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

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

PROVIDER: PRJNA412372 | ENA |

REPOSITORIES: ENA

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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

Tracking of dCas9-methyltransferase footprints

Project description:In normal mammalian development cytosine methylation is essential, and is directed to specific regions of the genome. Despite notable advances in mapping the genome-wide distribution, studying the direct contribution of DNA methylation to gene regulation has been limited by the lack of tools for its precise manipulation. Thus, combining the targeting capability of CRISPR/Cas9 systems with an epigenetic modifier has attracted interest in the scientific community. In contrast to profiling the genome-wide cleavage of a nuclease competent Cas9, tracing the global activity of a dead Cas9-methyltransferase is challenging within a highly modified genome. To overcome this, we utilized an engineered, methylation depleted but maintenance competent mouse ES cell line and find a surprisingly ubiquitous nuclear activity of dCas9-methyltransferases. Our results provide new insights into the targeted regulation of DNA methylation and point to an important difference between genetic and epigenetic editing tools that require unique experimental considerations.

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Tracking of dCas9-methyltransferase footprints

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Project description: Not available

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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

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Project description:O-GlcNAc is a nutritionally and metabolically relevant post-translational modification on thousands of nucleocytoplas-mic proteins. O-GlcNAcylation level dynamically responds to environmental cues in temporal and spatial dimensions, leading to discrete signaling transductions and physiological effects. Spatiotemporal regulation of O-GlcNAcylation level is essential for O-GlcNAc functional interrogation and manipulation of cell behaviors for desired outcomes, which is yet challenging owing to limited approaches. Here we achieved spatiotemporal O-GlcNAc reduction in live cells by designing a 4-hydroxytamoxifen (4-HT)-triggered O-GlcNAcase (OGA) activation strategy. After rational engineering and optimiza-tion, OGA variants bearing an intein located to different subcellular regions were generated and validated, whose deglyco-sidase activity can be turned on by 4-HT in a time- and dose-dependent manner. Finally, we demonstrated the dual func-tionality of 4-HT on breast cancer cells expressing the engineered OGA, which accelerated cell death with a lower dosage and thus mitigated the likelihood of acquiring drug resistance. Altogether, our strategy facilitates the precise regulation and functional understandings of O-GlcNAcylation in live cells.

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