Project description:We report the generation of CRISPR-dCas9 DNA methyltransferases to mediate targeted DNA methylation. Using the dCas9-BFP-DNMT3A and dCas9-BFP-DNMT3B methyltransferases, we have demonstrated that these two methyltransferase can mediate targeted methylation in three human genes tested: uPA, TGFBR3, and CDKN2A in human HEK293T cells. We also showed that these methyltransferases could mediate gene inhibition. five samples co-transfected with five uPA sgRNAs and each of the four dCas9 fusions, or control transfection with pUC19 plasmid

2016-06-01 | E-GEOD-74935 | biostudies-arrayexpress

Preclinical targeted therapy for retinoblastoma using EpCAM Aptamer siRNA chimeras

Project description:siRNAs have played a major role in cancer drug discovery, but their potential is hampered due to off-target effects. Thus, delivery systems like RNA aptamers have been used to enhance the specific delivery of these siRNAs to cancer stem cells. We report the efficacy of three different EpCAM aptamer siRNA chimeras, which were investigated both in vitro and in vivo for their ability to reduce cancer cell progression. Using these chimeras, we demonstrated specific gene knockdown in EpCAM positive cells which ultimately led to the apoptosis. To study the efficacy of these aptamer chimeras in vivo, retinoblastoma xenografts bearing NCC Rb C 51 cells were created for the first time. Systemic administration of these aptamer chimeras reduced tumour growth to about 50%. We further investigated the central Role of PLK1 in Cancer Progression and demonstrated the anti-cancer effects of targeted EpCAM siPLK1 approach. Using SILAC-Mass spectrometry analysis, we showed that silencing PLK 1 gene can lead to p53 mediated cell cycle arrest. Thus, we establish EpCAM-siRNA chimeras as potential markers for targeted anti-cancer applications, which paves a platform for efficient second line of therapies in addition to existing chemotherapy options.

2022-03-02 | PXD008221 | Pride

Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein

Project description:Targeted DNA methylation in vivo using an engineered dCas9-MQ1 fusion protein

| PRJNA355391 | ENA

Elongation roadblocks mediated by dCas9 across human genes modulate transcription and nascent RNA processing

Project description:Non-cleaving Cas9 (dCas9) is widely employed to manipulate specific gene loci often with scant regard for unintended transcriptional effects. We demonstrate here that dCas9 mediates precise transcriptional pausing followed by transcription termination and potential alternative polyadenylation. In contrast, alternative splicing is unaffected, likely requiring more sustained alteration to RNA polymerase II elongation speed. The effect on transcription is orientation-specific, with pausing only being induced when dCas9-associated guide RNA anneals to the non-template strand. Targeting template strand induces minimal effects on transcription elongation and thus provides a neutral approach to recruit dCas9 linked effector domains to specific gene regions. In essence we evaluate molecular effects of targeting dCas9 to mammalian transcription units. In so doing we also provide new information on transcriptional elongation by RNA polymerase II and coupled pre-mRNA processing.

2023-05-16 | GSE228798 | GEO

Precise modulation of genome functions and live tracking of genome regions in CRISPR/dCas9 knockin mice

Project description:we demonstrated ex vivo and in vivo validation of dCas9 KI functions such as transcription

2017-09-28 | GSE104331 | GEO

ChIP-seq analysis of dCas9 chromatin occupancy by dCas9/sgRNA-mediated CAPTURE

Project description:ChIP-seq analysis of dCas9 chromatin occupancy by dCas9/sgRNA-mediated CAPTURE

| PRJNA387472 | ENA

Expression data from Tregs purified from WT-CD3KO or IIKO-CD3KO chimeras

Project description:Disruption of TCR /MHC class II interactions leads rapidly to alterations of the common CD4 Treg transcriptional signature Self-deprived, non-functional Tregs were compare to fully functional Tregs by microarrays. CD3ε-/- mice were lethally irradiated and their immune system reconstituted with WT mouse (WT-CD3KO chimeras) or IIKO mouse (IIKO-CD3KO) Bone Marrow cells. Twenty eight days later, peripheral Tregs from these chimeras were purified for RNA extraction and hybridization on Affymetrix microarrays.

2011-02-09 | E-GEOD-27151 | biostudies-arrayexpress

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