Fig. 2: ATENA enables selective targeting of a G4 in the c-MYC promoter. | Nature Communications

Fig. 2: ATENA enables selective targeting of a G4 in the c-MYC promoter.

From: Chemically modified CRISPR-Cas9 enables targeting of individual G-quadruplex and i-motif structures, revealing ligand-dependent transcriptional perturbation

Fig. 2: ATENA enables selective targeting of a G4 in the c-MYC promoter.The alternative text for this image may have been generated using AI.

a (left) Schematic representation (created with BioRender, https://BioRender.com/s7u38i3) of the CAPA assay. (right) CAPA assay data on MCF7-dCas9-Halo cells treated with Cl-PyPDSn and followed by fluorophore incubation (Mean ± SD, n = 3). Data were analyzed with FlowJo software. b Schematic illustration of the c-MYC promoter with the annotated G4 (MYC-G4), sgRNA targeting region (black triangles), and their relative distance in bp from the MYC-G4. c RT-qPCR for c-MYC expression in MCF7-dCas9-Halo cells transfected with the indicated sgRNAs and incubated for 48 h in the presence of (2.5 µM) Cl-PyPDS2 or DMSO (mock). Mean ± SD, n = 3, biological replicates, each with three technical replicates. d RT-qPCR for c-MYC expression in MCF7 cells stably expressing dCas9-Halo transfected with sgRNAMYC+58 and incubated for 24 h in the presence of either Cl-PyPDS2 (2.5 µM) or Cl-OG (5 µM). Mean ± SD, n = 3, biological replicates, each with two technical replicates. e RT-qPCR for P1-driven c-MYC expression in MCF7 cells stably expressing dCas9-Halo transfected with the indicated sgRNAs and incubated for 48 h in the presence of (2.5 µM) Cl-PyPDS2. Mean ± SD, n = 3, biological replicates, each with three technical replicates. f BG4 CUT&Tag-qPCR for MCF7 cells stably expressing dCas9-Halo transfected with either sgRNAMYC-19 or sgRNA NTC and treated with DMSO (mock) or (2.5 µM) Cl-PyPDS2. BG4 accessibility was analyzed for c-MYC and normalized to three G4s in control gene sites (RPA3, MAZ, RBBP4). n = 2, biological replicates, each with three technical replicates for BG4 and one for the negative (no BG4 treatment). g RT-qPCR for P1-dependent c-MYC expression in MCF7 cells stably expressing dCas9-Halo transfected with sgRNAMYC-19 or sgRNA NTC and incubated for 48 h in the presence of (2.5 µM) Cl-PhenDC32. Mean ± SD, n = 3, biological replicates, each with three technical replicates. The expression values are represented as fold change (\({2}^{-\Delta \Delta {C}_{t}}\)) with respect to the mock (DMSO-treated) and normalized for the housekeeping gene GAPDH; data are the mean of n = number of independent biological samples. Statistical significance was calculated using a Welch-corrected two-tailed t test in GraphPad Prism; p-value: ns > 0.05, *≤0.05, **≤0.01, ***≤0.001, ****≤0.0001. Source data are provided as a Source Data file.

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