(A) Scatterplot for the primary screening assay for the Pictet-Spengler library. Dots in yellow, blue, and green represent DMSO controls, small molecules, and unlabeled 12PAM DNA competitor, respectively.

(B) Chemical structures of BRD7087, BRD5799, and BRD3539.

(C) BLI binding plots for BRD3539 and SpCas9:gRNA complex. The BLI experiment was performed using 1 μM of BRD3539 on streptavidin sensors followed by association with different concentrations of the SpCas9:gRNA complex and subsequent dissociation. Response data were plotted along the y axis, and the concentration of SpCas9:gRNA complex was plotted along the x axis. A global 2:1 (small molecule:protein) model was used to plot the steady state and determine the binding constant. The two binding plots correspond to two biological replicates.

(D) Binding competition between BRD3539 and biotin in BLI. BLI sensogram showing the interaction of streptavidin sensors loaded either with BRD3539 or biotin and SpCas9:gRNA RNP complex. BRD3539 (1 μM) or biotin (10 μM) in a 20 mM Tris buffer (pH 7.4, 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 0.01% Tween) was loaded onto the streptavidin sensors. In the competition assay, streptavidin sensors were pre-loaded with 10 μM of biotin followed by loading 1 μM of BRD3539. The SpCas9:gRNA complex concentration was varied from 0.25‒1 μM. Competitive BLI experiment with BRD3539 in the presence of 10-fold excess biotin.

(E) Background-subtracted BLI responses of BRD3539 with SpCas9:gRNA RNP complex in the presence of 10-fold excess biotin as the competitor in a 20 mM Tris buffer (pH 7.4, 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT, 0.01% Tween). The SpCas9:gRNA complex concentration was varied from 0.25‒1 μM.

(F) Aggregation behavior of BRD7087. Aggregate formation for BRD7087 was determined using dynamic light scattering, between 40‒100 μM in PBS. BRD7087 forms aggregates beyond 80 μM. Aggregate size distribution plots are an average of 10 individual reads. BRD7087 did not show any detectable aggregation up to ∼60 μM.

(G) Determination of BRD7087 solubility in PBS. The solubility of BRD7087 in PBS was determined by mass spectroscopy after a 24 h incubation at room temperature. Antipyrine and clotrimazole are positive and negative controls, respectively.

(H) NMR binding of BRD7087 to the SpCas9:gRNA complex. 19F NMR titration plot for BRD7087 with the SpCas9:gRNA complex in 20 mM Tris buffer (pH 7.4, 100 mM KCl, 5 mM MgCl 2 , 1 mM DTT). BRD7087 (50 μM) was titrated against increasing concentrations of the SpCas9:gRNA (0.75‒1.75 μM) complex.

(I) Cell viability of U2OS.eGFP.PEST cells in the presence of the small molecules. Cell viability was determined by measuring the ATP content of U2OS.eGFP.PEST cells upon incubation with BRD7087 or BRD5779 (5‒20 μM) for 24 h. Error bars represent ± s.d. across technical replicates (n = 3).

(J) Cell viability of HEK293T cells in the presence of the small molecules. Cell viability was determined by measuring the ATP content of HEK293T cells upon incubation with BRD7087 or BRD5779 (5‒20 μM) for 24 h. Error bars represent ± s.d. across technical replicates (n = 3).

(K) Dose-dependent inhibition of SpCas9 by BRD5779 in U2OS.eGFP.PEST cells. Compound was tested in a concentration range of 5‒20 μM with a 1.2 fold dilution. U2OS.eGFP.PEST cells were nucleofected with SpCas9- and gRNA-expressing plasmids and were incubated with the indicated concentration of compound for 24 h before imaging. Error bars represent ± s.d. across technical replicates (n = 4). ∗p ≤ 0.0001 for the small molecule at 20 μM compared to DMSO. (unpaired t test, two-tailed)

(L) Representative images of the eGFP-disruption assay. U2OS.eGFP.PEST cells were nucleofected with either SpCas9 alone or SpCas9- and eGFP-targeting gRNA plasmids and were treated with either the vehicle alone or the small molecule. Left panel represents cells nucleofected with SpCas9 alone. Middle panel represents cells nucleofected with SpCas9- and eGFP-targeting gRNA-expressing plasmids and treated with vehicle. Right panel represents cells nucleofected with SpCas9- and eGFP-targeting gRNA and treated with 15 μM of BRD7087 for 24 h. Scale bar = 100 μm.

(M) Effect of inhibitors on eGFP protein expression in U2OS.eGFP.PEST cells. The western blot analysis of eGFP protein expression in U2OS.eGFP.PEST cells was performed in the presence of DMSO and the inhibitors BRD5779 or BRD7087. Cells were incubated with either BRD5779 or BRD7087 at the indicated concentrations for 24 h before they were harvested and processed for western blotting. No change was observed in eGFP expression levels in the presence or absence of the compounds.

(N) Auto-fluorescence of U2OS.eGFP.PEST cells treated with the inhibitors in the eGFP-disruption assay. Cells were imaged in the RFP channel with the same exposure time as that used for the GFP channel in the eGFP-disruption assay. Small-molecule-treated cells showed a maximum of ∼1% auto-fluorescing population, indicating no significant contribution of auto-fluorescence. Error bars represent ± s.d. across technical replicates (n = 4).

(O) Dose-dependent inhibition of SpCas9 by the inhibitors in the mKate2-disruption assay. HEK293T cells were transfected with a single plasmid encoding SpCas9, gRNA, and mKate2 (T1gRNA). Cells transfected with a plasmid encoding SpCas9, mKate2, and a non-targeting gRNA (CgRNA) were used as the positive control. Cells transfected with T1gRNA were incubated either in the presence of DMSO or the inhibitors (1.5‒5 μM) for 24 h. Error bars represent ± s.d. across technical replicates (n = 3).

(P) Representative images of the mKate2-disruption assay. Representative images of untreated HEK293T cells and cells transfected with T1gRNA or CgRNA. The nuclei were counterstained with DAPI, and the expression level of mKate2 was measured using the red channel. Top panels represent untreated cells or cells transfected with the indicated plasmid and incubated with DMSO. Bottom panels represent cells transfected with T1gRNA and incubated with BRD7087 at the indicated concentrations. Scale bar = 100 μm.

(Q) Dose-dependent inhibition of SpCas9-mediated NHEJ. HEK293T cells were transfected with a plasmid encoding SpCas9, gRNA, and another plasmid encoding the reporter mCherry-Stop Codon (TAG)-GFP. Transfected cells were incubated with either DMSO or the small molecules (2‒10 μM) for 24 h. Error bars represent ± s.d. across technical replicates (n = 3).

(R) Dose-dependent inhibition of dSpCas9-based transcriptional activation of the HBG1 gene in HEK293FT cells. Cells were transfected with dSpCas9, MS2.p65.HSF1.GFP plasmids, and either the RFP or HBG1 gRNA plasmid and were incubated in the presence of the small molecules at the indicated concentration for 48 h before RT-qPCR analysis. Error bars represent ± s.e.m. for technical replicates (n = 6).

(S) Dose-dependent inhibition of the SpCas9(A840H)-cytidine deaminase conjugate (BE3) targeting the EMX1 gene in HEK293T cells. Small molecules preincubated with BE3:gRNA ribonucleoprotein were delivered to HEK293T cells and incubated in the presence of either DMSO or small molecules at the indicated concentration for 72 h. The cells were then harvested and processed for DNA sequencing to evaluate the extent of C5→T5 or C6→T6 conversion. Error bars represent ± s.d. across biological replicates (n = 3).