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

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.