Figure 2.

Development of an inducible CRISPR system for adipocyte genome editing. A, diagram of an inducible CRISPR KO system in which Cas9 expression is under the control of a tight tetracycline response element (TRE). Expression of the first single guide RNA is driven by a mouse U6 (mU6) promoter, whereas expression of the second sgRNA is driven by a human U6 (hU6) promoter. NLS, nuclear localization signal. B, timeline of adipocyte differentiation and dox-induced genome editing. Dox was added to preadipocytes at a final concentration of 2 μg/ml to induce Cas9 expression 24 h before addition of an adipocyte differentiation mixture. Unless indicated otherwise, all functional assays were performed using differentiated adipocytes. C, representative immunoblots showing the expression of the indicated proteins in WT or Exoc7 KO adipocytes. Two independent samples are shown for each cell line. M.W., molecular weight.