Fig. 4: Chemical modification of synthetic GNE-3 sgRNAs enhances target gene activation.

a Structural diagram of a full sgRNA with GNE-3 scaffold highlighting 5′/3′ ends (black box). Magnified view of sgRNA 5′/3′ end regions highlighting unmodified (orange) or modified (teal) nucleotides. Modified sgRNAs contain 2′-O-methyl (m)/ phosphorothioate (*) linker modifications. b, c Assessment of CRISPR mediated gene activation by unmodified or modified sgRNAs in a CAG-CRISPRa-sel engineered K562 population and assessed 3 days post-sgRNA delivery. b Gene expression displayed by representative flow cytometry histograms in populations electroporated with unmodified (top row, orange) or modified (bottom row, teal) GNE-3 sgRNAs. Stained cells electroporated with a non-targeting synthetic sgRNA overlaid in gray. c Median fluorescence intensity (MFI) of K562 populations stained with antibodies for the indicated genes (PD-L1, CD14, CD2, CXCR4) and normalized to a population of stained cells electroporated with a non-targeting sgRNA (top). Percentage of cells positive by antibody staining (bottom). Background staining of a cell population electroporated with a non-targeting control sgRNA indicated with a dashed horizontal line. n = 5 electroporation replicates per condition. Data are presented as mean values +/− SD. Statistical significance determined by an unpaired two-tailed t-test with a Welch’s correction. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. Source data are provided as a Source Data file.