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Böhm, Sybille, Victoria Splith, Lisa Maria Riedmayr, René Dominik Rötzer, Gilles Gasparoni, Karl J. V. Nordström, Johanna Elisabeth Wagner, et al. "A gene therapy for inherited blindness using dCas9-VPR–mediated transcriptional activation." Science Advances 6, no. 34 (August 2020): eaba5614. http://dx.doi.org/10.1126/sciadv.aba5614.
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Catalytically inactive dCas9 fused to transcriptional activators (dCas9-VPR) enables activation of silent genes. Many disease genes have counterparts, which serve similar functions but are expressed in distinct cell types. One attractive option to compensate for the missing function of a defective gene could be to transcriptionally activate its functionally equivalent counterpart via dCas9-VPR. Key challenges of this approach include the delivery of dCas9-VPR, activation efficiency, long-term expression of the target gene, and adverse effects in vivo. Using dual adeno-associated viral vectors expressing split dCas9-VPR, we show efficient transcriptional activation and long-term expression of cone photoreceptor-specific M-opsin (Opn1mw) in a rhodopsin-deficient mouse model for retinitis pigmentosa. One year after treatment, this approach yields improved retinal function and attenuated retinal degeneration with no apparent adverse effects. Our study demonstrates that dCas9-VPR–mediated transcriptional activation of functionally equivalent genes has great potential for the treatment of genetic disorders.
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Schüller, Andreas, Lisa Wolansky, Harald Berger, Lena Studt, Agnieszka Gacek-Matthews, Michael Sulyok, and Joseph Strauss. "A novel fungal gene regulation system based on inducible VPR-dCas9 and nucleosome map-guided sgRNA positioning." Applied Microbiology and Biotechnology 104, no. 22 (October 2, 2020): 9801–22. http://dx.doi.org/10.1007/s00253-020-10900-9.
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Abstract Programmable transcriptional regulation is a powerful tool to study gene functions. Current methods to selectively regulate target genes are mainly based on promoter exchange or on overexpressing transcriptional activators. To expand the discovery toolbox, we designed a dCas9-based RNA-guided synthetic transcription activation system for Aspergillus nidulans that uses enzymatically disabled “dead” Cas9 fused to three consecutive activation domains (VPR-dCas9). The dCas9-encoding gene is under the control of an estrogen-responsive promoter to allow induction timing and to avoid possible negative effects by strong constitutive expression of the highly active VPR domains. Especially in silent genomic regions, facultative heterochromatin and strictly positioned nucleosomes can constitute a relevant obstacle to the transcriptional machinery. To avoid this negative impact and to facilitate optimal positioning of RNA-guided VPR-dCas9 to targeted promoters, we have created a genome-wide nucleosome map from actively growing cells and stationary cultures to identify the cognate nucleosome-free regions (NFRs). Based on these maps, different single-guide RNAs (sgRNAs) were designed and tested for their targeting and activation potential. Our results demonstrate that the system can be used to regulate several genes in parallel and, depending on the VPR-dCas9 positioning, expression can be pushed to very high levels. We have used the system to turn on individual genes within two different biosynthetic gene clusters (BGCs) which are silent under normal growth conditions. This method also opens opportunities to stepwise activate individual genes in a cluster to decipher the correlated biosynthetic pathway. Keypoints • An inducible RNA-guided transcriptional regulator based on VPR-dCas9 was established in Aspergillus nidulans. • Genome-wide nucleosome positioning maps were created that facilitate sgRNA positioning. • The system was successfully applied to activate genes within two silent biosynthetic gene clusters.
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Blanas, Athanasios, Lenneke A. M. Cornelissen, Maximilianos Kotsias, Joost C. van der Horst, Henri J. van de Vrugt, Hakan Kalay, Daniel I. R. Spencer, Rad P. Kozak, and Sandra J. van Vliet. "Transcriptional activation of fucosyltransferase (FUT) genes using the CRISPR-dCas9-VPR technology reveals potent N-glycome alterations in colorectal cancer cells." Glycobiology 29, no. 2 (October 22, 2018): 137–50. http://dx.doi.org/10.1093/glycob/cwy096.
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AbstractAberrant fucosylation in cancer cells is considered as a signature of malignant cell transformation and it is associated with tumor progression, metastasis and resistance to chemotherapy. Specifically, in colorectal cancer cells, increased levels of the fucosylated Lewisx antigen are attributed to the deregulated expression of pertinent fucosyltransferases, like fucosyltransferase 4 (FUT4) and fucosyltransferase 9 (FUT9). However, the lack of experimental models closely mimicking cancer-specific regulation of fucosyltransferase gene expression has, so far, limited our knowledge regarding the substrate specificity of these enzymes and the impact of Lewisx synthesis on the glycome of colorectal cancer cells. Therefore, we sought to transcriptionally activate the Fut4 and Fut9 genes in the well-known murine colorectal cancer cell line, MC38, which lacks expression of the FUT4 and FUT9 enzymes. For this purpose, we utilized a physiologically relevant, guide RNA-based model of de novo gene expression, namely the CRISPR-dCas9-VPR system. Induction of the Fut4 and Fut9 genes in MC38 cells using CRISPR-dCas9-VPR resulted in specific neo-expression of functional Lewisx antigen on the cell surface. Interestingly, Lewisx was mainly carried by N-linked glycans in both MC38-FUT4 and MC38-FUT9 cells, despite pronounced differences in the biosynthetic properties and the expression stability of the induced enzymes. Moreover, Lewisx expression was found to influence core-fucosylation, sialylation, antennarity and the subtypes of N-glycans in the MC38-glycovariants. In conclusion, exploiting the CRISPR-dCas9-VPR system to augment glycosyltransferase expression is a promising method of transcriptional gene activation with broad application possibilities in glycobiology and oncology research.
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Josipović, Goran, Vanja Tadić, Marija Klasić, Vladimir Zanki, Ivona Bečeheli, Felicia Chung, Akram Ghantous, et al. "Antagonistic and synergistic epigenetic modulation using orthologous CRISPR/dCas9-based modular system." Nucleic Acids Research 47, no. 18 (August 14, 2019): 9637–57. http://dx.doi.org/10.1093/nar/gkz709.
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Abstract Establishing causal relationship between epigenetic marks and gene transcription requires molecular tools, which can precisely modify specific genomic regions. Here, we present a modular and extensible CRISPR/dCas9-based toolbox for epigenetic editing and direct gene regulation. It features a system for expression of orthogonal dCas9 proteins fused to various effector domains and includes a multi-gRNA system for simultaneous targeting dCas9 orthologs to up to six loci. The C- and N-terminal dCas9 fusions with DNMT3A and TET1 catalytic domains were thoroughly characterized. We demonstrated simultaneous use of the DNMT3A-dSpCas9 and TET1-dSaCas9 fusions within the same cells and showed that imposed cytosine hyper- and hypo-methylation altered level of gene transcription if targeted CpG sites were functionally relevant. Dual epigenetic manipulation of the HNF1A and MGAT3 genes, involved in protein N-glycosylation, resulted in change of the glycan phenotype in BG1 cells. Furthermore, simultaneous targeting of the TET1-dSaCas9 and VPR-dSpCas9 fusions to the HNF1A regulatory region revealed strong and persistent synergistic effect on gene transcription, up to 30 days following cell transfection, suggesting involvement of epigenetic mechanisms in maintenance of the reactivated state. Also, modulation of dCas9 expression effectively reduced off-target effects while maintaining the desired effects on target regions.
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Ding, Xiao, Lu Yu, Luo Chen, Yujie Li, Jinlun Zhang, Hanyan Sheng, Zhengwei Ren, et al. "Recent Progress and Future Prospect of CRISPR/Cas-Derived Transcription Activation (CRISPRa) System in Plants." Cells 11, no. 19 (September 28, 2022): 3045. http://dx.doi.org/10.3390/cells11193045.
