Fig. 5: Evaluation of CRISPRa synthetic guide formats across 2 cell lines.

a Structure diagrams of a commercially available, chemically modified, 2-part synthetic gRNA containing a single MS2 aptamer loop (top-orange); a modified, 2-part Format 1 synthetic gRNA containing a GNE-3 scaffold (center-maroon); or a modified sgRNA with a GNE-3 scaffold (bottom-teal). Blue shaded boxes highlight the MS2 aptamer-containing GNE-3 tetraloop and gray shaded boxes indicate the MS2-apatmer on stemloop 2. b–e CRISPR-mediated transcriptional activation of a CD2 target gene in two CAG-CRISPRa-sel-engineered cell lines (K562 or 293T) by electroporated modified, synthetic gRNAs in the formats depicted in a. CD2 target activation by 3 spacer sequences in an engineered K562 cell line assessed by flow cytometry. CD2 expression displayed by representative histograms overlaid with a control population (b) or summarized by median fluorescence intensity normalized to a non-targeting control (c-upper) or percent positive (c-lower). Percent positive of a stained control population treated with a non-targeting sgRNA are indicated by a dashed horizontal line. d, e CD2 target activation by synthetic gRNA formats as in b-c but in a 293T cell line. Flow cytometry performed 3 days after synthetic guide delivery. Data are presented as mean values +/− SD. Statistical comparison between guide formats was performed by an unpaired one-way ANOVA adjusted for multiple comparisons. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. n = 6 electroporation replicates for K562, n = 5 transfection replicates for 293T. m = 2′-O methyl. * = phosphorothioate linker. Source data are provided as a Source Data file.