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 primary inhibitor screening and identification of FDA-approved drugs to inhibit the endonuclease activity of Cas9 with a purified enzyme-based activity assay. A The primary inhibitor screening for suppressing the endonuclease activity of SpyCas9 and the schematic diagram for the in vitro enzyme assay, bacteria, mammalian cells, or animal -based studies of Cas9 inhibitors. After a drug screening of 4607 compounds at 200 μM in the activity assay of Cas9, 31 compounds were found to dose-dependently inhibit the activity of Cas9 with IC 50 values less than 200 μM. Five reprensentative leads were selected for the efficacy and selectivity studies on purified enzyme, bacteria, mammalian cell or animal levels. B The effect of five leads as well as AcrIIA4 on the cleavage activity of Spy Cas9 to the circular plasmid. Pamoic acid, epirubicin, dalbavancin, carbenoxolone or docusate at the indicated concentrations was incubated with a complex of Spy Cas9 (375 nM) and sgRNA (400 nM) and tested under the standard assay conditions (Additional file 1:Fig.S1B-C;see the “Methods” section). The pcDNA3.1/CT- GFP plasmid (300 ng) containing a protospacer sequence (N20: 5’-CCAATTCTTGTTGAATTAGA-3’) and a 5’-TGG PAM (Additional file 1: Table S4) was used in the assay. AcrIIA4, a known anti-CRISPR protein inhibitor of Cas9 , was also tested under the standard assay conditions with an exception of using 22 °C for the assay instead of 37 °C, which was reported for AcrIIA4 to show a better inhibition to Spy Cas9 . In TBE buffer, the linear form of pcDNA3.1/CT- GFP plasmid (~ 7200 bp) migrates faster than the covalently closed circular DNA (cccDNA) form of the plasmid, which was reported previously and also validated by us (Additional file 1:Fig.S1B).Means ± SDs ( n = 3, biological replicates). C The effect of five leads as well as AcrIIA4 on the cleavage activity of SpyCas9 to the linear oligonucleotide substrate. Pamoic acid, epirubicin, dalbavancin, carbenoxolone, or docusate as well as AcrIIA4 was tested under the assay conditions described above ( B ) using a FAM-labeled 87 bp oligonucleotide as the substrate (10 nM; linear substrate, thereafter), which bears the same sequence of protospacer and PAM to the pcDNA3.1/CT- GFP plasmid ( B ). Eighty seven base pairs, the 5′-FAM-labeled substrate. Thirty base pairs, the FAM-labeled cleaved 5′-terminal fragment. Means ± SDs ( n = 3, biological replicates). D , E The effect of five leads on the enzymatic activity of SauCas9. Pamoic, epirubicin, dalbavancin, carbenoxolone, or docusate at the indicated concentrations were incubated with the complex of SauCas9 (100 nM) and sgRNA (100 nM) before the addition of the pcDNA3.1/CT- GFP plasmid (300 ng, D ) or a FAM-labeled 87 bp oligonucleotide (10 nM, E ). The two forms of DNA substrate of this assay share the same sequence of protospacer (N21, 5′-CTGGAGTTGTCCCAATTCTTG-3′) and 5′-TTGAAT PAM (Additional file 1: Table S4). The original images for circular plasmid and linear oligonucleotide are shown in the Additional file 1: Fig. S3F and S3G, respectively. Means ± SDs ( n = 3, biological replicates). DNA only, pcDNA3.1/CT- GFP plasmid ( B ), or 87 bp linear DNA ( C ) in the presence of assay buffer (see the “ ” section). The optical density of the DNA band for the linear form (agarose gels, B ) or for the 30 bp product (polyacrylamide gels, C ), as well as the area containing the linear, open circular, and supercoil bands of the plasmid ( B ) or the area containing the 87 bp and 30 bp products ( C ) was quantified using ImageJ software (NIH, Bethesda, MD). The cleavage activity of Cas9 was calculated by dividing the band density of the linear form ( B ) or 30 bp product ( C ) by the density of total area in the same lane (100%) and is shown as a percentage below the gel. The cleavage activity was normalized to that of the DMSO group (100%), as shown in the right panel. Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05; ** p < 0.01; *** p < 0.001. The experiments were independently repeated three times, and representative results are presented

Article Snippet: HEK293FT wt cells or HEK293FT cells stably expressing GFP were seeded at a volume of 1 ml with ~ 2 × 10 5 cells per well in 12-well plates for 1 day, and 1 μg of PX459- Spy Cas9 (Addgene, #62,988) together with 1 μg of pGL3-sg GFP or empty vector (Additional file 1: Table S2) were then transiently transfected into the cells using LipofectamineTM 3000 transfection reagent according to the manufacturer’s protocol (Thermal Fisher, L3000008).

