Extended Data Fig. 5: Counter assays to validate BRD7586 in cells.
From: A general approach to identify cell-permeable and synthetic anti-CRISPR small molecules
(a, b) Immunoblotting analysis of SpCas9 expression in (a) HEK293T cells (data represent mean from 2 independent experiments) or (b) U2OS.eGFP.PEST cells (error bars represent mean ± SD from 3 independent experiments) transfected with SpCas9 plasmid and incubated with BRD7586 for 24 h. For BRD7586 at 20 µM compared to DMSO in U2OS.eGFP.PEST cells, p = 0.49 (unpaired t-test, two-tailed). (c) Changes in the fluorescence intensity from U2OS.eGFP.PEST cells in the presence of BRD7586. Cells were treated with the compound for 24 h, and the fluorescence intensity was measured to calculate the fraction of eGFP-positive populations. Means from 3 independent experiments are shown. Due to the low SD, error bars cannot be shown. For BRD7586 at 20 µM compared to DMSO, p = 0.14 (unpaired t-test, two-tailed). (d) Dose-dependent inhibition of SpCas9 or LbCas12a by BRD7586 in the eGFP disruption assay using RNP delivery methods (U2OS.eGFP.PEST cells, 24 h). Error bars represent mean ± SD from 3 independent experiments. For SpCas9, p = 8.3 × 10−5 with BRD7586 at 15 µM compared to DMSO. For LbCas12a, p = 0.023 with BRD7586 at 15 µM compared to DMSO (unpaired t-test, two-tailed).
