Fig. 1: Circular guide RNAs (cgRNAs) with increased stability activate reporter genes in the CRISPR/Cas12f system. | Nature Communications

Fig. 1: Circular guide RNAs (cgRNAs) with increased stability activate reporter genes in the CRISPR/Cas12f system.

From: Engineered circular guide RNAs enhance miniature CRISPR/Cas12f-based gene activation and adenine base editing

Fig. 1

a Schematic representation of circular gRNA for CRISPR/Cas12f. b Reverse transcription PCR (RT-PCR) confirmation of RNA circularization in cells. c RT-PCR analysis revealed the abundance of circular RNAs in cells. HEK293T cells were transfected with indicated plasmids encoding normal, linear or circular RNAs, and 48 h post-transfection, RNA was harvested for RT-PCR analysis. d RT-PCR analysis of RNA stability. Cells transfected with gRNAs were treated with actinomycin D for 1, 3, 6, 9, and 18 h, starting 24 h post-transfection. e Fluorescence-activated Cell Sorting (FACS) analysis revealing mNeonGreen expression in reporter cells three days post-transfection with G3 or G5 gRNA plasmids. f Quantification of activation efficiency by the ratio of mNeonGreen-positive cells in FACS assays. For c–f n = 3 independent experiments, and data are presented as mean values ± SD. Normal, normal gRNAs driven by the polymerase III promoter U6; Circular, circular gRNA driven by the polymerase III promoter U6; Linear, linear gRNA having the same base residues as the circular gRNA; NC, non-targeting gRNA which transfected gRNA plasmids do not target any site in the human genome or transcriptome. **p < 0.01, ***p < 0.001, two-sided Student’s t-test. Source data are provided as a Source Data file.

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