dCas9-metabolic enzyme fusions modulate global and locus-specific gene expression
Kellen V Biesbrock1,2, Spencer A Haws1,2, Harshini Cormaty1,3,4
1Wisconsin Institute for Discovery, University of Wisconsin-Madison, Madison, WI, 53715, USA.
Biorxiv : the Preprint Server for Biology
|May 18, 2026
Summary
We developed CRISPR metabolite (CRISPRm) to control nuclear metabolite levels, revealing how these changes impact gene expression and chromatin regulation. This tool links cellular metabolism directly to epigenetic modifications and gene transcription.
Area of Science:
- Molecular Biology
- Epigenetics
- Metabolic Regulation
Background:
- Cellular metabolism provides essential co-substrates for chromatin-modifying enzymes, linking metabolism to chromatin regulation.
- Nuclear localization of metabolic enzymes suggests nucleus-specific metabolite availability regulates chromatin state.
- Understanding this metabolism-epigenome axis is crucial for deciphering gene regulation.
Purpose of the Study:
- To develop and utilize a novel CRISPR-based tool (CRISPRm) for assessing nucleus-specific metabolic perturbations.
- To investigate how modulating nuclear co-substrate levels influences chromatin function and gene expression.
- To explore the direct link between nuclear metabolite availability and epigenetic modifications.
Main Methods:
- Developed CRISPR metabolite (CRISPRm), a modular dCas9-effector platform.
- Engineered five dCas9-metabolic enzyme fusions (dCas9-ACSS2, -NMNAT1, -MAT2A, -GDH, -AHCY) to modulate nuclear co-substrate levels.
- Assessed global gene expression changes and targeted promoter modulation in HEK293T cells.
Main Results:
- CRISPRm fusions induced distinct global gene expression changes, with dCas9-ACSS2 (acetyl-CoA) and dCas9-NMNAT1 (NAD+) showing opposing effects.
- Targeting CRISPRm to promoters enhanced transcriptional modulation, with specific chromatin feature enrichment (H3K9ac, H3K18ac, H3K27ac, H3K4me3, p300) at sensitive loci.
- dCas9-MAT2A (SAM) and dCAS9-GDH (alpha-ketoglutarate) demonstrated unique sensitivities, with dCAS9-AHCY (SAH) showing unexpected regulatory effects.
Conclusions:
- CRISPRm is a powerful tool for studying nuclear metabolic regulation of transcription.
- Perturbations in nuclear co-substrates modulate specific chromatin-modifying enzymes, leading to targeted transcriptional responses.
- The study provides strong evidence for the direct impact of nuclear metabolite availability on epigenetic states and gene expression.
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