Fig. 2: Relative CRISPR activation efficiency of sgRNAs with an optimized MS2 aptamer-containing scaffold.

a Structure diagram of the MS2-aptamer containing tetraloop in the 2.0 sgRNA format¹³ (left) or an optimized tetraloop structure (right and Supplementary Fig. 3). The optimized GNE-3 tetraloop contains an additional stem extension and removal of a polyU tract²¹. In addition, the bulge region connecting the MS2 aptamer and stem extension region 1 in the 2.0 format has been removed. b Flow cytometric analysis of target gene activation by sgRNAs with either a 2.0 (orange) or GNE-3 (teal) scaffold context. Representative histograms of analyzed K562 CRISPRa-sel populations infected with five distinct spacer sequences targeting the promoter proximal region of PD-L1 (top) or CD2 (bottom). Populations infected with a non-targeting sgRNA sequence overlaid (gray). c Activation of 6 target genes by GNE-3 sgRNAs normalized to the activation efficiency of the same spacer sequence in a 2.0 format (dashed line). Normalized gene activation was evaluated in Zeocin selected populations by qRT-PCR (left) at day 14 post-sgRNA infection or by flow cytometry (right) at day 10 post-sgRNA infection. Data are presented as mean values +/− SD. n = 4 independent biological replicates per sgRNA. # indicates an inactive spacer where neither 2.0 nor GNE-3 sgRNAs activated the target gene (cutoff <1.5-fold over control). Source data are provided as a Source Data file.