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Genome editing technology has become one of the hottest research areas in recent years. Among diverse genome editing tools, the Clustered Regularly Interspaced Short Palindromic Repeats/CRISPR-associated proteins system (CRISPR/Cas system) has exhibited the obvious advantages of specificity, simplicity, and flexibility over any previous genome editing system. In addition, the emergence of Cas9 mutants, such as dCas9 (dead Cas9), which lost its endonuclease activity but maintains DNA recognition activity with the guide RNA, provides powerful genetic manipulation tools. In particular, combining the dCas9 protein and transcriptional activator to achieve specific regulation of gene expression has made important contributions to biotechnology in medical research as well as agriculture. CRISPR/dCas9 activation (CRISPRa) can increase the transcription of endogenous genes. Overexpression of foreign genes by traditional transgenic technology in plant cells is the routine method to verify gene function by elevating genes transcription. One of the main limitations of the overexpression is the vector capacity constraint that makes it difficult to express multiple genes using the typical Ti plasmid vectors from Agrobacterium. The CRISPRa system can overcome these limitations of the traditional gene overexpression method and achieve multiple gene activation by simply designating several guide RNAs in one vector. This review summarizes the latest progress based on the development of CRISPRa systems, including SunTag, dCas9-VPR, dCas9-TV, scRNA, SAM, and CRISPR-Act and their applications in plants. Furthermore, limitations, challenges of current CRISPRa systems and future prospective applications are also discussed.
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Riedmayr, Lisa M., Klara S. Hinrichsmeyer, Nina Karguth, Sybille Böhm, Victoria Splith, Stylianos Michalakis, and Elvir Becirovic. "dCas9-VPR-mediated transcriptional activation of functionally equivalent genes for gene therapy." Nature Protocols 17, no. 3 (February 7, 2022): 781–818. http://dx.doi.org/10.1038/s41596-021-00666-3.
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Ewen-Campen, Ben, Donghui Yang-Zhou, Vitória R. Fernandes, Delfina P. González, Lu-Ping Liu, Rong Tao, Xingjie Ren, et al. "Optimized strategy for in vivo Cas9-activation in Drosophila." Proceedings of the National Academy of Sciences 114, no. 35 (August 14, 2017): 9409–14. http://dx.doi.org/10.1073/pnas.1707635114.
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While several large-scale resources are available for in vivo loss-of-function studies in Drosophila, an analogous resource for overexpressing genes from their endogenous loci does not exist. We describe a strategy for generating such a resource using Cas9 transcriptional activators (CRISPRa). First, we compare a panel of CRISPRa approaches and demonstrate that, for in vivo studies, dCas9-VPR is the most optimal activator. Next, we demonstrate that this approach is scalable and has a high success rate, as >75% of the lines tested activate their target gene. We show that CRISPRa leads to physiologically relevant levels of target gene expression capable of generating strong gain-of-function (GOF) phenotypes in multiple tissues and thus serves as a useful platform for genetic screening. Based on the success of this CRISRPa approach, we are generating a genome-wide collection of flies expressing single-guide RNAs (sgRNAs) for CRISPRa. We also present a collection of more than 30 Gal4 > UAS:dCas9-VPR lines to aid in using these sgRNA lines for GOF studies in vivo.
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Lainšček, Duško, Lucija Kadunc, Mateja Manček Keber, Iva Hafner Bratkovič, Rok Romih, and Roman Jerala. "Delivery of an Artificial Transcription Regulator dCas9-VPR by Extracellular Vesicles for Therapeutic Gene Activation." ACS Synthetic Biology 7, no. 12 (December 4, 2018): 2715–25. http://dx.doi.org/10.1021/acssynbio.8b00192.
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Brancazio, S., C. Baez, L. Shan, T. H. Burdo, and R. Kaminski. "PP 4.12 – 00141 Enhancement of IL15/IL15RA signaling in immune cells using CRISPR-dCas9-VPR platform." Journal of Virus Eradication 8 (December 2022): 100220. http://dx.doi.org/10.1016/j.jve.2022.100220.
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Hu, Wenbo, Xiaogang Wang, Sanyuan Ma, Zhangchuan Peng, Yang Cao, and Qingyou Xia. "CRISPR-Mediated Endogenous Activation of Fibroin Heavy Chain Gene Triggers Cellular Stress Responses in Bombyx mori Embryonic Cells." Insects 12, no. 6 (June 13, 2021): 552. http://dx.doi.org/10.3390/insects12060552.
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The silkworm Bombyx mori is an economically important insect, as it is the main producer of silk. Fibroin heavy chain (FibH) gene, encoding the core component of silk protein, is specifically and highly expressed in silk gland cells but not in the other cells. Although the silkworm FibH gene has been well studied in transcriptional regulation, its biological functions in the development of silk gland cells remain elusive. In this study, we constructed a CRISPRa system to activate the endogenous transcription of FibH in Bombyx mori embryonic (BmE) cells, and the mRNA expression of FibH was successfully activated. In addition, we found that FibH expression was increased to a maximum at 60 h after transient transfection of sgRNA/dCas9-VPR at a molar ratio of 9:1. The qRT-PCR analysis showed that the expression levels of cellular stress response-related genes were significantly up-regulated along with activated FibH gene. Moreover, the lyso-tracker red and monodansylcadaverine (MDC) staining assays revealed an apparent appearance of autophagy in FibH-activated BmE cells. Therefore, we conclude that the activation of FibH gene leads to up-regulation of cellular stress responses-related genes in BmE cells, which is essential for understanding silk gland development and the fibroin secretion process in B. mori.
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Bui, Michelle, Elena Dalla Benetta, Yuemei Dong, Yunchong Zhao, Ting Yang, Ming Li, Igor A. Antoshechkin, et al. "CRISPR mediated transactivation in the human disease vector Aedes aegypti." PLOS Pathogens 19, no. 1 (January 19, 2023): e1010842. http://dx.doi.org/10.1371/journal.ppat.1010842.
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As a major insect vector of multiple arboviruses, Aedes aegypti poses a significant global health and economic burden. A number of genetic engineering tools have been exploited to understand its biology with the goal of reducing its impact. For example, current tools have focused on knocking-down RNA transcripts, inducing loss-of-function mutations, or expressing exogenous DNA. However, methods for transactivating endogenous genes have not been developed. To fill this void, here we developed a CRISPR activation (CRISPRa) system in Ae. aegypti to transactivate target gene expression. Gene expression is activated through pairing a catalytically-inactive (‘dead’) Cas9 (dCas9) with a highly-active tripartite activator, VP64-p65-Rta (VPR) and synthetic guide RNA (sgRNA) complementary to a user defined target-gene promoter region. As a proof of concept, we demonstrate that engineered Ae. aegypti mosquitoes harboring a binary CRISPRa system can be used to effectively overexpress two developmental genes, even-skipped (eve) and hedgehog (hh), resulting in observable morphological phenotypes. We also used this system to overexpress the positive transcriptional regulator of the Toll immune pathway known as AaRel1, which resulted in a significant suppression of dengue virus serotype 2 (DENV2) titers in the mosquito. This system provides a versatile tool for research pathways not previously possible in Ae. aegypti, such as programmed overexpression of endogenous genes, and may aid in gene characterization studies and the development of innovative vector control tools.
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Yilmaz, Ebru, Alisan Kayabolen, Semra Cemre Atalar, Buket Yigit, Burge Ulukan, Mujdat Zeybel, and Tugba Bagci-Onder. "Abstract 6255: CRISPRa-mediated induction of apoptosis in therapy-resistant glioblastoma cells via derepression of pro-apoptotic genes." Cancer Research 82, no. 12_Supplement (June 15, 2022): 6255. http://dx.doi.org/10.1158/1538-7445.am2022-6255.