Techniques: Activity Assay, Purification, Enzyme Activity Assay, In Vitro, Enzymatic Assay, Bacteria, Drug discovery, Plasmid Preparation, Incubation, Sequencing, CRISPR, Inhibition, Labeling, Software

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 structure and inhibitory effect of representative Cas9 inhibitors

Article Snippet: HEK293FT wt cells or HEK293FT cells stably expressing GFP were seeded at a volume of 1 ml with ~ 2 × 10 5 cells per well in 12-well plates for 1 day, and 1 μg of PX459- Spy Cas9 (Addgene, #62,988) together with 1 μg of pGL3-sg GFP or empty vector (Additional file 1: Table S2) were then transiently transfected into the cells using LipofectamineTM 3000 transfection reagent according to the manufacturer’s protocol (Thermal Fisher, L3000008).

Techniques: In Vitro, Purification, Enzymatic Assay, Bacteria, Cell Based Assay

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 biochemical mechanisms of five representative leads on inhibiting Spy Cas9. A Surface plasmon resonance assay analysis of the binding of five representative leads to Spy Cas9. Spy Cas9 at a concentration of 100 μg/mL was immobilized onto the surface of the CM5 sensor chip and then employed to analyse the binding of pamoic acid, dalbavancin or carbenoxolone as well as epirubicin and docusate (Additional file 1:Fig. S4D). The equilibrium affinity constant (K D ) values were calculated with BIAcore evaluation software (version 3.1). B The pre-incubation of pamoic acid or carbenoxolone, but not dalbavancin, epirubicin or docusate, with Spy Cas9 ribonucleoprotein enhances their inhibitory effects on the activity of Cas9. Pamoic acid or carbenoxolone at the indicated concentrations were pre-incubated with Cas9: sgRNA (375 nM: 400 nM) for 15 min before adding the GFP plasmid (condition a of left panel), or exposed to the enzyme together with the plasmid without a pre-incubation (condition b ). Then, the samples were tested under the standard assay conditions. Means ± SDs ( n = 3, technical replicates). The results for other inhibitors were slowed in the Additional file 1: Fig. S4E. C-D Pamoic acid or carbenoxolone, but not dalbavancin, docusate or epirubicin, specifically prevents the binding of DNA substrate to Spy Cas9 in DNA EMSA assays. dCas9 in the presence of sgRNA ( C or right panelof D ) or in the absence (left panelof D ) was incubated with the 5’-FAM labeled linear DNA substrate or plasmid and the inhibitor under the indicated sampling order, before a separation on a 6% native polyacrylamide gel (upper panel of C for the 87 bp oligonucleotide; see the “ ” section) or a 1% native agarose gel (lower panel of C for the plasmid). The polyacrylamide gels were stained with 1 × SYBR™ Gold (the right panel of C and D ) or were imaged for fluroresence of FAM under an excitation of 488 nm on a ChemiDoc scanner (left and upper panels of C ; left panel of D ), while the agarose gel was dying with EB and illuminated under UV (lower panels of C ). The lower and right panel of C , the plasmid DNA (the lower and left panel) treated with the indicated compounds in the absence of dCas9 and sgRNA. E Dalbavancin binds to free sgRNA or dCas9: sgRNA complex in a sgRNA EMSA assay. The free sgRNA (left panel) or sgRNA captured with dCas9 (right panel) was incubated with the inhibitor before a staining with 1 × SYBR™ Gold. F Epirubicin as well as doxorubicin or cisplatin directly binds to free DNA substrates. The plasmid (300 ng, equal to 5 nM) or oligonucleotide (10 nM) was pre-incubated with the compounds before adding of 5 × loading buffer containing 1.2% SDS. The samples were heated for 5 min at 95 °C and separated on respective gels (see above). G Oligonucleotide containing 8 × PAM rescues the destabilization of pamoic acid or carbenoxolone on Cas9 ribonucleoprotein in a thermal shift assay. The complex of Cas9 and sgRNA at 1 μM was incubated with 200 μM pamoic acid, 100 μM carbenoxolone or 50 μM dalbavancin for 15 min in the presence of the oligonucleotide containing of 8 × PAM at a molar ratio of 0.5:1, 1:1, 2:1 or 5:1 to Cas9 (see the “ ” section). The melting curves of Cas9 were showed in the Additional file 1:Fig.S5D and the unfolding transition midpoint temperature (Tm) of Cas9 was calculated by the GraphPad software (version 8.4, San Diego, CA). Means ± SDs ( n = 3, biological replicates). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test; ns, no significance; ** p < 0.01; *** p < 0.001. H The dose-dependent inhibitory effect of compounds on the cleavage of target strand ssDNA by Cas9. The activity assay was performed using 5’-FAM labeled 87 bp single stand oligonucleotide that is reverse and complement to the anti- GFP sgRNA and without the sequence of PAM (Additional file 1: Table S3-4), instead of the annealed double strand oligonucleotide. The original image was shown in the Additional file 1:Fig.S5F and the normalized data are shown here. Means ± SDs ( n = 3, technical replicates). DNA only, pcDNA3.1/CT- GFP plasmid ( B , lower panel of C and upper panel of F ), or 87 bp linear DNA (upper panel of C, D and lower panel of F ) in the presence of assay buffer. The cleavage activity of Cas9 was determined and is shown as a percentage of the DMSO group (100%; right panel of B or H ). All experiments were independently repeated at least twice, and representative results are presented

