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Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP)-complexes

ABSTRACT: Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP)-complexes

PROVIDER: PRJNA1216001 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR32133323_1.fastq.gz	Fastqsanger.gz
	SRR32133323_2.fastq.gz	Fastqsanger.gz
	SRR32133324_1.fastq.gz	Fastqsanger.gz
	SRR32133324_2.fastq.gz	Fastqsanger.gz
	SRR32133325_1.fastq.gz	Fastqsanger.gz

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Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP)-complexes

Project description:For genome editing, the use of CRISPR ribonucleoprotein (RNP) complexes is well established and often the superior choice over plasmid-based or viral strategies. RNPs containing dCas9 fusion proteins, which enable the targeted manipulation of transcriptomes and epigenomes remain significantly less accessible. Here, we describe the production, delivery and optimization of second generation CRISPRa RNPs (dRNPs). We characterize the transcriptional and cellular consequences of dRNP treatments in a variety of human target cells and show that the uptake is very efficient. The targeted activation of genes demonstrates remarkable potency, even for genes that are strongly silenced, such as developmental master transcription factors. In contrast to DNA based CRISPRa strategies, gene activation is immediate and characterized by a sharp temporal precision. We also show, that dRNPs allow very high target multiplexing, enabling undiminished gene activation of multiple genes simultaneously. Applying these insights, we find that intensive target multiplexing at single promoters synergistically elevates gene transcription. Finally, we demonstrate in human stem and differentiated cells that the preferable features of dRNPs allow to instruct and convert cell fates efficiently without the need for DNA delivery or viral vectors. Please cite: Tobias Schmidt, Maximilian Wiesbeck, Luisa Egert, Thi-Tram Truong, Anna Danese, Lukas Voshagen, Simon Imhof, Matilde Iraci Borgia, Deeksha, Andrea M Neuner, Anna Köferle, Arie Geerlof, André Santos Dias Mourão, Stefan H Stricker, Efficient DNA- and virus-free engineering of cellular transcriptomic states using dCas9 ribonucleoprotein (dRNP) complexes, Nucleic Acids Research, Volume 53, Issue 6, 11 April 2025, gkaf235, https://doi.org/10.1093/nar/gkaf235

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