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Abstract Induction of apoptosis in cancer cells via de-repressing dormant pro-apoptotic genes might be a successful therapeutic strategy. In this study, we utilized several dCas9-mediated CRISPR activation (CRISPRa) systems to activate two key pro-apoptotic genes DR4 and XAF1 in glioblastoma cells. CRISPRa is used to activate endogenous genes via either recruiting transcription factors or epigenetic modifiers to regulatory regions in the genome. Previously conducted studies showed that VPR, Suntag, and SAM systems are the most effective transcription factor-based systems for activating several genes in a variety of cell lines. However, epigenetic modifier-based systems that involve the recruitment of TET demethylases have more permanent effects. Therefore, we hypothesized that combinations of these systems may induce stronger activation. We systematically tested various CRISPRa systems and their combinations to activate XAF1 and DR4, which are known to be epigenetically silenced in drug-resistant glioblastoma cells. Inducing the expression of these genes, we aimed to observe apoptosis in glioblastoma cells either at a basal state or upon treatment with the pro-apoptotic ligand TRAIL. By transcriptomic alteration and cell viability analyses, we demonstrated that the combinatorial system including dCas9-TET1 and MS2-p65-hsf1 was the most efficient one for the activation of DR4 and XAF1 genes in both 293T and glioblastoma cells. Another system in which multiple TET1 enzymes were recruited to the gene promoter together with MS2-p65-hsf1 was found to be effective similarly, suggesting that the promoter demethylation is critical for the activation of these genes. Activation of these genes with the most efficient systems caused apoptosis and also sensitized glioblastoma cells to TRAIL treatment. Our preliminary work on measuring methylation levels of several CpG sites in the promoter regions of DR4 is in line with our observations of cell-based phenotypes. Together, our study indicates that the combination of transcription factor-based and demethylation-based CRISPR activation systems can induce pro-apoptotic gene expression in cancer cells and lead cancer cells to undergo apoptosis. Future applications of this methodology might involve the development of cancer-specific pro-apoptotic therapies. [E.Y. and A.K. contributed equally to this work.] Citation Format: Ebru Yilmaz, Alisan Kayabolen, Semra Cemre Atalar, Buket Yigit, Burge Ulukan, Mujdat Zeybel, Tugba Bagci-Onder. CRISPRa-mediated induction of apoptosis in therapy-resistant glioblastoma cells via derepression of pro-apoptotic genes [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6255.
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Yang, Zhifen, and Francesco Marincola. "154 Context-dependent reversible modulation of cJUN expression by CAR T cells for cancer treatment." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A164. http://dx.doi.org/10.1136/jitc-2021-sitc2021.154.
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BackgroundOverexpression of canonical AP-1 factor cJUN was shown to prevent CAR T cell exhaustion and improve anti-tumor potency in vivo (1). However, its clinical utilization is limited by potential for transformation and oncogenic risk. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-339) that delivers context-dependent upregulation of endogenous cJUN increasing CAR-T cell resistance to exhaustion.MethodsRB-339 is a CAR T cell engineered to conditionally turn on the transcription of the cJUN endogenous gene. The circuit includes a lentiviral construct encoding an anti-HER2 (4D5) single chain variable fragment, with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and a single guide RNA (sgRNA) targeting the promoter region of cJUN. A second construct encodes linker for activation of T cells, complexed to nuclease-deactivated/dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) via a TEV-cleavable linker (LdCV). Activation of CAR allows the release of dCas9 for nuclear localization and conditionally and reversibly induces the expression of cJUN. RB-339 was compared in vitro to control (cRB-339, lacking the cJUN sgRNA) and CAR-T cells engineered to constitutively express cJun.ResultsRB-339 induced cJUN upregulation upon stimulation with HER2-coated beads and this resulted in significantly elevated expression over a 6-day time course compared to the control cRB-339 (figure 1A-B). When HER2-coated beads were removed at day 3, cJUN expression returned to baseline parallel to cRB-339. The conditional upregulation of cJUN in RB-339 contrasted with the constitutive overexpression in the transgene carrying cells that was irrespective of antigen stimulation (figure 1C). Upon exposure to HER2+ FaDu cancer cells, RB-339 peaked at day 2 and declined afterwards when FaDu cells were killed at day 3; cJUN increased again at day 4 upon restimulation with FaDu cells at day 3 (figure 2). Such a dynamic induction of cJUN resulted in significantly enhanced CAR-T cells proliferation in RB-339 compared to the respective conventional CAR-T cells or cRB-339 (figure 3).Abstract 154 Figure 1Conditional upregulation of cJUN by RB-339 in vitro. RB-339 and its control cRB-339 were stimulated by HER2-coated or BSA-coated beads for either six days or three days followed by removal of beads at day 3 (figure 1A-B). Intracellular expression of cJUN was detected at indicated time points. Intracellular cJUN expression in overexpressed cJUN-CAR (figure 1C)Abstract 154 Figure 2Kinetics of cJUN upregulation in RB-339 upon exposure to HER2+ FaDu tumor cells. RB-339 and its control cRB-339 were stimulated by FaDu tumor cells for six days with or without restimulation at day 3Abstract 154 Figure 3Conditional upregulation of cJUN resulted in enhanced CAR-T proliferation in RB-339 in vitro after 6-day co-culture with FaDu tumor cells, compared to conventional HER2 CAR or cRB-339ConclusionsWe conclude that CAR-T engineered to conditionally express the canonical AP-1 factor cJUN increases expansion potential similarly to CAR-T cells engineered to constitutively express the cJun transgene. However, the context-dependent upregulation of cJUN limits the risk of oncogenic transformation. We are currently combining inducible and reversible cJUN and IL-12 upregulation for the generation of the next RB-339 product.ReferenceLynn RC, Weber EW, et al. c-Jun overexpression in CAR T cells induces exhaustion resistance. Nature 2019; 576(7786):293–300.
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Yang, Zhifen, Maggie Bobbins, Hana Choi, Ofir Stefanson, Jin Yang, Khristina Magallanes, Bing Wang, Lei Stanley Qi, and Francesco Marincola. "765 Contextual secretion of nanoscale interleukin (IL)-12 by CAR T cells for the treatment of cancer." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A813. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0765.
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BackgroundInterleukin(IL)-12 activates T cells and macrophages pivoting the switch that turns chronic into acute inflammation and results in cancer rejection. However, despite formidable antitumor effects in preclinical models, its clinical utilization is limited by severe systemic toxicity. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-2-12) that purposefully induces minimally effective doses of IL-12 for autocrine activation of CAR-T.MethodsRB-2-12 is a CAR T cell engineered to express the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. The circuit includes a lentiviral constructs encoding an anti-HER2 (4D5) single chain variable fragment, with CD28 and CD3ζ co-stimulatory domains linked to a tobacco etch virus (TEV) protease and two single guide RNAs (sgRNA) targeting the promoter region for IL-12A orL-12B. A second constructs encodes linker for activation of T cells, complexed to nuclease-deactivated/dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) via a TEV-cleavable linker (LdCV). Activation of CAR brings CAR-TEV in proximity to LdCV releasing dCas9 for nuclear localization to the regulatory regions and conditionally and reversibly induce nanoscale expression of the p70 heterodimer. RB-2-12 was compared in vitro to control (cRB-2-12, lacking the IL-12 sgRNAs).ResultsRB-2-12 induced autocrine production of low concentrations of IL-12 upon exposure to HER2+ FaDu cancer cells resulting in significantly enhanced production of interferon (IFN)-γ, cytotoxic activity and proliferation (figure 1a). These effects were comparable to co-culturing conventional HER2-specific CAR-T cells with a modified FaDu cell line expressing high doses of IL-12 (figure 1b).Abstract 765 Figure 1Conditional autocrine release of nanoscale-dose p70/IL-12 byConditional autocrine release of nanoscale-dose p70/IL-12 by RB-2-12 resulting in enhanced IFN-γ production, cytotoxicity and proliferation in vitro after three days of exposure to FaDu cells (figure 1a). Constitutive high dose release of p70-IL-12 by HER2+ Fadu cells engineered to constitutively express IL-12 (FaDu/IL-12) and its effect on IFN-γ secretion, cytotoxicity and proliferation of conventional HER-2-specific CAR T-cells three days after exposure to FaDu or FaDu/IL-12 cancer cells (figure 1b). N.T. = non-transduced T cells; NOsg = cRB-2-12 CAR-T cells missing the sgRNAs for the two IL-12 subunits; IL12sg = complete product incorporating the full CRISPRa functions.ConclusionsWe have previously shown that tandem suppression of PD-1 expression upon HER-2 CAR activation using CRISPR interference enhances anti-cancer properties of CAR-T cells in vivo against HER2-FaDu xenografts by promoting their persistence and long-term tumor colonization (companion abstract submitted to SITC annual meeting). We hypothesize that addition of a Th1 polarizing component such as IL-12 will exponentially increase the efficacy of reprogrammed CAR-T cells by combining enhancement of effector functions to cellular fitness. At the same time, the autocrine effects of nanoscale IL-12 production limit the risk of off-tumor leakage and systemic toxicity. Such cumulative synthetic biology approaches are currently investigated in vitro and in vivo model systems. Current work is testing the effectiveness of RB-2-12 in vivo against FaDu xenografts.AcknowledgementsNoneTrial RegistrationN.A.Ethics ApprovalNot ApplicableConsentNot ApplicableReferencesNone
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Yang, Zhifen, and Francesco Marincola. "153 Nanoscale, antigen-dependent, IL-12 delivery by CAR T cells plus PD-L1 blockade for cancer treatment." Journal for ImmunoTherapy of Cancer 9, Suppl 2 (November 2021): A162—A163. http://dx.doi.org/10.1136/jitc-2021-sitc2021.153.
