single ms2 aptamer loop  (Revvity)

Journal: Nature Communications

Article Title: A versatile, high-efficiency platform for CRISPR-based gene activation

doi: 10.1038/s41467-023-36452-w

Figure Lengend Snippet: a Structure diagram of the MS2-aptamer containing tetraloop in the 2.0 sgRNA format (left) or an optimized tetraloop structure (right and Supplementary Fig. ). The optimized GNE-3 tetraloop contains an additional stem extension and removal of a polyU tract . 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.

Article Snippet: Commercially available modified, synthetic 2-part guide RNAs containing a single MS2 aptamer loop purchased from Horizon Inc. ( https://horizondiscovery.com/ ).

Techniques: Activation Assay, Infection, Sequencing, Quantitative RT-PCR, Flow Cytometry

Journal: Nature Communications

Article Title: A versatile, high-efficiency platform for CRISPR-based gene activation

doi: 10.1038/s41467-023-36452-w

Figure Lengend Snippet: 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.

Article Snippet: Commercially available modified, synthetic 2-part guide RNAs containing a single MS2 aptamer loop purchased from Horizon Inc. ( https://horizondiscovery.com/ ).

Techniques: Modification, CRISPR, Activation Assay, Flow Cytometry, Expressing, Fluorescence, Staining, Electroporation, Transfection