Details

Autor(en) / Beteiligte

• Jensen, Trine I
• Mikkelsen, Nanna S
• Gao, Zongliang
• Foßelteder, Johannes
• Pabst, Gabriel
• Axelgaard, Esben
• Laustsen, Anders
• König, Saskia
• Reinisch, Andreas
• Bak, Rasmus O

Titel

Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi

Ist Teil von

• Genome research, 2021-11, Vol.31 (11), p.2120-2130

Ort / Verlag

United States

Erscheinungsjahr

2021

Quelle

MEDLINE

Beschreibungen/Notizen

• Targeted transcriptional activation or interference can be induced with the CRISPR-Cas9 system (CRISPRa/CRISPRi) using nuclease-deactivated Cas9 fused to transcriptional effector molecules. These technologies have been used in cancer cell lines, particularly for genome-wide functional genetic screens using lentiviral vectors. However, CRISPRa and CRISPRi have not yet been widely applied to ex vivo cultured primary cells with therapeutic relevance owing to a lack of effective and nontoxic delivery modalities. Here we develop CRISPRa and CRISPRi platforms based on RNA or ribonucleoprotein (RNP) delivery by electroporation and show transient, programmable gene regulation in primary cells, including human CD34 hematopoietic stem and progenitor cells (HSPCs) and human CD3 T cells. We show multiplex and orthogonal gene modulation using multiple sgRNAs and CRISPR systems from different bacterial species, and we show that CRISPRa can be applied to manipulate differentiation trajectories of HSPCs. These platforms constitute simple and effective means to transiently control transcription and are easily adopted and reprogrammed to new target genes by synthetic sgRNAs. We believe these technologies will find wide use in engineering the transcriptome for studies of stem cell biology and gene function, and we foresee that they will be implemented to develop and enhance cellular therapeutics.