Article Snippet: HEK293FT wt cells or HEK293FT cells stably expressing GFP were seeded at a volume of 1 ml with ~ 2 × 10 5 cells per well in 12-well plates for 1 day, and 1 μg of PX459- Spy Cas9 (Addgene, #62,988) together with 1 μg of pGL3-sg GFP or empty vector (Additional file 1: Table S2) were then transiently transfected into the cells using LipofectamineTM 3000 transfection reagent according to the manufacturer’s protocol (Thermal Fisher, L3000008).

Techniques: SPR Assay, Binding Assay, Concentration Assay, Software, Incubation, Activity Assay, Plasmid Preparation, Labeling, Sampling, Agarose Gel Electrophoresis, Staining, Thermal Shift Assay, Sequencing

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 selectivity and structure–activity relationship of Cas9 inhibitor. A The effect of pamoic acid or carbenoxolone on inhibiting the activity of Cas9-VQR (upper) or SpRY-Cas9 (lower) mutant. Compounds were tested under the standard conditions with using the Cas9-VQR (D1135V/R1335Q/T1337R) or SpRY-Cas9 (A61R/L1111R/D1135L/S1136W/G1218K/E1219Q/N1317R/A1322R/R1333P/R1335Q/T1337R; NEB, M0669T) and the FAM-labeled 87 bp GFP oligonucleotide with a 5’-NGA or 5’-NAG PAM (Additional file 1: Table S2 and S4) , the latter of which is the preferred 5’-NHN PAM (H = A, C, or T) for SpRY-Cas9. The original images are shown in the Additional file 1: Fig. S6B. Means ± SDs ( n = 3, biological replicates). B The effect of pamoic acid or carbenoxolone on inhibiting the cleavage of GFP plasmids containing a 5’-NAG or 5’-NGA PAM by Spy Cas9. The original images are shown in the Additional file 1: Fig. S6D. Means ± SDs ( n = 3, technical replicates). C The effect of pamoic acid or carbenoxolone on the activity of Spy Cas9 nickase. Compounds were tested under the standard conditions using the wt Spy Cas9 or nickase, i.e. Cas9 D10A or Cas9 H840A mutant, and the linear GFP substrate. The original images were shown in the Additional file 1: Fig. S6E. Means ± SDs ( n = 3, biological replicates). D The structure-activity relationship of pamoic acid on inhibiting the activity of Spy Cas9 (see Additional file 1:Fig. S6F for the original images). The common structural moiety between the Asp60-Glu70 dipeptide (right panel) and pamoic acid is shown in orange. E The effect of compounds on the activity of Fno Cas12a or Lba Cas12a, Type V-A CRISPR/Cas enzyme with a respective 5’-TTN PAM and a 5'-TTTV PAM. The commercially available Fno Cas12a (left panel) or Lba Cas12a (right panel) CRISPR/Cas enzymes (#32106-03 or #32108-03 of ToloBio, Shanghai, China) were incubated with indicated compounds for 15 min before loading the pre-mixture of DNA substrate (DNA methyltransferase 1 fragment) and crRNA (see the “ ” section). The 825 bp substrate or 525 bp and 300 bp products of DNA methyltransferase 1 was separated on 1% agarose gel and stained with EB. The optical density of the DNA bands was determined with ImageJ and the cleavage activity was showed as percentages ( n = 2, biological replicates; see the “ ” section). F The effect of Cas9 inhibitors on the activity of EcoRI or KpnI endonuclease. Compounds were tested in assays containing 375 nM Cas9 or 0.5 μl EcoRI or KpnI (7.5 U for EcoRI and 10 U for KpnI per assay) and 300 ng ~2.5 kb linear DNA (Additional file 1: Fig. S3C). The original images were shown in the Additional file 1: Fig. S7A. ATA, aurintricarboxylic acid (Additional file 1: Table S1 and Additional file 1: Fig. S2). Statistical analysis was performed using one-way ANOVA with Tukey’s multiple comparisons test; * p < 0.05; ** p < 0.01; *** p < 0.001. All experiments were independently repeated at least twice, and representative results are presented

