Extended Data Fig. 8: Screening dCas12a crRNAs for activating endogenous Oct4.
From: Multiplexed genome regulation in vivo with hyper-efficient Cas12a
a, Schematic of dCas12a crRNAs (red) targeting promoters of Oct4 and their relative positions to known dCas9 sgRNAs31 that are functional (black) or non-functional (grey) for activating Oct4. Arrows indicate sense or antisense binding of crRNAs/sgRNAs to the target DNA. The genomic position of the first ‘T’ in PAM (relative to TSS, which is ‘0’) are shown for each crRNA targeting to the Oct4 promoter. b, Immunostaining of Oct4. White scale bar, 100 µm. c, Inset shows merge with brightfield to demonstrate nuclear localization of mCherry (hyperdCas12a) and target (Oct4), since BFP on crRNA plasmid precludes the use of an additional nuclear dye. Yellow scale bar, 50 µm. d. Quantification of panel b, where 100-600 cells for each condition were quantitated over one screening experiment with multiple fields of view. The exact number of cells for each condition are listed in the Source Data for Extended Data Fig. 8. e. Immunostaining of Oct4 after activation by paired crRNA consisting of the two most potent crRNAs (O1 + O2), which shows lack of additive effect. White scale bar, 100 µm. f. Quantification in panels e, where 200-700 cells for each condition were quantitated over one screening experiment with multiple fields of view. For box-and-whisker plots, the box shows 25-75% (with bar at median, dot at mean), and whiskers encompass 10-90%, with individual data points shown for the lowest and highest 10% of each dataset. The exact number of cells for each condition is listed in the Source Data for Extended Data Fig. 8.
