Relevant bibliographies by topics / DCas9-VPR

Academic literature on the topic 'DCas9-VPR'

Author: Grafiati
Published: 10 December 2022
Last updated: 28 January 2023

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Journal articles on the topic "DCas9-VPR"

1

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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2

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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3

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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4

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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5

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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6

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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7

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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8

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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9

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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10

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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