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BackgroundInterleukin(IL)-12 activates T cells pivoting the switch that turns lingering inflammation into acute inflammation and cancer rejection. However, its clinical utilization is limited by severe systemic toxicity. IL-12 is a potent inducer of PD-1 expression in T cells. Here, we present a conditional, antigen-dependent, non-editing CRISPR-activation (CRISPRa) circuit (RB-312) that delivers nanoscale doses of IL-12 for autocrine activation of CAR-T cells. RB-312 was also tested in combination with PD-L1 blocking antibody (atezolizumab).MethodsRB-312 is a CAR T cell engineered to express the IL-12 heterodimer via conditional transcription of its two endogenous subunits p35 and p40. The circuit includes two lentiviral constructs with one encoding HER2-specific chimeric antigen receptor and two sgRNAs targeting IL-12A or IL-12B and the other encoding linker for activation of T cells, complexed to dead Cas9 (dCas9)-VP64-p65-Rta transcriptional activator (VPR) (LdCV). Activation of CAR allows the release of dCas9 for nuclear localization and hence conditionally and reversibly induces the secretion of IL-12 p70 heterodimer.ResultsRB-312 induced low concentrations of IL-12 upon exposure to HER2+ FaDu cancer cells engineered to overexpress PD-L1 and this resulted in significantly enhanced production of IFN-γ, cytotoxicity and CAR-T proliferation (figure 1A). These effects were comparable to co-culturing conventional HER2 CAR with FaDu cells modified to express high doses of IL-12 (figure 1B). In vivo administration of RB-312 significantly enhanced survival of mice carrying FaDu xenografts compared to mice treated with the respective conventional HER2 CAR or cRB-312 (control lacking the IL-12 sgRNAs, figure 2A). RB-312 induced a strong upregulation of PD-1 in CAR-T cells in vivo (figure 2B). The critical role of the PD-1/PD-L1 interaction was demonstrated in vitro by comparing RB-312 proliferation when exposed to FaDu overexpressing PD-L1 or PD-L1 knock out cells (figure 3A). Indeed, combined treatment of RB-312 and atezolizumab resulted in significant reduction in tumor growth (figure 3B and C) and significantly enhanced survival (figure 3D).Abstract 153 Figure 1Conditional autocrine release of nanoscale-dose p70/IL-12 by RB-312 resulting in enhanced IFN-γ production in vitro after three days of exposure to HER2+ FaDu cells (figure 1A), and the level of IFN-γ production was comparable to co-culturing conventional HER2-specific CAR-T cells with a modified FaDu cell line engineered to constitutively express high doses of IL-12 (FaDu/IL-12, figure 1B)Abstract 153 Figure 2Intra-tumoral administration of RB-312 extended survival in mice carrying FaDu xenografts compared to NT (non-transduced T cells), HER2 CAR (conventional HER2 CAR-T cells) and cRB-312 CAR-T cells missing the sgRNAs for the two IL-12 subunits (figure 2A). Analysis of necropsy material demonstrated that PD-1 expression was dramatically increased in RB-312 compared with the respective control cRB-312 (figure 2B)Abstract 153 Figure 3RB-312 cellular function in vivo. PD-L1 expression by FaDu cell lines is a critical mechanism of repression of RB-312 function. In vitro CAR-T proliferation of RB-312 upon stimulation with FaDu tumor cells (orange solid lines) or FaDu/PD-L1 knockout tumor cells (orange dashed lines) over 6-day time course (figure 3A). In vivo efficacy of intra-tumoral RB-312 against FaDu tumor cells with (orange solid lines) or without (orange dashed lines) addition of PD-L1 blocking antibody atezolizumab (administered intravenously at 5 mg/kg twice per week), as shown by tumor growth followed till day 29 and scatter plot at day 29 (figure 3B), tumor growth spider plots (figure 3C) and Kaplan-Meier survival curve (figure 3D)ConclusionsWe concluded that addition of a Th1 polarizing component such as IL-12 exponentially increases the efficacy of reprogrammed CAR-T cells by combining enhancement of effector functions to cellular fitness. The autocrine effects of nanoscale IL-12 production limit the risk of off-tumor leakage and systemic toxicity. Here, we tested the combination of PD-1/PD-L1 blockade with IL-12-induced CAR-T cell activation demonstrated dramatic synergistic effects. We are currently evaluating the intrinsic combination of IL-12 delivery and PD-L1 resistance for the next generation of RB-312 product eliminating the need for systemic checkpoint blockade.
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Guo, Jimmy A., Jennifer Su, Ananya Jambhale, Julien Dilly, Connor J. Hennessey, Carina Shiau, Patrick Yu, et al. "Abstract A052: Systematic dissection of transcriptional states in pancreatic cancer." Cancer Research 82, no. 22_Supplement (November 15, 2022): A052. http://dx.doi.org/10.1158/1538-7445.panca22-a052.
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Abstract Transcriptional states in pancreatic cancer can stratify patients by response to chemotherapy and clinical outcomes. These include the classical and basal-like states as well as a newly identified neural-like progenitor (NRP) state, which we have previously found to be enriched in primary patient tumors treated with neoadjuvant chemotherapy and radiotherapy. While several transcription factor drivers of classical and basal-like identity have been described, key regulators of the NRP state are unknown. Through in silico approaches, we identified candidate transcription factors of the NRP state, including GLIS3, a Krüppel-like zinc finger protein that mediates neuroendocrine fate during pancreatic development and differentiation of human embryonic stem cells into posterior neural progenitor cells. Our understanding of biologic and clinically-relevant attributes of transcriptional cell states remains limited by state-specific biases in various preclinical models. Existing human cell lines maintained as two-dimensional cultures tend to preferentially represent the basal-like state, whereas human three-dimensional organoid models grown in standard culture conditions best reflect the classical state. These phenotypes are therefore impacted by culture conditions as well as underlying genetic features. Furthermore, most murine pancreatic cancer models do not fully reflect the classical vs. basal-like state heterogeneity observed in humans. To enable systematic study of the classical, basal-like and NRP phenotypes, we developed isogenic KP (KrasG12D/+;Trp53FL/FL) murine organoids with a germline dCas9-VPR system to enable facile overexpression of state-specific transcription factors through CRISPR activation approaches. Quantitative PCR, RNA-sequencing, and proteomics confirmed Gata6, deltaN Trp63, and Glis3 as drivers of classical, basal-like, and NRP identity, respectively. DeltaN Trp63 organoids were further differentiated by loss of luminal morphology. Pairwise comparisons of global transcriptional alterations suggest the greatest similarities between the Gata6- and Glis3-overexpressed models, which is consistent with enhanced associations between classical and NRP states in patient tumors. Finally, although basal-like and NRP states are associated with poorer response to multi-agent chemotherapy, state-specific therapeutic sensitivities to other treatments remain incompletely defined. We therefore performed drug sensitivity assays with a panel of targeted therapies and unveiled state-specific sensitivities. These data were corroborated by drug sensitivity profiling of human patient-derived organoids and cell lines. Taken together, these results suggest a framework for defining cell state-specific vulnerabilities that may aid in stratifying and treating pancreatic cancer patients with new therapies. Citation Format: Jimmy A. Guo, Jennifer Su, Ananya Jambhale, Julien Dilly, Connor J. Hennessey, Carina Shiau, Patrick Yu, Steven Wang, Junning Wang, Laleh Abbassi, James Neiswender, Tate Bertea, Annan Yang, Qijia Yu, Peter Westcott, Jason Schenkel, Daniel Y. Kim, Hannah I. Hoffman, Grissel Cervantes Jaramillo, Giselle A. Uribe, Westley W. Wu, Arnav Mehta, David Ting, Julian A. Pacheco, Amy Deik, Clary Clish, Francisca Vazquez, Brian Wolpin, Aviv Regev, William A. Freed-Pastor, Joseph D. Mancias, Tyler Jacks, William L. Hwang, Andrew J. Aguirre. Systematic dissection of transcriptional states in pancreatic cancer [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr A052.
