Fig. 2: Benchmarking of HEAL performance against state-of-the-art CRISPRa systems in mammalian cells and mice.
From: A compact and inducible dCas12f-based CRISPRa platform for programmable in vivo gene activation
a Schematics for endogenous gene activation with the HEAL (top), dCasMINI (middle), or dCpf1 (bottom) systems. In the HEAL system, transactivators are recruited to the MS2 aptamers within the sgRNA, whereas transactivators are fused directly to the CRISPR-dCas proteins in the dCasMINI and dCpf1 systems. b–f Evaluation of endogenous gene activation by HEAL in comparison with dCasMINI and dCpf1 systems in mammalian cells. B16F10 or HEK-293T cells were co-transfected with plasmids encoding HEAL, dCasMINI, or dCpf1 systems targeting different endogenous genes. QPCR assays of Ucp1 (b) and Tslp (c) mRNA levels in B16F10 cells, as well as RHOXF2 (d), UCP1 (e), and HBG (f) transcription in HEK-293T cells were performed at 48 h post-transfection. In panels (b–f) colors indicate different systems: light blue, HEAL; orange-brown, dCasMINI; charcoal brown, dCpf1. g Schematic for endogenous gene activation with the HEAL or dCas9-SAM systems. In both systems, transactivators are recruited to the MS2 aptamers within the sgRNA. h–k Comparison of endogenous gene activation levels between the HEAL and dCas9-SAM systems. B16F10 or HEK-293T cells were co-transfected with plasmids encoding HEAL or dCas9-SAM systems targeting different endogenous genes. QPCR-based measurements of Tslp (h) and Ucp1 (i) transcription in B16F10 cells, as well as HBG (g) and UCP1 (k) in HEK-293T cells at 48 h post-transfection. In panels (h–k), colors indicate different systems: light blue, HEAL; beige, dCas9-SAM. l Schematic of the timeline for assessing endogenous gene activation in mice. Plasmids encoding the dCpf1, dCasMINI, HEAL, or dCas9-SAM system were delivered to mouse livers via hydrodynamic tail vein injection. NT, non-targeting. m Relative Ucp1 mRNA expression in mouse livers following activation by the dCpf1, dCasMINI, HEAL, or dCas9-SAM systems. Data in (b–f, h–k) are means ± s.d.; n = 3 independent experiments, with 3 technical replicates for each. Data in (m) are means ± s.e.m.; n = 6 mice. Statistical significance was determined by two-way ANOVA (b–f, m) or one-way ANOVA (h–k); ns, not significant, *P <  0.05, **P <  0.01, ***P <  0.001, ****P <  0.0001. Source data are provided as a Source Data file.
