Fig. 3: dCasCMA-mediated targeted degradation of endogenous Pd-l1 mRNA.
From: Programmable targeted RNA degradation via dCas13d-directed chaperone-mediated autophagy (dCasCMA)
a Schematic of the gRNA design for targeting the Pd-l1 mRNA. b WB analysis of total PD-L1 levels in CT26 cells treated with dCCTM and different gRNAs after 36 hours. (n = 3, biological replicates). c Quantitative analysis of PD-L1 protein expression levels shown in (b), gPdl1-A p = 0.0004; gPdl1-B p = 0.0008; gPdl1-C p < 0.0001; gPdl1-D p < 0.0001. d qPCR analysis of Pd-l1 mRNA levels in CT26 cells 36 h after dCCTM/gRNA transfection, gPdl1-A p < 0.0001; gPdl1-B p < 0.0001; gPdl1-C p < 0.0001; gPdl1-D p < 0.0001. e Time-course comparison of Pd-l1 mRNA knockdown efficiency between dCCTM/gPdl1-D and siPdl1, (0 h p = 0.9761;36 h p = 0.5124; 48 h p = 0.4019; 60 h p = 0.9157). f Downregulation of metastasis-related mRNAs (Pik3, β-catenin, Wip, N-cadherin, Vimentin) following Pd-l1 mRNA degradation induced by dCCTM/gPdl1-D, (Pik3 p = 0.0015; β-catenin p = 0.005; Wip p = 0.0083; N-cadherin p = 0.0013; Vimentin p = 0.0005). g Schematic illustration of the wound-healing assay for studying the migration ability of the cells under different conditions. Created in BioRender. Wen, H. (2025) https://BioRender.com/41b43vf. h, i Confocal images and cell migration rates of CT26 cells treated with dCCTM/NT and dCCTM/gPdl1-D. Scale bar, 500 μm. (Mock vs. dCCTM + NT, p = 0.9593; Mock vs. dCCTM + gPdl1-D, p = 0.0004). j Relative NEAT1 RNA expression levels in cells transfected with plasmids expressing dCCTM/dCCTM-NCS proteins and the indicated gRNA, measured by qPCR, (dCCTM + NT vs. dCCTM + gNEAT1, p = 0.6466; dCCTM-NCS + NT vs. dCCTM-NCS + gNEAT1, p = 0.0001). Data represent mean ± SD (n = 3, biological replicates). Statistical significance was determined by one-way ANOVA (c, d, h and j) or unpaired two-tailed Student’s t-test (e, f). ns, not significant; **p < 0.01, ***p < 0.001. Source data are provided as a Source Data file.