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Hwang, William L., Jennifer Su, Jimmy A. Guo, Carina Shiau, Jaimie L. Barth, Hannah I. Hoffman, Prajan Divakar, et al. "Abstract C052: Identifying mediators of perineural invasion in pancreatic cancer using spatial transcriptomics." Cancer Research 82, no. 22_Supplement (November 15, 2022): C052. http://dx.doi.org/10.1158/1538-7445.panca22-c052.
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Abstract Intratumoral nerves play important and versatile roles in cancer initiation, progression, recurrence, treatment-resistance, metastasis, morbidity, and mortality for many malignancies but the diverse molecular mechanisms underlying tumor-nerve crosstalk remain largely unknown. One of the differentiating hallmarks of pancreatic ductal adenocarcinoma (PDAC) is an exceptionally high frequency of perineural invasion (PNI), a histopathologic manifestation of tumor-nerve crosstalk whereby cancer cells recruit, migrate towards, and envelop or invade peripheral nerves. Evidence for some neurochemicals/neurotrophins involved in PNI have been uncovered, but most of the underlying work was limited by a lack of cell-type specificity, spatial context, and fragmented focus on individual pathways. To address these shortcomings, we set out to comprehensively identify cell-type specific genes spatially linked to PNI in patient tumors and then dissect the functional roles of these genes through live imaging of dorsal root ganglia (DRG) sensory neurons incubated in conditioned media from cancer cell organoids overexpressing candidate genes via CRISPR activation (CRISPRa). First, we performed whole transcriptome digital spatial profiling (NanoString GeoMx) on twelve custom tissue microarrays (n=288 cores) derived from intratumorally-matched malignant regions with and without PNI in primary resected PDAC specimens (n=31 patients). Differential gene expression (DE) analysis (FDR < 0.001) for PNI demonstrated that for malignant cells there were 271 enriched and 65 depleted genes, and for fibroblasts there were 16 enriched and 27 depleted genes. We further evaluated associations between PNI and expression of malignant subtypes previously identified from single-nucleus RNA-seq applied to 43 primary resected PDAC specimens. We found that malignant cells engaged in PNI were enriched in the mesenchymal, basaloid and neural-like progenitor (NRP) subtypes and depleted in the classical subtype. To test these associations functionally, we generated isogenic murine organoid lines (KrasG12D/+;Trp53FL/FL;R26-dCas9-VPR) overexpressing subtype-driving transcription factors and collected conditioned media. DRG sensory neurons demonstrate enhanced and suppressed growth kinetics when grown in NRP and classical conditioned media, respectively; mesenchymal and basal-like conditioned media do not appear to influence growth kinetics. These results suggest that while mesenchymal, basaloid, and NRP cells likely all play a role in cancer cell invasion of nerves, NRP cells may have an additional role in tumor-nerve tropism. Additional experiments exploring the functional effects of the top enriched and depleted genes from the DE analysis are ongoing. We anticipate that this study will provide a high-resolution understanding of critical intercellular interactions in the PDAC tumor microenvironment that facilitate PNI and tumor-nerve crosstalk more broadly to guide novel therapeutic strategies. Citation Format: William L. Hwang, Jennifer Su, Jimmy A. Guo, Carina Shiau, Jaimie L. Barth, Hannah I. Hoffman, Prajan Divakar, Jason W. Reeves, Eric Miller, Grissel Cervantes-Jaramillo, William Freed-Pastor, Vanessa Funes, Jennifer Y. Wo, Theodore S. Hong, Carlos Fernandez-del Castillo, Lei Zheng, Andrew J. Aguirre, David T. Ting, Mari Mino-Kenudson, Tyler Jacks. Identifying mediators of perineural invasion in pancreatic cancer using spatial transcriptomics [abstract]. In: Proceedings of the AACR Special Conference on Pancreatic Cancer; 2022 Sep 13-16; Boston, MA. Philadelphia (PA): AACR; Cancer Res 2022;82(22 Suppl):Abstract nr C052.
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Ortega-Yáñez, Andrea, Samantha Cruz-Ruiz, Martha Vázquez, and Mario Zurita. "Different transcriptional responses by the CRISPRa system in distinct types of heterochromatin in Drosophila melanogaster." Scientific Reports 12, no. 1 (July 9, 2022). http://dx.doi.org/10.1038/s41598-022-15944-7.
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AbstractTranscription factors (TFs) activate gene expression by binding to elements close to promoters or enhancers. Some TFs can bind to heterochromatic regions to initiate gene activation, suggesting that if a TF is able to bind to any type of heterochromatin, it can activate transcription. To investigate this possibility, we used the CRISPRa system based on dCas9-VPR as an artificial TF in Drosophila. dCas9-VPR was targeted to the TAHRE telomeric element, an example of constitutive heterochromatin, and to promoters and enhancers of the HOX Ultrabithorax (Ubx) and Sex Combs Reduced (Scr) genes in the context of facultative heterochromatin. dCas9-VPR robustly activated TAHRE transcription, showing that although this element is heterochromatic, dCas9-VPR was sufficient to activate its expression. In the case of HOX gene promoters, although Polycomb complexes epigenetically silence these genes, both were ectopically activated. When the artificial TF was directed to enhancers, we found that the expression pattern was different compared to the effect on the promoters. In the case of the Scr upstream enhancer, dCas9-VPR activated the gene ectopically but with less expressivity; however, ectopic activation also occurred in different cells. In the case of the bxI enhancer located in the third intron of Ubx, the presence of dCas9-VPR is capable of increasing transcription initiation while simultaneously blocking transcription elongation, generating a lack of functional phenotype. Our results show that CRISPRa system is able to activate transcription in any type of heterochromatin; nevertheless, its effect on transcription is subject to the intrinsic characteristics of each gene or regulatory element.
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Bergenholm, David, Yasaman Dabirian, Raphael Ferreira, Verena Siewers, Florian David, and Jens Nielsen. "Rational gRNA design based on transcription factor binding data." Synthetic Biology, July 27, 2021. http://dx.doi.org/10.1093/synbio/ysab014.
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Abstract The clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 system has become a standard tool in many genome engineering endeavors. The endonuclease-deficient version of Cas9 (dCas9) is also a powerful programmable tool for gene regulation. In this study, we made use of Saccharomyces cerevisiae transcription factor (TF) binding data to obtain a better understanding of the interplay between TF binding and binding of dCas9 fused to an activator domain, VPR. More specifically, we targeted dCas9–VPR toward binding sites of Gcr1–Gcr2 and Tye7 present in several promoters of genes encoding enzymes engaged in the central carbon metabolism. From our data, we observed an upregulation of gene expression when dCas9–VPR was targeted next to a TF binding motif, whereas a downregulation or no change was observed when dCas9 was bound on a TF motif. This suggests a steric competition between dCas9 and the specific TF. Integrating TF binding data, therefore, proved to be useful for designing guide RNAs for CRISPR interference or CRISPR activation applications.
