Fig. 2: Type I–F PaeCascade could activate endogenous genes in human cells.
From: Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells
a Quantitative PCR analysis of HBB transcription level in HEK293T cells transfected with type I–F PaeCascade 2-vector systems and crRNA targeting HBB. Left: schematic illustration of different type I–F PaeCascade VPR activators generated from 2-vector systems in Fig. 1f. Gray: Csy1; red: Csy2; blue: Csy3; yellow: Csy4; orange flag: VPR. Different fusions of PaeCascade subunits resulted in different locations and copy numbers of VPR. HEK293T cells were transfected with PaeCascade 2-vector systems and crRNA vector. 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. b Quantitative PCR analysis of gene transcription levels in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR 2-vector system) targeting different regions upstream the transcriptional start site (TSS) of six genes (HBB, HBG, SOX2, OCT4, IL1B, and IL1R2). n.d.: not determined. c Histogram showing the normalized mean transcription activating levels of HBB, HBG, SOX2, OCT4, IL1B, and IL1R2 induced by type I–F PaeCascade VPR (Csy3-VPR 2-vector system) transcription activator targeting different regions upstream of TSS. For normalization of data from different TSS among different genes, data in the same gene were processed by percentage normalization with 100% defined by the sum of all values in data set. The normalized values of all six genes were pooled and plotted as box & whiskers plot with min to max option. The median value is displayed as the center of the data set, and is derived using the lower and upper quartile values. The maximum and minimum values are displayed as whiskers. d The efficiency of target gene activation as a function of basal transcript levels. Data from (b) were plotted by fold changes comparing to negative control and relative basal transcript level of HBB, HBG, SOX2, OCT4, IL1B, and IL1R2. Each dot represented the mean relative activation level of each crRNA from the three replications in (b). Ctrl: non-targeting crRNA control. Data in (a, b) represented three biological repeats and displayed as mean ± S.E.M. Statistical significance was calculated using one-way ANOVA (*P < 0.05; **P < 0.01; ***P < 0.001). Source data are provided as a Source Data file.
