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Updated: Oct 23, 2025
CRISPR Epigenome Editing in Human Cells using Plasmid DNA Transfection and mRNA Nucleofection Delivery
Published on: May 30, 2025
Targeted regulation of transcription in primary cells using CRISPRa and CRISPRi
Trine I Jensen1, Nanna S Mikkelsen1, Zongliang Gao1
1Department of Biomedicine, Aarhus University, 8000 Aarhus C., Denmark.
Abstract:
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.
Insights
CRISPR activation (CRISPRa) and interference (CRISPRi) now effectively regulate gene expression in primary cells. This new RNA or ribonucleoprotein delivery method is non-toxic and programmable for therapeutic applications.
Area of Science:
- Molecular Biology
- Gene Editing
- Cellular Therapeutics
Background:
- CRISPR-Cas9 systems (CRISPRa/CRISPRi) enable targeted gene regulation by fusing nuclease-deactivated Cas9 to effector molecules.
- Previous applications were limited to cancer cell lines, often using lentiviral vectors.
- Effective and non-toxic delivery to ex vivo primary cells for therapeutic use remained a challenge.
Purpose of the Study:
- To develop novel CRISPRa and CRISPRi platforms for transient, programmable gene regulation in primary cells.
- To establish effective and non-toxic delivery modalities for these systems.
- To explore applications in stem cell biology and cellular therapeutics.
Main Methods:
- Development of CRISPRa and CRISPRi platforms utilizing RNA or ribonucleoprotein (RNP) delivery.
- Electroporation as the primary delivery method for primary cells.
- Application in human CD34+ hematopoietic stem and progenitor cells (HSPCs) and human CD3+ T cells.
Main Results:
- Demonstrated transient and programmable gene regulation in primary cells, including HSPCs and T cells.
- Achieved multiplex and orthogonal gene modulation using multiple sgRNAs and diverse CRISPR systems.
- Successfully manipulated HSPC differentiation trajectories using CRISPRa.
Conclusions:
- The developed platforms offer simple, effective, and transient transcriptional control in primary cells.
- These systems are easily adaptable and reprogrammable for new gene targets.
- The technology holds significant potential for advancing stem cell research, gene function studies, and developing enhanced cellular therapeutics.
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