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Klinnert, Sarah, Alex Chemnitzer, Peter Rusert, and Karin J. Metzner. "Systematic HIV-1 promoter targeting with CRISPR/dCas9-VPR reveals optimal region for activation of the latent provirus." Journal of General Virology 103, no. 6 (June 7, 2022). http://dx.doi.org/10.1099/jgv.0.001754.
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CRISPR/dCas9-based activation systems (CRISPRa) enable sequence-specific gene activation and are therefore of particular interest for the ‘shock and kill’ cure approach against HIV-1 infections. This approach aims to activate the latent HIV-1 proviruses in infected cells and subsequently kill these cells. Several CRISPRa systems have been shown to specifically and effectively activate latent HIV-1 when targeted to the HIV-1 5′LTR promoter, making them a promising ‘shock’ strategy. Here, we aimed to evaluate the dCas9-VPR system for its applicability in reversing HIV-1 latency and identify the optimal gRNA target site in the HIV-1 5′LTR promoter leading to the strongest activation of the provirus with this system. We systematically screened the HIV-1 promoter by selecting 14 specific gRNAs that cover almost half of the HIV-1 promoter from the 3′ half of the U3 until the beginning of the R region. Screening in several latently HIV-1 infected cell lines showed that dCas9-VPR leads to a high activation of HIV-1 and that gRNA-V and -VII induce the strongest activation of replication competent latent provirus. This data indicates that the optimal activation region in the HIV-1 promoter for the dCas9-VPR system is located −165 to −106 bp from the transcription start site and that it is consistent with the optimal activation region reported for other CRISPRa systems. Our data demonstrates that the dCas9-VPR system is a powerful tool for HIV-1 activation and could be harnessed for the ‘shock and kill’ cure approach.
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Karbassi, Elaheh, Alessandro Bertero, Shin Kadota, Paul Fields, Lil Pabon, Hans Reinecke, Stephen Hauschka, and Charles E. Murry. "Abstract 352: Optimization of a CRISPR Activation Human Pluripotent Stem Cell Line for Screening of Candidate Regulators of Cardiomyocyte Maturation." Circulation Research 127, Suppl_1 (July 31, 2020). http://dx.doi.org/10.1161/res.127.suppl_1.352.
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Human pluripotent stem cell-derived cardiomyocyte (hPSC-CM) applications for cell therapy and disease modeling are limited due to the cells’ lack of resemblance structurally and functionally to adult cardiomyocytes. To understand hPSC-CM maturation, we characterized two established approaches to mature cardiomyocytes—long term culture (aging of cells in a dish) and in vivo transplantation to an infarcted adult rat heart. RNA sequencing of hPSC-CMs from these systems demonstrated that in vivo transplantation is much more effective in maturing hPSC-CMs, inducing a more adult-like cardiac gene program (e.g. upregulation of TNNI3, MYL2, SCN5A ), compared to cells kept in culture up to one year. Using this dataset, we identified candidate drivers of hPSC-CM maturation, including transcription factors and chromatin regulators, that we hypothesize are necessary to program hPSC-CMs to an adult-like state. To test the relationship between transcription factor regulation and hPSC-CM maturation, we developed a constitutive CRISPR activation (CRISPRa) pluripotent stem cell line to upregulate these transcriptional regulators upon addition of guide RNAs (gRNA). This cell line expresses nuclease-deficient Cas9 fused to the transcriptional activator VPR (dCas9-VPR), driven by the strong CAG promoter and targeted to the AAVS1 safe harbor site. In pluripotent stem cells, target genes are upregulated up to 150-fold when gRNA is present; however, after differentiation into cardiomyocytes, dCas9-VPR transgene expression is silenced, and dCas9-VPR levels are insufficient to activate gRNA-targeted genes. To optimize CRISPRa for cardiomyocyte applications, we are generating alternative stem cell lines with dCas9-VPR targeted to the human ROSA26 safe harbor site or driven by a cardiac-specific troponin T promoter, testing the regulation of transgene expression mediated by safe harbor site or promoter respectively. The characterization of these CRISPRa cell lines provides insights into CRISPR expression regulation and genome engineering strategies for applications in stem cells and hPSC-CMs. We will use this system to screen for maturation regulators and identify key combinations that are effective in programming hPSC-CMs towards an adult-like state.
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Mózsik, László, Mirthe Hoekzema, Niels A. W. de Kok, Roel A. L. Bovenberg, Yvonne Nygård, and Arnold J. M. Driessen. "CRISPR-based transcriptional activation tool for silent genes in filamentous fungi." Scientific Reports 11, no. 1 (January 13, 2021). http://dx.doi.org/10.1038/s41598-020-80864-3.
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AbstractFilamentous fungi are historically known to be a rich reservoir of bioactive compounds that are applied in a myriad of fields ranging from crop protection to medicine. The surge of genomic data available shows that fungi remain an excellent source for new pharmaceuticals. However, most of the responsible biosynthetic gene clusters are transcriptionally silent under laboratory growth conditions. Therefore, generic strategies for activation of these clusters are required. Here, we present a genome-editing-free, transcriptional regulation tool for filamentous fungi, based on the CRISPR activation (CRISPRa) methodology. Herein, a nuclease-defective mutant of Cas9 (dCas9) was fused to a highly active tripartite activator VP64-p65-Rta (VPR) to allow for sgRNA directed targeted gene regulation. dCas9-VPR was introduced, together with an easy to use sgRNA “plug-and-play” module, into a non-integrative AMA1-vector, which is compatible with several filamentous fungal species. To demonstrate its potential, this vector was used to transcriptionally activate a fluorescent reporter gene under the control of the penDE core promoter in Penicillium rubens. Subsequently, we activated the transcriptionally silent, native P. rubens macrophorin biosynthetic gene cluster by targeting dCas9-VPR to the promoter region of the transcription factor macR. This resulted in the production of antimicrobial macrophorins. This CRISPRa technology can be used for the rapid and convenient activation of silent fungal biosynthetic gene clusters, and thereby aid in the identification of novel compounds such as antimicrobials.
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Cui, Xuelian, Chao Zhang, Zhifang Xu, Shuaibin Wang, Xin Li, Erica Stringer-Reasor, Sejong Bae, et al. "Dual CRISPR interference and activation for targeted reactivation of X-linked endogenous FOXP3 in human breast cancer cells." Molecular Cancer 21, no. 1 (February 7, 2022). http://dx.doi.org/10.1186/s12943-021-01472-x.
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Abstract Background Unlike autosomal tumor suppressors, X-linked tumor suppressors can be inactivated by a single hit due to X-chromosome inactivation (XCI). Here, we argue that targeted reactivation of the non-mutated allele from XCI offers a potential therapy for female breast cancers. Methods Towards this goal, we developed a dual CRISPR interference and activation (CRISPRi/a) approach for simultaneously silencing and reactivating multiple X-linked genes using two orthogonal, nuclease-deficient CRISPR/Cas9 (dCas9) proteins. Results Using Streptococcus pyogenes dCas9-KRAB for silencing XIST and Staphylococcus aureus dCas9-VPR for activating FOXP3, we achieved CRISPR activation of FOXP3 in various cell lines of human female breast cancers. In human breast cancer HCC202 cells, which express a synonymous heterozygous mutation in the coding region of FOXP3, simultaneous silencing of XIST from XCI led to enhanced and prolonged FOXP3 activation. Also, reactivation of endogenous FOXP3 in breast cancer cells by CRISPRi/a inhibited tumor growth in vitro and in vivo. We further optimized CRISPRa by fusing dCas9 to the demethylase TET1 and observed enhanced FOXP3 activation. Analysis of the conserved CpG-rich region of FOXP3 intron 1 confirmed that CRISPRi/a-mediated simultaneous FOXP3 activation and XIST silencing were accompanied by elevated H4 acetylation, including H4K5ac, H4K8ac, and H4K16ac, and H3K4me3 and lower DNA methylation. This indicates that CRISPRi/a targeting to XIST and FOXP3 loci alters their transcription and their nearby epigenetic modifications. Conclusions The simultaneous activation and repression of the X-linked, endogenous FOXP3 and XIST from XCI offers a useful research tool and a potential therapeutic for female breast cancers.
