Journal: Cell & bioscience

Article Title: CRISPRa-mediated FOXP3 gene upregulation in mammalian cells.

doi: 10.1186/s13578-019-0357-0

Figure Lengend Snippet: Fig. 2 Targeted up and down-regulation of the FOXP3 transcript levels in HEK293 cells. a Schematic presentation of the transcriptional regulation with CRISPR–dCas9. b, c HEK293 cells were transfected with plasmids encoding sgRNAs targeting six distinct regulatory regions of the FOXP3 gene (see Fig. 1) and cotransfected with plasmids encoding dCas9-based activators (VP16 or VPR) or a repressor (KRAB). Fold expression of FOXP3 was analyzed in respect to a control sample transfected with a random sgRNA (rnd) and normalized to a house-keeping gene (GAPDH). A mixture of four sgRNAs was used for each region (sgRNA sequences are listed in Table 1). RNA was isolated from samples 48 h post transfection. Experiments were repeated 3 times. b Results using dCas9-VP16 or VPR activators show that regulatory target sites downstream (CNS3, CNS1) and upstream (Cage, Cage2) of core promoter can result in higher activation of FOXP3 transcription than sgRNA targeting core promoter. c Results using dCas9-KRAB demonstrate that FOXP3 basal transcription can be further downregulated using fusion of dCas9 with a repressor domain KRAB

Article Snippet: The following primary antibodies were used for western blotting: Mouse monoclonal to GAPDH diluted 1:2000, purchased from Proteintech (#60004-1-Ig), Mouse monoclonal [236A/E7] to FOXP3 from Abcam (1:1000) and Goat Anti-Mouse IgG (H+L)-HRP diluted 1:3000 (Jackson ImmunoResearch #115-035-003).

Techniques: CRISPR, Transfection, Expressing, Control, Isolation, Activation Assay