🧬 PubMed RNA Editing Daily Digest

We previously reported that poly (N-vinylacetamide-co-acrylic acid) coupled with d-octaarginine (VP-R8) efficiently introduces proteins and nucleic acids into cells. Based on these results, we hypothesized that VP-R8 can introduce a complex composed of guide RNA and Cas9 (RNP complex) into cells and induce genome editing mediated by the CRISPR-Cas9 system. We tested this hypothesis using a mouse dendritic cell line and mouse primary dendritic cells. The RNP complexes formed by guide RNA consisting of CRISPR RNA (crRNA), fluorescently labeled trans-activating crRNA (tracrRNA), and GFP-fused Cas9 were introduced into a mouse dendritic cell line using VP-R8 or control transfection reagents. Cells transfected using VP-R8 exhibited higher fluorescence than those transfected with other transfection reagents, indicating that VP-R8 efficiently introduced the RNP complex into the mouse dendritic cell line. Genome editing of the target DNA was detected in cells transfected with the RNP complex using VP-R8 and dominant relative to other transfection reagents. We also observed that VP-R8 effectively delivered RNP complexes consisting of single-guide RNA and Cas9 and induced genome editing in the dendritic cell line. Additionally, VP-R8 efficiently delivered RNP complexes into mouse primary dendritic cells and induced genome editing of the functional gene without producing early inflammatory cytokines. Thus, VP-R8 shows potential as a transfection tool to generate dendritic cells with specific gene regions deleted by genome editing via the CRISPR-Cas9 system. This approach aims to elucidate the detailed molecular mechanisms of dendritic cell function for its application to vaccines.

Both metabolic dysregulation and the immunosuppressive tumor microenvironment of pancreatic ductal adenocarcinoma (PDAC) contribute to the recalcitrance of this lethal disease to treatment. Accordingly, we aimed to identify and characterize a target that elicits an anticancer response through both disrupting cancer cell redox homeostasis and increasing the immunogenicity of PDAC. First, mitochondrial metabolic dependencies in PDAC were identified by using a CRISPR-Cas9 screening system with a custom single-guide RNA library. Functional validation analyses revealed GFER, a mitochondrial FAD-dependent sulfhydryl oxidase, as an essential regulator of tumor growth. In vitro and in vivo methodologies demonstrated that GFER depletion perturbed redox homeostasis and stimulated tumor immunogenicity, including sensitization to immune checkpoint blockade. In patient-derived xenograft models of PDAC, the growth-inhibitory response induced by GFER depletion was mediated by an altered oxidative balance that released damaged mitochondrial DNA into the cytoplasm of tumor cells, leading to the activation of the cGAS-STING pathway and expression of type I IFNs. This effect was recapitulated in a mouse immunocompetent syngeneic PDAC model, in which GFER depletion suppressed tumor growth and promoted T-cell infiltration to enhance tumor-killing effects. Consequently, GFER depletion significantly increased the antitumor efficacy of immune checkpoint blockade. Overall, these findings identify GFER as a critical node for both mitochondrial redox homeostasis and immunomodulation in PDAC and reveal a therapeutic opportunity for sensitizing PDAC to immune checkpoint blockade. GFER is essential for mitochondrial redox balance and suppressing tumor immunogenicity in pancreatic tumors, with the combination of GFER inhibition with immune checkpoint blockade resulting in a strong antitumor response.

The U6 promoter plays a pivotal role in the CRISPR/Cas9 system by driving the transcription of single guide RNA (sgRNA), which directs Cas9 to achieve precise genome editing. Endogenous U6 promoters typically exhibit superior transcriptional activation efficiency compared to exogenous counterparts, thereby enhancing the efficacy of genome editing. However, the endogenous U6 promoter in kenaf (