ptarget-sg ssea  (Addgene inc)

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 genome editing on SSEA locus by Spy Cas9 or Sau Cas9 in bacteria. A The schematic diagram of two-plasmid-based bacterial survival assay for detecting the activity of Spy Cas9 in bacteria. pCas plasmid (Addgene #62225,Additional file 1: Table S2) was transformed into the E. coli MG1655 before an electroporation with pTarget plasmids coding for sgRNA targeting SSEA (pT-sg SSEA , Additional file 1: Table S4) or empty vector (pT-sg Control ; pTargetF, Addgene #62226). Then, the effect of compounds was evaluated by counting the number of survival colonies (see the “ ” section). B The effect of Cas9 inhibitors on the activity of Spy Cas9 in the two-plasmid-based bacterial survival assay. Compounds were incubated with the electroporated bacteria ( A ) for 1.5 h at 32 °C before spreading on LB plates with kanamycin and spectinomycin antibiotics. After an overnight incubation, the image of cultured plate was taken (Additional file 1:Fig.S7D), and the number of colonies (indicated below the plate image) was quantified with a Colony Counter software (Tanon, China), and expressed as the percentages of their respective control at the same concentration (the pT-sg Control electroporated strain treated with the compound, 100%; Additional file 1:Fig.S7D). Means ± SDs ( n = 3, biological replicates). Statistical analyses were performed using two-way ANOVA with Bonferroni posttests; *p < 0.05; **p < 0.01; *** p < 0.001. C The schematic diagram of the genome editing assay for monitoring the activity of Spy Cas9 or Sau Cas9 in the presence of homologous repair template DNA and in bacteria. pCas plasmid was first transformed into MG1655 strain before an electroporation to pTarget plasmids carrying a pair of homologous repair template DNA that is missing the 1-243 bp of SSEA gene (see the “ ” section) and a coding sequence for Spy Cas9 or Sau Cas9 sgRNA (pT-sg SSEA (867 bp)). Then, the E. coli cells were incubated with the compounds before genotyping with PCR for analyzing the efficiency of genome editing at the SSEA . 909 bp, the size of PCR product from a strain, in which 1-243 bp of SSEA is missing; 1152 bp, the size of PCR product from a strain with wt SSEA gene. D The effect of Cas9 inhibitors on the genome editing activity of Spy Cas9 (left panel) or Sau Cas9 (right panel) in the presence of a homologous recombination repair template. Compounds were incubated with the electroporated bacteria ( C ) for 20 h at 32 °C, and the SSEA in the treated bacteria was amplified with PCR and analyzed on a 1% agarose. The original images for PCR-based genome typing were shown in the Additional file 1:Fig.S8B. Means ± SDs ( n = 3, biological replicates). The ddH 2 O (for dalbavancin or carbenoxolone) or DMSO (pamoic acid or epirubicin) treated groups, 100%. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test; *** p < 0.001. All experiments were independently repeated at least twice, and representative results are presented

Article Snippet: Next, 300 ng of pTargetF (Addgene, #62,226) or pTarget-sg SSEA (Additional file 1: Table S2) was electroporated into the pCas competent cells in a 0.1-cm Gene Pulser®/MicroPulserTM Electroporation Cuvette (Bio-Rad, #1,652,089) with Gene Pulser XcellTM Electroporation System (Bio-Rad, #165–2660) under conditions of 1800 V and 5.2 ms.

Techniques: Bacteria, Plasmid Preparation, Clonogenic Cell Survival Assay, Activity Assay, Transformation Assay, Electroporation, Control, Incubation, Cell Culture, Software, Concentration Assay, Sequencing, Homologous Recombination, Amplification

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 genome editing on SSEA locus by Spy Cas9 or Sau Cas9 in bacteria. A The schematic diagram of two-plasmid-based bacterial survival assay for detecting the activity of Spy Cas9 in bacteria. pCas plasmid (Addgene #62225,Additional file 1: Table S2) was transformed into the E. coli MG1655 before an electroporation with pTarget plasmids coding for sgRNA targeting SSEA (pT-sg SSEA , Additional file 1: Table S4) or empty vector (pT-sg Control ; pTargetF, Addgene #62226). Then, the effect of compounds was evaluated by counting the number of survival colonies (see the “ ” section). B The effect of Cas9 inhibitors on the activity of Spy Cas9 in the two-plasmid-based bacterial survival assay. Compounds were incubated with the electroporated bacteria ( A ) for 1.5 h at 32 °C before spreading on LB plates with kanamycin and spectinomycin antibiotics. After an overnight incubation, the image of cultured plate was taken (Additional file 1:Fig.S7D), and the number of colonies (indicated below the plate image) was quantified with a Colony Counter software (Tanon, China), and expressed as the percentages of their respective control at the same concentration (the pT-sg Control electroporated strain treated with the compound, 100%; Additional file 1:Fig.S7D). Means ± SDs ( n = 3, biological replicates). Statistical analyses were performed using two-way ANOVA with Bonferroni posttests; *p < 0.05; **p < 0.01; *** p < 0.001. C The schematic diagram of the genome editing assay for monitoring the activity of Spy Cas9 or Sau Cas9 in the presence of homologous repair template DNA and in bacteria. pCas plasmid was first transformed into MG1655 strain before an electroporation to pTarget plasmids carrying a pair of homologous repair template DNA that is missing the 1-243 bp of SSEA gene (see the “ ” section) and a coding sequence for Spy Cas9 or Sau Cas9 sgRNA (pT-sg SSEA (867 bp)). Then, the E. coli cells were incubated with the compounds before genotyping with PCR for analyzing the efficiency of genome editing at the SSEA . 909 bp, the size of PCR product from a strain, in which 1-243 bp of SSEA is missing; 1152 bp, the size of PCR product from a strain with wt SSEA gene. D The effect of Cas9 inhibitors on the genome editing activity of Spy Cas9 (left panel) or Sau Cas9 (right panel) in the presence of a homologous recombination repair template. Compounds were incubated with the electroporated bacteria ( C ) for 20 h at 32 °C, and the SSEA in the treated bacteria was amplified with PCR and analyzed on a 1% agarose. The original images for PCR-based genome typing were shown in the Additional file 1:Fig.S8B. Means ± SDs ( n = 3, biological replicates). The ddH 2 O (for dalbavancin or carbenoxolone) or DMSO (pamoic acid or epirubicin) treated groups, 100%. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test; *** p < 0.001. All experiments were independently repeated at least twice, and representative results are presented

Article Snippet: Similarly, pTarget-sg SSEA (867 bp)- Sau was constructed by cloning a pair of annealed oligonucleotides encoding a 21-bp spacer of sg SSEA (No. 37–38, Additional file 1: Table S3-4) at the SapI site in a plasmid, which was assembled with the PCR products from pX601 miniCMV-SaCas9 (Addgene, #107,055, Additional file 1: Table S2; a gift from Dr. Alex Hewitt, School of Medicine, University of Tasmania) and pTargetF (867 bp) by using Seamless Assembly (No. 39–42, Additional file 1: Table S3).

Techniques: Bacteria, Plasmid Preparation, Clonogenic Cell Survival Assay, Activity Assay, Transformation Assay, Electroporation, Control, Incubation, Cell Culture, Software, Concentration Assay, Sequencing, Homologous Recombination, Amplification