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Mijakovac, Anika, Julija Jurić, Wendy M. Kohrt, Jasminka Krištić, Domagoj Kifer, Kathleen M. Gavin, Karlo Miškec, et al. "Effects of Estradiol on Immunoglobulin G Glycosylation: Mapping of the Downstream Signaling Mechanism." Frontiers in Immunology 12 (May 25, 2021). http://dx.doi.org/10.3389/fimmu.2021.680227.
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Glycans attached to immunoglobulin G (IgG) directly affect this antibody effector functions and regulate inflammation at several levels. The composition of IgG glycome changes significantly with age. In women, the most notable change coincides with the perimenopausal period. Aiming to investigate the effect of estrogen on IgG glycosylation, we analysed IgG and total serum glycomes in 36 healthy premenopausal women enrolled in a randomized controlled trial of the gonadotropin-releasing hormone analogue (GnRHAG) leuprolide acetate to lower gonadal steroids to postmenopausal levels and then randomized to transdermal placebo or estradiol (E2) patch. The suppression of gonadal hormones induced significant changes in the IgG glycome, while E2 supplementation was sufficient to prevent changes. The observed glycan changes suggest that depletion of E2 primarily affects B cell glycosylation, while liver glycosylation stays mostly unchanged. To determine whether previously identified IgG GWAS hits RUNX1, RUNX3, SPINK4, and ELL2 are involved in downstream signaling mechanisms, linking E2 with IgG glycosylation, we used the FreeStyle 293-F transient system expressing IgG antibodies with stably integrated CRISPR/dCas9 expression cassettes for gene up- and downregulation. RUNX3 and SPINK4 upregulation using dCas9-VPR resulted in a decreased IgG galactosylation and, in the case of RUNX3, a concomitant increase in IgG agalactosylation.
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Pakalniškytė, Dalia, Tanja Schönberger, Benjamin Strobel, Birgit Stierstorfer, Thorsten Lamla, Michael Schuler, and Martin Lenter. "Rosa26-LSL-dCas9-VPR: a versatile mouse model for tissue specific and simultaneous activation of multiple genes for drug discovery." Scientific Reports 12, no. 1 (November 10, 2022). http://dx.doi.org/10.1038/s41598-022-23127-7.
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AbstractTransgenic animals with increased or abrogated target gene expression are powerful tools for drug discovery research. Here, we developed a CRISPR-based Rosa26-LSL-dCas9-VPR mouse model for targeted induction of endogenous gene expression using different Adeno-associated virus (AAV) capsid variants for tissue-specific gRNAs delivery. To show applicability of the model, we targeted low-density lipoprotein receptor (LDLR) and proprotein convertase subtilisin/kexin type 9 (PCSK9), either individually or together. We induced up to ninefold higher expression of hepatocellular proteins. In consequence of LDLR upregulation, plasma LDL levels almost abolished, whereas upregulation of PCSK9 led to increased plasma LDL and cholesterol levels. Strikingly, simultaneous upregulation of both LDLR and PCSK9 resulted in almost unaltered LDL levels. Additionally, we used our model to achieve expression of all α1-Antitrypsin (AAT) gene paralogues simultaneously. These results show the potential of our model as a versatile tool for optimized targeted gene expression, alone or in combination.
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Yang, Jiangeng, An Xia, Huajie Zhang, Qi Liu, Hongke You, Daoyuan Ding, Yonghua Yin, and Bo Wen. "Up-Regulating ERIC by CRISPR-dCas9-VPR Inhibits Cell Proliferation and Invasion and Promotes Apoptosis in Human Bladder Cancer." Frontiers in Molecular Biosciences 8 (March 29, 2021). http://dx.doi.org/10.3389/fmolb.2021.654718.
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LncRNAs are defined as non-coding RNAs that are longer than 200 nucleotides in length. The previous studys has shown that lncRNAs played important roles in the regulation of gene expression and were essential in mammalian development and disease processes. Inspired by the observation that lncRNAs are aberrantly expressed in tumors, we extracted RNA from Bladder urothelial carcinoma and matched histologically normal urothelium from each patient and bladder carcinoma cell lines. Then, we reversed transcribed them into cDNA.Last, we investigated the expression patterns of ERIC by the fluorescence quantitative PCR in bladder cancer tissues and cell lines. CRISPR-dCas9-VPR targeting ERIC plasmid was transfected into T24 and 5637 cells, and cells were classified into two groups: negative control (NC) and ERIC overexpression group. MTT assay, transwell assay, and flow cytometry were performed to examine changes in cell proliferation, invasiveness, and apoptosis. We found that the expression of ERIC was down-regulated in bladder urothelial carcinoma compared to matched histologically normal urotheliam. The differences of the expression of this gene were large in the bladder cancer lines. Compared with the negative control group, the ERIC overexpression group showed significantly decreased cell proliferation rate (t = 7.583, p = 0.002; t = 3.283, p = 0.03) and invasiveness (t = 11.538, p < 0.001; t = 8.205, p = 0.01); and increased apoptotic rate (t = −34.083, p < 0.001; t = −14.316, p < 0.001). Our study lays a foundation for further study of its pathogenic mechanism in bladder cancer.
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Garcia-Perez, Elena, Borja Diego-Martin, Alfredo Quijano-Rubio, Elena Moreno-Giménez, Sara Selma, Diego Orzaez, and Marta Vazquez-Vilar. "A copper switch for inducing CRISPR/Cas9-based transcriptional activation tightly regulates gene expression in Nicotiana benthamiana." BMC Biotechnology 22, no. 1 (March 24, 2022). http://dx.doi.org/10.1186/s12896-022-00741-x.
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Abstract Background CRISPR-based programmable transcriptional activators (PTAs) are used in plants for rewiring gene networks. Better tuning of their activity in a time and dose-dependent manner should allow precise control of gene expression. Here, we report the optimization of a Copper Inducible system called CI-switch for conditional gene activation in Nicotiana benthamiana. In the presence of copper, the copper-responsive factor CUP2 undergoes a conformational change and binds a DNA motif named copper-binding site (CBS). Results In this study, we tested several activation domains fused to CUP2 and found that the non-viral Gal4 domain results in strong activation of a reporter gene equipped with a minimal promoter, offering advantages over previous designs. To connect copper regulation with downstream programmable elements, several copper-dependent configurations of the strong dCasEV2.1 PTA were assayed, aiming at maximizing activation range, while minimizing undesired background expression. The best configuration involved a dual copper regulation of the two protein components of the PTA, namely dCas9:EDLL and MS2:VPR, and a constitutive RNA pol III-driven expression of the third component, a guide RNA with anchoring sites for the MS2 RNA-binding domain. With these optimizations, the CI/dCasEV2.1 system resulted in copper-dependent activation rates of 2,600-fold and 245-fold for the endogenous N. benthamiana DFR and PAL2 genes, respectively, with negligible expression in the absence of the trigger. Conclusions The tight regulation of copper over CI/dCasEV2.1 makes this system ideal for the conditional production of plant-derived metabolites and recombinant proteins in the field.
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Schoger, Eric, Lavanya M. Iyer, Claudia Noack, Cheila Rocha, Mareike Jassyk, Orr Shomroni, Gabriela Salinas, and Laura C. Zelarayan. "Abstract 498: Krueppel-like Factor 15 Modulation: A Crispr-based Re-establishment of Homeostatic Transcription in Diseased Mouse and Human Cardiomyocytes." Circulation Research 127, Suppl_1 (July 31, 2020). http://dx.doi.org/10.1161/res.127.suppl_1.498.
