RESEARCH ARTICLE
Open Access

CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study

Hiroshi Takamura1, Yuki Nakamura2, Rina Fujimoto3, Keisuke Morita1, Ayaka Shimizu1, Takeshi Yamamoto4*ORCID 

¹ Department of Molecular and Cellular Medicine, Graduate School of Medicine, Kyoto
University, Kyoto, Japan.
² Department of Genome Biology, Graduate School of Medicine, Kyoto University, Kyoto, Japan.
3 Department of Immunology and Genomic Medicine, Graduate School of Medicine, Kyoto
University, Kyoto, Japan.
4 Department of Experimental Pathology, Graduate School of Medicine, Kyoto University, Kyoto,
Japan.
DOI: 10.18081/ajbm.2026.3.294

Publication History: Received 19 May 2026 • Revised 20 June 2026 • Accepted 22 July 2026 • Available online 27 August 2026
Copyright: © 2026 Yamamoto et al. This is an open-access article under a Creative Commons license (CC BY 4.0).
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ABSTRACT

Background

Persistent NF-κB-driven inflammatory transcription promotes inflammasome activation, oxidative stress, mitochondrial dysfunction, and regulated cell death. Although CRISPR-based epigenome editing enables locus-specific transcriptional regulation without permanent modification of the underlying DNA sequence, its capacity to protect against inflammatory cell injury in vivo remains incompletely defined.

Objective

To determine whether dCas9-KRAB-mediated epigenetic repression of Nfkb1 attenuates inflammatory cell injury and to define the molecular mechanisms linking targeted chromatin remodeling with cytoprotection.

Method

A nuclease-inactive CRISPR/dCas9-KRAB system targeting the Nfkb1 regulatory region was evaluated in LPS-stimulated RAW 264.7 macrophages and a murine model of systemic LPS-induced inflammation. Non-targeting sgRNA served as the CRISPR control. Target engagement was assessed by RT-qPCR, immunoblotting, and H3K9me3 ChIP-qPCR. Inflammatory cytokines, NLRP3/caspase-1/GSDMD signaling, intracellular and mitochondrial reactive oxygen species, NRF2-associated antioxidant responses, mitochondrial membrane potential, cell viability, membrane injury, apoptosis, and pyroptosis-associated endpoints were evaluated. In vivo outcomes included circulating cytokines, histopathological injury, neutrophilic infiltration, tissue inflammasome signaling, TUNEL positivity, biochemical indices of organ injury, and survival.

Results

Targeted dCas9-KRAB repression increased H3K9me3 enrichment at the Nfkb1 regulatory locus and reduced Nfkb1 transcription by 64.8% compared with the LPS + non-targeting CRISPR group (P<0.001), accompanied by an approximately 57% reduction in NF-κB p65 phosphorylation. Expression of Tnf, Il1b, and Il6 decreased by 58.6%, 61.9%, and 55.3%, respectively, while Nlrp3 expression decreased by 52.7% (all P<0.001). NLRP3, cleaved caspase-1, and GSDMD-N were significantly reduced, with corresponding decreases in IL-1β/IL-18 release and membrane injury. Targeted repression reduced intracellular ROS by 46.2%, restored NRF2-associated antioxidant signaling, and preserved mitochondrial membrane potential. Cell viability increased from 63.5 ± 6.1% to 85.7 ± 5.4%, while apoptosis decreased from 34.2 ± 4.9% to 16.8 ± 3.5% (P<0.001). In vivo, targeted Nfkb1 repression reduced circulating TNF-α, IL-1β, IL-6, and IL-18 by approximately 45%–56%, suppressed tissue inflammasome and cell-death signaling, reduced histopathological injury by approximately 56%, and decreased TUNEL positivity from 36.9 ± 5.8% to 17.5 ± 4.1% (P<0.001). These molecular changes were accompanied by reduced multiorgan injury and improved survival.

Conclusion

Locus-specific CRISPR/dCas9-KRAB-mediated repression of Nfkb1 attenuated inflammatory cell injury through coordinated suppression of NF-κB-dependent transcription, NLRP3 inflammasome activation, oxidative and mitochondrial stress, and apoptotic and pyroptotic signaling. The concordant in vitro and in vivo findings identify targeted epigenetic reprogramming as a potential strategy for controlling pathological inflammation without permanent alteration of genomic DNA.

Keywords: CRISPR; dCas9-KRAB; epigenome editing; Nfkb1; NF-κB; NLRP3 inflammasome; pyroptosis; oxidative stress; inflammatory cell injury.

Recommended Citation

Takamura H, Nakamura Y, Fujimoto R, Morita M, Shimizu A, Yamamoto T. CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study. Advanced Journal of Biomedicine & Medicine. 2026;14(3):294-334. doi:10.18081/ajbm.2026.3.294

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Creative Commons Attribution 4.0 International License
This work is licensed under a Creative Commons Attribution 4.0 International License.

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Takamura H, Nakamura Y, Fujimoto R, Morita K, Shimiz A, Yamamoto T (2026). CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study. Advanced Journal of Biomedicine & Medicine, 14(3), 294-334. https://doi.org/10.18081/ajbm.2026.3.294
Takamura H, Nakamura Y, Fujimoto R, Morita K, Shimiz A, Yamamoto T. " CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study." Advanced Journal of Biomedicine & Medicine, vol. 14, no. 3, 2026, pp. 294-334. DOI: 10.18081/ajbm.2026.3.294.
Takamura H, Nakamura Y, Fujimoto R, Morita K, Shimiz A, Yamamoto T. CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study. AJBM. 2026;14(3):294-334. DOI: 10.18081/ajbm.2026.3.294. PMID: .
Takamura H, Nakamura Y, Fujimoto R, Morita K, Shimiz A, Yamamoto T 2026, " CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study", Advanced Journal of Biomedicine & Medicine, vol. 14, no. 3, pp. 294-334.
@article{takamura2026, title={ CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study}, author={Takamura H, Nakamura Y, Fujimoto R, Morita K, Shimiz A, Yamamoto T}, journal={Advanced Journal of Biomedicine & Medicine}, volume={14}, number={3}, pages={294-334}, year={2026}, doi={10.18081/ajbm.2026.3.294} }
TY - JOUR AU - Takamura H, Nakamura Y, Fujimoto R, Morita K, Shimiz A, Yamamoto T TI - CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study JO - American Journal of Biomedicine VL - 14 IS - 3 SP - 294-334 PY - 2026 DO - 10.18081/ajbm.2026.3.294 ER -
%0 Journal Article %A Takamura H, Nakamura Y, Fujimoto R, Morita K, Shimiz A, Yamamoto T %T CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study %J American Journal of Biomedicine %V 14 %N 3 %P 294-334 %D 2026 %R 10.18081/ajbm.2026.3.294 %M
Takamura H, Nakamura Y, Fujimoto R, Morita K, Shimiz A, Yamamoto T (2026). CRISPR-Mediated Epigenetic Reprogramming Attenuates Inflammatory Cell Injury Through Targeted Transcriptional Modulation: An Experimental in Vitro and in Vivo Study. Advanced Journal of Biomedicine & Medicine, 14(3), 294-334. https://doi.org/10.18081/ajbm.2026.3.294

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