Journal: Genome Biology

Article Title: Pamoic acid and carbenoxolone specifically inhibit CRISPR/Cas9 in bacteria, mammalian cells, and mice in a DNA topology-specific manner

doi: 10.1186/s13059-025-03521-w

Figure Lengend Snippet: The effect of inhibitors on suppressing the editing of exogenous GFP gene, endogenous COSMC or EMX1 loci by Spy Cas9, Sau Cas9 or BE4 in HEK293FT cells. A The schematic diagram of cellular assays for analyzing the effects of inhibitors on the activity of Spy Cas9, Sau Cas9 or BE4 for the cleavage of GFP , COSMC or EMX1 gene in HEK293FT cells. The cells were transfected with Lipofectamine™ 3000 Transfection Reagent (Thermal Fisher, L3000008) in the presence of plasmids containing Spy Cas9, Sau Cas9 or their sgRNAs before an incubation with the compound for 24 h. After the colony selection with puromycin, genomic DNA of the cells was then extracted, and the target gene was amplified with PCR (see the “ ” section). The editing efficiency of Cas9 was analyzed with flow cytometry, TIDE, EditR or T7EI method [ – ]. B Flow cytometry analysis of the effect of compounds on the disruption of GFP by Spy Cas9 in HEK293FT cells. The cells stably expressing of GFP were transiently transfected with the PX459- Spy Cas9 (Addgene #62988) and pGL3 plasmids coding anti- GFP sgRNA (the blue line) or control sgRNA (the green line) before an incubation with the compound for 24 h ( A ). The GFP fluorescent from the treated cells was analyzed with LSR Fortessa flow cytometer (BD Biosciences, Franklin Lakes, NJ), and the gating strategy and corresponding histograms are shown in the Additional file 1:Fig.S9A-B. The quantitative data was shown as the percentage of the group treated with control sgRNA (100%). Means ± SDs ( n = 3, biological replicates). C The effect of compounds on the editing of endogenous COSMC or EMX1 by Spy Cas9 in HEK293FT wt cells. The cells were transfected with PX459- Spy Cas9 plasmid coding anti- COSMC , anti- EMX1 sgRNA (for the spacer sequences, see Additional file 1: Table S4) or control sgRNA (the Spy Cas9 only group, Additional file 1:Fig.S9D),before the treatment with the indicated compounds for 24 h ( A ). The target DNA was accordingly amplified with PCR before subjected to Sanger sequencing (Additional file 1:Fig.S9E-Gand Additional file 1:Fig.S10A-C). The editing efficiency of Cas9 was analyzed with the TIDE method (https://tide.nki.nl/), normalized with the DMSO (for pamoic acid; final concentrations, 0.4%) or ddH 2 O (for dalbavancin or carbenoxolone) -treated groups (100%) and shown. AcrIIA4 was used as a positive control (Additional file 1:Fig.S10D). Means ± SDs ( n = 3, biological replicates). D T7EI mismatch detection assay analysis of the inhibitory effect of compounds on editing of COSMC by Spy Cas9 in HEK293FT cells. The amplified PCR products (541 bp; input) from the extracted genomic DNA of the control (the left four lanes of each gel) or edited HEK293FT cells ( C ), which have been treated with DMSO or ddH 2 O (the middle four lanes), 100 μM pamoic acid, 50 μM dalbavancin or 50 μM carbenoxolone (the right four lanes), were incubated with T7EI or without the treatment at 37 °C for 15 min before an analysis the COSMC site with 2% agarose gels (see the “ ” section). The percentage of Indel was calculated by the formula dividing the band density of fragments to that of total input and normalized with the background value . E The effect of compounds on the base editing of EMX1 by BE4 base editor in HEK293FT cells. The cells were co-transfected with the BE4 and pGL3 plasmids, the latter coding an anti- EMX1 sgRNA that is the same as the one used for Spy Cas9 (Additional file 1: Table S4), and treated with the compounds for 24 h ( A ). The amplified PCR products from the EMX1 site were accordingly sequenced (Additional file 1:Fig.S11E-F), and the converting rates of C 5 or C 6 base in the N20 sequence were analyzed with EditR ( https://moriaritylab.shinyapps.io/editr_v10/;upperpanels ) [ , ]. The converting rate at the C 5 and C 6 position were normalized with the respective control (100%) and the base editing efficiency was shown as percentages for both the C 5 and C 6 sites (lower panels). Means ± SDs ( n = 3, biological replicates). F The effect of compounds on the editing of COSMC by Sau Cas9 in HEK293FT cells. The cells were co-transfected with pX601- Sau Cas9 (Addgene #107055) and pGL3 plasmids containing an anti- COSMC sgRNA (Additional file 1: Table S2 and S4), before the treatment with the compounds for 24 h ( A ). The editing efficiency of Cas9 was analyzed with the TIDE method ( https://tide.nki.nl/ ) and presented as percentages of control (100%). The raw sequencing results were shown in the Additional file 1:Fig.S12A-C. Means ± SDs ( n = 3, biological replicates). G The comparison for the effects of pamoic acid or carbenoxolone on the Spy Cas9-mediated disruption of GFP plasmids with a 5’-NGG or 5’-NAG PAM. The HEK293FT wt cells were transiently co-transfected with the PX459- Spy Cas9 and pCDH- GFP plasmids, which contains a 5’-NGG PAM or an in situ mutated 5’-NAG PAM (see the “ ” section) and were treated with the compounds 6 h post the transfection and for 24 h. The GFP fluorescent from the cells was accordingly quantified with flow cytometer and the data was shown as presentages of the control (100%; for the raw histograms, see Additional file 1:Fig.S12E; see the “ ” section). Means ± SDs ( n = 6, biological replicates). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test ( B , C , E or F ) or two-way ANOVA with Bonferroni posttests ( G ). * p < 0.05, ** p < 0.01, *** p < 0.001. All experiments were independently repeated at least twice, and representative results are presented

Article Snippet: To assess the inhibitory effect of AcrIIA4, a known protein inhibitor of Spy Cas9, HEK293FT cells were co-transfected with the PX459- Spy Cas9 plasmid containing sg COSMC , sg EMX1 , or sg Control with the CMV-NLS-AcrIIA4 plasmid (Addgene, #113,037) using LipofectamineTM 3000 before standard treatment and analysis as described above.

Techniques: Activity Assay, Transfection, Incubation, Selection, Amplification, Flow Cytometry, Disruption, Stable Transfection, Expressing, Control, Plasmid Preparation, Sequencing, Positive Control, Detection Assay, Comparison, In Situ

Journal: Cell Reports Medicine

Article Title: Targeting pancreatic cancer glutamine dependency confers vulnerability to GPX4-dependent ferroptosis

doi: 10.1016/j.xcrm.2025.101928

Figure Lengend Snippet:

Article Snippet: Rabbit anti-WDR5 , Proteintech , Cat# 15544-1-AP; RRID: AB_2257220.

Techniques: Virus, CRISPR, Recombinant, Protease Inhibitor, Lysis, Ligation, Reverse Transcription, SYBR Green Assay, Glo Assay, Sample Prep, DNA Extraction, Enzyme-linked Immunosorbent Assay, Multiple Displacement Amplification, Flow Cytometry, Software