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Schmdit, T. ; Wiesbeck, M. ; Egert, L. ; Truong, T.-T. ; Danese, A.* ; Voshagen, L.* ; Imhof, S.* ; Iraci Borgia, M.* ; Deeksha ; Neuner, A.M. ; Köferle, A. ; Geerlof, A. ; Mourao, A. ; Stricker, S.H.

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

Nucleic Acids Res. 53:gkaf235 (2025)

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	Open Access Gold

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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.

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Publication type Article: Journal article

Document type Scientific Article

Corresponding Author

Keywords Gene-expression; In-vivo; Rna; Crispr; Activation; Delivery; System; Cas9; Platform; Protein

ISSN (print) / ISBN 0305-1048

e-ISSN 1362-4962

Journal Nucleic Acids Research

Quellenangaben Volume: 53, Issue: 6, Article Number: gkaf235

Publisher Oxford University Press

Publishing Place Great Clarendon St, Oxford Ox2 6dp, England

Non-patent literature Publications

Reviewing status Peer reviewed

Institute(s) Institute of Stem Cell Research (ISF)
Institute of Structural Biology (STB)

Grants Humanities Research Council of Canada
natural sciences and engineering research council of canada
CIHR
REGENERAR
Helmholtz Zentrum Munchen
International Helmholtz-Edinburgh Research School for Epigenetics
EpiCrossBorders