Fig. 4: Near homogenous activation of mcdFLASH-HIF by CRISPRoff knockdown of VHL. | Nature Communications

Fig. 4: Near homogenous activation of mcdFLASH-HIF by CRISPRoff knockdown of VHL.

From: dFLASH; dual FLuorescent transcription factor activity sensor for histone integrated live-cell reporting and high-content screening

Fig. 4

a Clonal (1) mcdFLASH-HIF lines derived post-hygromycin (HygroB) selection were transduced first with the (2) sgRNA vector targeting the VHL transcriptional start site, followed by puromycin selection (Puro). This pool was subsequently transduced with the (3) CRISPRoffv2.1 virus and selected with blasticidin (BlastS) prior to flow cytometry (on day 5 and 10 post BlastS selection). b The (1) dFLASH vector with the HRE enhancer was transduced, as were 2 variants of the CRISPRoffv2.1 vector with either (3A) EF1Ī± promoter or (3B) SFFV promoter driving the dCas9 expression cassette. 3A denotes Dnmt3A, 3L denotes Dnmt3L, XTEN80 denotes an 80 amino acid linker sequence for improved protein half-life.Ā a, bĀ created in BioRender. Peet, D. (2025) https://BioRender.com/l71s669. c, d Flow cytometry for dFLASH-HIF induction in response to the CRISPRoffv2.1 VHL knockdown relative to parental line (ctrl) with (c) EF1Ī± or (d) SFFV expression constructs after 10ā€‰days of selection. Data are representative of nā€‰=ā€‰2 independent experiments.

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