Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi

TY - JOUR

T1 - Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi

AU - Jensen, Trine I

AU - Mikkelsen, Nanna S

AU - Gao, Zongliang

AU - Foßelteder, Johannes

AU - Pabst, Gabriel

AU - Axelgaard, Esben

AU - Laustsen, Anders

AU - König, Saskia

AU - Reinisch, Andreas

AU - Bak, Rasmus O

N1 - © 2021 Jensen et al.; Published by Cold Spring Harbor Laboratory Press.

PY - 2021/11

Y1 - 2021/11

N2 - 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.

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.

KW - ACTIVATION

KW - HIGHLY EFFICIENT

U2 - 10.1101/gr.275607.121

DO - 10.1101/gr.275607.121

M3 - Journal article

C2 - 34407984

SN - 1088-9051

VL - 31

SP - 2120

EP - 2130

JO - Genome Research

JF - Genome Research

IS - 11

ER -