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. Author manuscript; available in PMC: 2017 Apr 7.
Published in final edited form as: Cell Stem Cell. 2016 Mar 10;18(4):541–553. doi: 10.1016/j.stem.2016.01.022

Figure 6. CRISPRi knockdown in differentiated cell types and cardiac-disease modeling.

Figure 6

(A) Using CRISPRi, MESP1 was knocked down by ~90% in polyclonal cardiac progenitors, and MYBPC3 and HERG were knocked down by ~90% and 60% in polyclonal iPS-CMs, respectively. (B) Immunostaining of day-35 lactate-purified iPS-CMs stained with antibodies against MYBPC3 (green) and ACTN2 (red). Using CRISPRi knockdown, loss of MYBPC3 was observed in over 85% of analyzed cells in a polyclonal population. Nuclei were counterstained with DAPI. Scale bar = 100 µm. (C) Western blot of day-35 lactate-purified iPS-CMs with antibodies against MYBPC3, ACTN2, and GAPDH. Using CRISPRi, MYBPC3 protein was knocked down by ~90%. (D) GCaMP fluorescence in iPS-CMs containing gRNA against HERG and cultured in doxycycline (red). Recordings show a prolonged beat duration compared to untreated controls (green). (E) Quantified ratio of the downstroke-to-upstroke duration of doxycycline-treated iPS-CMs shows a significant difference in untreated iPS-CMs containing a gRNA against HERG, but not in iPS-CMs containing gRNA against OCT4 (negative control). (F) Patch-clamp recordings from single iPS-CMs show prolonged action potential durations in doxycycline-treated samples containing HERG gRNA.

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