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An altered transcriptomic profile of cardiomyocytes is a hallmark of cardiac remodelling and heart failure. Re-activation of gene programs necessary for myocardial homeostasis and inhibition of deleterious signalling pathways, such as Wnt/beta-catenin signalling, is therefore therapeutically desirable, however challenging to achieve tissue-specifically. We identified Krueppel-like factor 15 (KLF15) as a cardiac specific inhibitor of Wnt/beta-catenin signalling, lost during heart failure progression in the mouse and human heart. To re-establish endogenous Klf15 expression, we designed a cardiomyocyte specific CRISPR activation mouse model expressing enzymatically inactive Cas9 (dCas9) fused to a transcriptional activator (VPR) under Myh6 promoter control. The dCas9VPR protein is directed by guide RNAs (gRNA), delivered by AAV9, to the promoter region of Klf15 and induces gene expression. We tested activation of Klf15 in the adult myocardium by injecting 15 weeks old Myh6-dCas9VPR mice systemically with AAV9 carrying Klf15 gRNAs and increasing Klf15 transcript levels 8 weeks later up to 1.6 fold compared to controls. Since we found a 0.5 fold change of Klf15 expression upon transverse aortic constriction compared to sham operated mice, we expect the CRISPR activation induced Klf15 activation to be sufficient to re-establish Klf15 expression. As a translational approach and since we demonstrated KLF15-WNT regulation in human cardiomyocytes, we integrated dCas9VPR into human induced pluripotent stem cells (hIPSC) by targeted genome editing at the AAVS1 safe harbour locus and confirmed dCas9VPR expression in hIPSC and in hIPSC-derived cardiomyocytes (hIPSC-CM). We tested KLF15 gRNAs in HEK293 cells and selected the most efficient candidate gRNAs to test Klf15 activation in dCas9VPR-IPSC resulting in an up to 1.7 fold activation compared to controls. For gene activation in dCas9VPR hIPSC-CM, we are currently testing lentivirus constructs to deliver up to 3 gRNAs simultaneously to tailor KLF15 expression. With these prerequisites, we aim to re-activate master regulators of normal cardiomyocyte function in failing cardiomyocytes at physiologically relevant levels, such as KLF15, to test potential therapeutic targets for the prevention of heart failure progression.
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Schoger, Eric, Kim Rosa, Cheila Rocha, Mareike Jassyk, Shirin Doroudgar, Oliver Mueller, Lukas Cyganek, Wolfram H. Zimmermann, and Laura C. Zelarayan. "Abstract MP217: Nuclease-deficient Cas9 Transcription Factors Restore Krueppel-like Factor 15 Expression In Stressed Mouse And Human Cardiomyocytes." Circulation Research 129, Suppl_1 (September 3, 2021). http://dx.doi.org/10.1161/res.129.suppl_1.mp217.
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Transcriptional changes in cardiomyocytes drive heart failure progression, however, precise control over endogenous gene expression remains challenging. The expression of Krueppel-like factor 15 ( KLF15 ), an evolutionary conserved nuclear and cardiomyocyte specific inhibitor of WNT/CTNNB1 signalling in the heart, is lost upon cardiac remodelling, and accompanied by aberrantly active WNT/CTNNB1 resulting in heart failure progression. We investigated KLF15 expression dynamics employing CRISPR/Cas9-based tools in mouse cardiomyocytes in vivo and in human induced pluripotent stem cell derived cardiomyocytes (hiPSC-CM) under the hypothesis that re-establishment of KLF15 levels in myocardial stress conditions prevents heart failure progression. Using a mouse model expressing enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) under Myh6 -promoter control, we activated Klf15 in a murine pressure overload model by transverse aortic constriction. Delivery of Klf15 gRNAs targeted to the Klf15 promoter region via AAV9 induced Klf15 expression sufficiently to re-normalize Klf15 expression to transcript levels comparable to sham surgery hearts. This was accompanied by reduced decrease of fractional shortening as well as reduced cardiomyocyte hypertrophy in stressed Klf15 re-activated hearts compared to non-trageted (NT) gRNA hearts (n=3-8 per group, echo data from 4 and 8 weeks post-surgery). We achieved titratable KLF15 activation in dCas9VPR transgenic hiPSC-CM by selection of single and multiple gRNAs (n=3-4 replicates) and used these cells to generate human engineered myocardium by combining hiPSC-CM and fibroblasts which we subjected to isometric contractions in order to induce mechanical stress, which resulted in KLF15 expressional decrease in line with our in vivo data. This transcriptional loss was rescued in CRISPR/dCas9VPR hiPSC-CM targeted to the KLF15 locus compared to controls (n=6-9/2/4 tissues per group/casting sessions/differentiations). Additionally, TGFB1 induced cardiomyocyte stress resulted in decreased KLF15 expression levels in 2D hiPSC-CM cultures which were rescued by dCas9VPR- KLF15 targeting (n=3 experiments). In conclusion, we report controllable gene activity by CRISPR/dCas9VPR to restore the loss of KLF15 in stressed mouse and human cardiomyocytes. We furthermore evaluate the potential to gain full control over gene dose titratability with these models to validate and define novel therapeutic targets for the prevention of heart failure progression.
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Schoger, E., L. Argyriou, W. H. Zimmermann, L. Cyganek, and L. C. Zelarayan. "Cutting-free application of CRISPR-mediated endogenous gene activation in human induced pluripotent stem cell derived cardiomyocytes and engineered human myocardium." European Heart Journal 42, Supplement_1 (October 1, 2021). http://dx.doi.org/10.1093/eurheartj/ehab724.3272.
Full textAbstract:
Abstract Background Imbalanced transcriptional networks characterize cardiomyocyte stress and result in cardiac remodelling. We hypothesize that re-establishing homeostatic gene networks in cardiomyocytes will prevent further tissue damage. To tackle this challenge, we applied CRISPR-based endogenous gene activation (CRISPRa) in vivo and in vitro. Methods We employ precision transcriptome editing tools based on CRISPR/Cas9 with enzymatically inactive Cas9 (dCas9) fused to transcriptional activators (VPR) to induce target gene expression by directing dCas9VPR to promoter regions by guide RNAs (gRNA). Results Homozygous CRISPRa hiPSC cell lines were generated by targeted integration of a CAG promoter driven dCas9VPR-T2A-tdTomato expression cassette into the AAVS1 locus by CRISPR/Cas9 editing and homology directed repair. Expression of dCas9VPR was evaluated by immunoblotting and co-expressed reporter fluorescence in spontaneously beating hiPSC-CM. We previously identified a crosstalk between WNT signalling and Krueppel-like factor 15 (KLF15) necessary for controlling cardiac homeostasis. We designed and tested 8 non-overlapping gRNAs in the –400 bp region upstream of the KLF15 transcriptional start site (TSS) and tested individual gRNA effectiveness for gene activation in HEK293T cells. Five gRNAs were identified inducing KLF15 transcript levels between 2- and 5-fold compared to non-targeted (NT) gRNA transfected cells (n=3 experiments). The single most effective gRNA was transduced by lentiviral particles into CRISPRa hiPSC-CM increasing KLF15 transcript levels to 1.5-fold compared to NT-gRNA control. Synergistic effects of 3 instead of single gRNA increased KLF15 transcript levels by 3-fold compared to controls (n≥3 experiments). We hypothesized that dCas9VPR expression could be harnessed as an additional option for gene dose titration and we generated hiPSC lines with enhanced dCas9VPR expression (v2.0). We observed up to 5-fold KLF15 gene activation when triple gRNA and v2.0 were combined (n≥4 experiments). Engineered human myocardium (EHM) was generated consisting of CRISPRa cardiomyocytes, fibroblasts and collagen and we observed similar contractility in 4-week cultured EHM suggesting innocuous dCas9VPR and gRNA expression. CRISPRa component expression was maintained over the entire culture period as evaluated by dCas9VPR immunoblotting and KLF15 transcriptional activation (1.4 fold, v1.0 CRISPRa hiPSC-CM, n≥8 tissues) indicating sustained gene activition. Conclusions Targeted gene activation with CRISPR/Cas9 is a precise and effective tool for transcriptional activation in hiPSC-CM. We observed titratability of gene activation by 1.) dCas9VPR expression levels and 2.) single versus multiple gRNA use. We furthermore elucidated general rules for effective gRNA targeting within the 5' TSS of genes of interest which confirmed a dependency of baseline gene activity as a limiting factor for endogenous gene activation. Funding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): German Research Foundation (DFG) - Collaborative Research Center 1002German Center for Cardiovascular Research (DZHK)