ptarget plasmids  (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: Nucleic Acids Research

Article Title: Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons

doi: 10.1093/nar/gkaf149

Figure Lengend Snippet: PmcCAST TnsC forms a heptameric complex in the presence of ATP and DNA. ( A ) Schematic representation of the type I-B CAST system from Peltigera membranacea cyanobiont 210A . LE and RE denote left and right transposon ends. ( B ) Size-exclusion chromatography profiles of TnsC alone, in the presence of ATP, double-stranded (ds)DNA, or both ATP and dsDNA. ( C ) Representative negative-stain electron micrographs of TnsC in the presence or absence of dsDNA and/or ATP. Scale bar corresponds to 100 nm. ( D ) Single-particle cryo-EM reconstruction of the dsDNA- and AMPPNP-bound TnsC heptamer (from left to right: top, side and bottom views). ( E ) Structural model of the dsDNA- and AMPPNP-bound TnsC heptamer (from left to right: top, side and bottom views). Individual TnsC protomers are labeled with distinct superscript letters to differentiate each subunit. AMPPNP molecules are shown in space-fill representation.

Article Snippet: To remove the tRNA gene from the pTarget vector, a pTarget_ΔtRNA vector was created by Gibson assembly between two fragments amplified from the template pTarget (PmcCAST) (Addgene #168163) using the primers pTarget_ΔtRNA_1_f with pTarget_ΔtRNA_1_r, and pTarget_ΔtRNA_2_f with pTarget_ΔtRNA_2_r ( ).

Techniques: Size-exclusion Chromatography, Staining, Single Particle, Cryo-EM Sample Prep, Labeling

Journal: Nucleic Acids Research

Article Title: Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons

doi: 10.1093/nar/gkaf149

Figure Lengend Snippet: TnsC oligomerization, DNA binding and ATP hydrolysis are required for transposition. ( A ) Inter-protomer interfaces within the TnsC heptamer. Regions depicted in detail in subsequent panels are indicated with dashed boxes. ( B ) Detailed view of the ATPase catalytic pocket. Bound AMPPNP and interacting amino acid residues are shown in stick format. Mg 2+ ion is depicted as a green sphere. ( C ) RNA-guided transposition activity in E. coli of PmcCAST systems containing mutations in the catalytic pocket of TnsC, as quantified by ddPCR. Data are presented as mean ± s.d. of three biologically independent replicates ( n = 3), each measured in technical duplicates. Bars labeled as n.d. (“non-detectable”) indicate values below the detection limit of the assay. The positive control (“CTRL+”) corresponds to a ddPCR reaction that uses as DNA template an artificial pTarget plasmid with the transposon DNA from the pDonor plasmid inserted at the target site in the left end–right end (LE–RE) orientation. ( D – F ) Detailed views of the TnsC inter-protomer interfaces. ( G ) RNA-guided transposition efficiency in E. coli of PmcCAST systems containing mutations in the inter-protomer interfaces, quantified by ddPCR as in panel (C). ( H ) Detailed view of the DNA-binding interfaces. Only residues within 3.5 Å of the DNA are displayed. ( I ) RNA-guided transposition efficiency in E. coli of PmcCAST systems containing mutations in the DNA-binding interfaces, quantified by ddPCR as in panel (C).

Article Snippet: To remove the tRNA gene from the pTarget vector, a pTarget_ΔtRNA vector was created by Gibson assembly between two fragments amplified from the template pTarget (PmcCAST) (Addgene #168163) using the primers pTarget_ΔtRNA_1_f with pTarget_ΔtRNA_1_r, and pTarget_ΔtRNA_2_f with pTarget_ΔtRNA_2_r ( ).

Techniques: Binding Assay, Activity Assay, Labeling, Positive Control, Plasmid Preparation

Journal: Nucleic Acids Research

Article Title: Structural basis of TnsC oligomerization and transposase recruitment in type I-B CRISPR-associated transposons

doi: 10.1093/nar/gkaf149

Figure Lengend Snippet: The C-terminal hook in TnsAB interacts with heptameric TnsC. ( A ) Schematic diagram of the domain organization of type I-B PmcCAST TnsAB. ( B ) Coprecipitation of TnsC by amylose-immobilized TnsAB (full-length or C-terminal domain), fused to MBP) in the presence of different nucleotides (ADP, AMPPNP, ATP; bottom). I: 10% input; E: elution; C: TnsC-only control; C + AB: TnsC and MBP-TnsAB full-length sample; C + AB CTD : TnsC and MBP-TnsAB C-terminal domain sample. ( C ) Single-particle cryo-EM reconstruction of the heptameric TnsC–DNA–AMPPNP complex bound to six copies of the TnsAB hook (top and side views). Individual TnsC subunits are indicated with different colours. The TnsAB hooks densities are depicted in bright yellow. ( D ) Structural model of the TnsC–DNA–AMPPNP complex bound to six copies of the TnsAB hook (top and side views). Individual TnsC protomers are labeled with distinct superscript letters to differentiate each subunit. The TnsAB hooks are depicted in space-fill representation (bright yellow). ( E ) Detailed view of the TnsAB hook and the TnsC interface. ( F ) RNA-guided transposition activity in E. coli of PmcCAST systems containing mutations in the TnsAB hook and TnsC interface, as quantified by ddPCR. Data are presented as mean ± s.d. of three biologically independent replicates ( n = 3), each measured in technical duplicates. Bars labeled n.d. (“non-detectable”) indicate values below the detection limit of the assay. The positive control (“CTRL+”) corresponds to a ddPCR reaction that uses as DNA template an artificial pTarget plasmid with the transposon DNA from the pDonor plasmid inserted at the target site in the left end-right end (LE–RE) orientation.

Article Snippet: To remove the tRNA gene from the pTarget vector, a pTarget_ΔtRNA vector was created by Gibson assembly between two fragments amplified from the template pTarget (PmcCAST) (Addgene #168163) using the primers pTarget_ΔtRNA_1_f with pTarget_ΔtRNA_1_r, and pTarget_ΔtRNA_2_f with pTarget_ΔtRNA_2_r ( ).

Techniques: Control, Single Particle, Cryo-EM Sample Prep, Labeling, Activity Assay, Positive Control, Plasmid Preparation