RT Journal
A1 Jensen, Trine I.
A1 Mikkelsen, Nanna S.
A1 Gao, Zongliang
A1 Foßelteder, Johannes
A1 Pabst, Gabriel
A1 Axelgaard, Esben
A1 Laustsen, Anders
A1 König, Saskia
A1 Reinisch, Andreas
A1 Bak, Rasmus O.
T1 Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi
JF Genome Research 
JO Genome Research 
YR 2021 
FD November 01 
VO 31 
IS 11 
SP 2120 
OP 2130 
DO 10.1101/gr.275607.121 
UL http://genome.cshlp.org/content/31/11/2120.abstract 
AB 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.