires both loss-of-function and gain-of-function studies, which have been challenging in primary human cells. We now report genome-wide CRISPR activation (CRISPRa) and interference (CRISPRi) screens in primary human T cells to identify gene networks controlling interleukin-2 (IL-2) and interferon-γ (IFN-γ) production. Arrayed CRISPRa confirmed key hits and enabled multiplexed secretome characterization, revealing reshaped cytokine responses. Coupling CRISPRa screening with single-cell RNA sequencing enabled deep molecular characterization of screen hits, revealing how perturbations tuned T cell activation and promoted cell states characterized by distinct cytokine expression profiles. These screens reveal genes that reprogram critical immune cell functions, which could inform the design of immunotherapies.

Identifying Novel Regulators of Erythroid Maturation and Proliferation Using a Genome-Scale CRISPR Activation Screen

Biochem. Soc. Trans. (2020) 48 (5): 2253–2259 DOI: https://doi.org/10.1042/BST20200523The authors of this paper would like to make the following correction to the citations within the text:Reference 48 was a duplication of reference 12 and should have instead cited Bersuker, K., Hendricks, J.M., Li, Z. et al. (2019) The CoQ oxidoreductase FSP1 acts parallel to GPX4 to inhibit ferroptosis Nature575, 688–692. DOI: 10.1038/s41586-019-1705-2In the section entitled “The FSP1 ubiquinone system”The FSP1-ubiquinone axis was then characterized as a GSH/GPX4 independent ferroptosis-suppressing pathway that exemplifies an elegant system for the enzymatic regeneration of endogenous antioxidants [47].Should instead cite the following:The FSP1-ubiquinone axis was then characterized as a GSH/GPX4 independent ferroptosis-suppressing pathway that exemplifies an elegant system for the enzymatic regeneration of endogenous antioxidants [47, 48].In the section entitled “The tetrahydrobiopterin-DHFR system”More recently, another novel endogenous antioxidant system has been identified. Using CRISPR activation screens, a study identified tetrahydrobiopterin (BH4) as an essential metabolite supporting the proliferation of cancer cell lines challenged with the GPX4 inhibitor RSL3 [48].Should have citedMore recently, another novel endogenous antioxidant system has been identified. Using CRISPR activation screens, a study identified tetrahydrobiopterin (BH4) as an essential metabolite supporting the proliferation of cancer cell lines challenged with the GPX4 inhibitor RSL3 [11, 12].At the end of the article, the final sentenceHence, these studies demonstrate that DHFR should be regarded as an important regulator of ferroptosis, while its inhibitor methotrexate, a common chemotherapeutic agent, could be a promising therapeutic option for ferroptotic anti-cancer therapies [48]Should have readHence, these studies demonstrate that DHFR should be regarded as an important regulator of ferroptosis, while its inhibitor methotrexate, a common chemotherapeutic agent, could be a promising therapeutic option for ferroptotic anti-cancer therapies [12].

CRISPR Activation Screen to Optimize Chimeric Antigen Receptor (CAR) T Cell Immunophenotype

Type III cytokines interleukin (IL)-22 and IL-17 are expressed by both innate and adaptive immune cells to defend against extracellular pathogens, yet their dysregulation contributes to autoimmunity and malignancy. A deeper knowledge of IL-22/17 regulation is warranted to understand physiologic processes as well as disease pathogenesis, thereby elucidating druggable targets. Toward this goal, we applied genome-wide CRISPR activation (CRISPRa) and CRISPR inhibition (CRISPRi) screens to identify the collection of factors that positively or negatively regulate IL-17/22 expression in a murine type III innate lymphoid cell (ILC3) model. Following lentiviral transduction of a CRISPRi or CRISPRa guide RNA (gRNA) library, cells were stimulated and sorted by levels of intracellular IL-22/17 protein expression. Relative gRNA frequencies in each sequenced population revealed enrichment and depletion of potential regulators (“hits”), which were then tested with gene-specific gRNAs to validate or exclude them from further study. These genome-wide screens have illuminated hits from several biological pathways, ranging from transmembrane channels to RNA splicing. We identify previously known regulators, including IL23R, STAT3, and ZFP36, as well as novel potential regulators, including SLC family members and a nuclear protein called SON. Our screens provide a diverse array of candidate factors that govern expression of type III cytokines by ILC3s, with potential for extrapolation to other cytokine-expressing cells. Genome-wide gain and loss of function screens are powerful, complementary tools to facilitate regulatory pathway discovery.

