Generation of Inducible CRISPRi and CRISPRa Human Stromal/Stem Cell Lines for Controlled Target Gene Transcription during Lineage Differentiation

Figure 1

Generation of inducible CRISPRi and CRISPRa hMSCs. (a) Illustration and flow chart for generating inducible CRISPRi using dCAs9 TRE-KRAB and CRISPRa using dCas9 TRE-VP64. (b) Western blot analysis of expression of dCas9-KRAB-HA and dCas9-VP64-HA in the presence of increasing concentrations of doxycycline (Dox) in CRISPRi-hMSC and CRISPRa-hMSC, separately. The effect of the absence or presence of Dox on protein expression of dCas9-KRAB-HA (c) or dCas9-VP64-HA (d). CRISPRi-hMSC and CRISPRa-hMSC were cultured in cell culturing medium with or without Dox (0 or 1000 ng/ml) for 2 days, then washed twice by PBS and changed the cell culturing medium to the medium without Dox, with continuous culturing of the cells till day 6. Cell proteins were harvested on days 2, 3, 4, and 6 and subjected to Western blots to test the expression of dCas9-KRAB and dCas9-VP64 by HA antibody.

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