Fig. 4: crRNA engineering to enhance the activation of type I–F PaeCascade.
From: Repurposing type I–F CRISPR–Cas system as a transcriptional activation tool in human cells
a Quantitative PCR analysis of HBB, HBG, and SOX2 transcription levels in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR 2-vector system) with spacers in different lengths. Upper: schematic illustration of differences in Csy3 copy numbers in type I–F PaeCascade VPR (Csy3-VPR) with spacers in different lengths. Gray: Csy1; red: Csy2; blue: Csy3; yellow: Csy4; orange flag: VPR. The longer the spacer was, the more Csy3-VPR in type I–F PaeCascade. Lower: quantitative PCR analysis of HBB, HBG, and SOX2 transcription level in HEK293T cells cotransfected with type I–F PaeCascade VPR and crRNA targeting HBB, HBG, and SOX2. 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. b Quantitative PCR analysis of HBB, HBG, SOX2, OCT4, IL1B, and IL1R2 transcription levels in HEK293T cells transfected with type I–F PaeCascade VPR (Csy3-VPR 2-vector system) and crRNAs targeting −100 bp (crRNA1) and −200 bp (crRNA2) upstream of TSS in Fig. 2. Upper: schematic illustration of enhancing transcription level by two crRNAs targeting the same gene. Lower: quantitative PCR analysis of HBB, HBG, SOX2, OCT4, IL1B, and IL1R2 transcription levels in HEK293T cells. 2 crRNAs indicates two independent crRNA expression vectors. c Quantitative PCR analysis of HBG transcription level in HEK293T cells cotransfected with type I–F PaeCascade VPR (Csy3-VPR) and crRNA with different distances to crRNA2 (−200 bp upstream TSS in Fig. 2). 48 h post-transfection, cells were lysed for RNA extraction and quantitative PCR assay. Ctrl: non-targeting crRNA control. Data 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.
