Extended Data Fig. 6: Screening dCas12a crRNAs for activating endogenous Sox2.

a, Schematic of dCas12a crRNAs (red) targeting promoter of Sox2, and their relative positions to validated dCas9 sgRNAs³¹ that are functional (black) for activating Sox2. Arrows indicate sense or antisense binding of crRNAs/sgRNAs to target DNA. The genomic position of the first ‘T’ in PAM (relative to TSS, which is ‘0’) are shown for each crRNA targeting the Sox2 promoter. b, Immunostaining of Sox2 expression from activation by WT dCas12a–miniVPR with various Sox2 single crRNAs, compared to activation by dCas9-miniVPR (using a validated sgRNA, S84)³¹. Scale bar, 100 µm. c-d, Immunostaining of Sox2 expression and co-localization with BFP and mCherry for a pair of crRNAs (c) and a panel of ‘triplet’ crRNAs (d), demonstrating additive or synergistic effect when multiple crRNAs are used in tandem. Interestingly, addition of a third crRNA targeting a region between the paired crRNAs S1 and S2 decreases the level of activation. Inset (c) shows brightfield image to demonstrate nuclear localization of mCherry (hyperdCas12a) and target (Sox2), since BFP on crRNA plasmid precludes the use of an additional nuclear dye. White scale bar, 100 µm; yellow scale bar (within inset), 50 µm e-f. Automated quantitation of images in panels b-d. In panel e, 350-2000 cells for each condition were quantitated for one screening experiment with multiple fields of view. The exact number of cells for each condition is listed in the Source Data for Extended Data Fig. 6. NT, non-targeting crRNA. In panel f, 70-250 cells for each condition were quantitated for 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 are listed in the Source Data for Extended Data Fig. 6. NT, non-targeting crRNA.

Source data