Adoptive immune cell therapies, such as CAR T, have revolutionized how we can approach cancer therapy, however challenges remain to increase their efficacy, response rates, and resistance to dysfunction. The systematic investigation of gene programs governing human T cell function would be advantageous for reprogramming cell products for clinical benefit. Recent studies have demonstrated the power of CRISPR-based forward genetic screens in primary human T cells to nominate candidate perturbations for enhanced T cell function, however combinations of perturbations will likely be necessary to overcome the most challenging tumors. To enable the systematic and high-throughput interrogation of combinations of gain and loss-of-function perturbations, we have developed a bidirectional CRISPR activation and interference (CRISPRai) system, where pairs of genes can be simultaneously activated and repressed in primary human T cells. Leveraging hits from previous genome-wide CRISPRa and CRISPRi screens, we constructed a library of >66,000 sgRNA combinations and screened for regulators of acute T cell stimulation responses, including cytokine production and proliferation. Reassuringly, these screens identified expected gene epistasis interactions (e.g. rescue of PLCG1 repression by PLCG2 activation), while also discovering novel genetic interactions, providing mechanistic insights into the regulatory networks governing stimulation response. To further identify gene “building blocks” for evaluation as combinations, we completed genome-wide CRISPRa and CRISPRi screens for regulators of human CD8+ CAR T cell exhaustion. These screens identified known potential enhancements (e.g. gain of BATF, loss of MED12), and also uncovered novel regulators of T cell function, with CRISPRa discovering genes not normally expressed in T cells but whose overexpression could cause resistance to exhaustion by synthetic mechanisms. Gene expression programs controlled by perturbation of key regulators were mapped by pairing CRISPR with single-cell RNA-seq (Perturb-seq) and arrayed functional assays. Finally, a top gain-of-function candidate was evaluated for in vivo tumor control, using an antigen-inducible Synthetic Notch system. Together, this work demonstrates how advanced forward genetics approaches can discover and elucidate both natural and synthetic programs that regulate T cell states, and the nomination of perturbations to encode in logic-gated synthetic biology designs for cell therapies. Citation Format: Zachary Steinhart, Jeffrey Perera, Dmytro Dorovskyi, Luke Workley, Carinna Tran, Rosmely Hernandez, Zhongmei Li, Max Foisey, Daniel Goodman, Kole Roybal, Brian Shy, Alexander Marson. Deciphering natural and synthetic genetic programs governing human T cell function with combinatorial CRISPR activation and interference screens [abstract]. In: Proceedings of the AACR IO Conference: Discovery and Innovation in Cancer Immunology: Revolutionizing Treatment through Immunotherapy; 2025 Feb 23-26; Los Angeles, CA. Philadelphia (PA): AACR; Cancer Immunol Res 2025;13(2 Suppl):Abstract nr A026.

CRISPR activation screens map the genomic landscape of cancer glycome remodeling.

Defective assembly of primary cilia causes ciliopathies, but cilia disassembly and its role in disease remain poorly understood. From a genome-wide CRISPR activation (CRISPRa) screen for negative regulators of ciliary function, we find here that the F2R G protein–coupled receptor, sterile alpha and TIR motif-containing 1 (SARM1) hydrolase, ryanodine receptors, peri-centrosomal calcium signaling, and RhoA form a functional pathway that is necessary and sufficient for cilia disassembly. Highlighting the significance of this pathway, several components are somatically mutated in focal cortical dysplasia (FCD), a neurological disorder characterized by intractable epilepsy. Supporting the functional impact of these variants, patient-derived SARM1 and RhoA mutations potentiate cilia loss, and a RhoA variant impairs cortical development. Conversely, SARM1 inhibition restores cilia in cells with FCD-associated alterations. Together, our work identifies a pathway for cilia disassembly, implicates aberrant pathway activation as a feature of FCD-associated mutations, and illustrates the potential of CRISPRa screening to provide insight into diseases caused by somatic mutations.