Article Snippet: HEK293FT wt cells or HEK293FT cells stably expressing GFP were seeded at a volume of 1 ml with ~ 2 × 10 5 cells per well in 12-well plates for 1 day, and 1 μg of PX459- Spy Cas9 (Addgene, #62,988) together with 1 μg of pGL3-sg GFP or empty vector (Additional file 1: Table S2) were then transiently transfected into the cells using LipofectamineTM 3000 transfection reagent according to the manufacturer’s protocol (Thermal Fisher, L3000008).

Techniques: Activity Assay, Mutagenesis, Labeling, CRISPR, Incubation, Agarose Gel Electrophoresis, Staining

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: HEK293FT wt cells or HEK293FT cells stably expressing GFP were seeded at a volume of 1 ml with ~ 2 × 10 5 cells per well in 12-well plates for 1 day, and 1 μg of PX459- Spy Cas9 (Addgene, #62,988) together with 1 μg of pGL3-sg GFP or empty vector (Additional file 1: Table S2) were then transiently transfected into the cells using LipofectamineTM 3000 transfection reagent according to the manufacturer’s protocol (Thermal Fisher, L3000008).

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 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: HEK293FT wt cells or HEK293FT cells stably expressing GFP were seeded at a volume of 1 ml with ~ 2 × 10 5 cells per well in 12-well plates for 1 day, and 1 μg of PX459- Spy Cas9 (Addgene, #62,988) together with 1 μg of pGL3-sg GFP or empty vector (Additional file 1: Table S2) were then transiently transfected into the cells using LipofectamineTM 3000 transfection reagent according to the manufacturer’s protocol (Thermal Fisher, L3000008).

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: 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 effects of inhibitors on antagonizing the Cas9-mediated genome editing in a hydrodynamic-injection-based anti- PCSK9 mice model. A Scheme of experiments for the administration of compounds and construction of a Spy Cas9-mediated anti- PCSK9 mouse model by hydrodynamic injection. BALB/c mice were intraperitoneally injected with the compounds at doses of 10, 20, 40, 80 or 160 mg/kg on day 0, followed by a hydrodynamic injection of 90 mμg of PX459- Spy Cas9 plasmids encoding anti- PCSK9 sgRNA (Additional file 1: Table S4) or control (vehicle group), and 5-8 s into the tail vein. Blood was collected via orbital bleeding on day 2. Two additional cycles of intraperitoneal injection of compounds (at the same dose as the 1 st treatment), hydrodynamic injection of PX459- Spy Cas9 plasmids, or orbital bleeding to mice were performed on days 3-8 before the mice were sacrificed on day 8. B Pamoic acid, dalbavancin or carbenoxolone restored the circulatory amount of PCSK9 in a Spy Cas9-mediated anti- PCSK9 mouse model. After collecting the blood sample from the orbital sinus on Day 2 (1 st orbital blood collection), Day 5 (2 nd orbital blood collection) or Day 8 (3 rd orbital blood collection), the amount of plasma PCSK9 was determined by Mouse Proprotein Convertase 9/ PCSK9 Quantikine ELISA Kit (R&D, MPC900, Minneapolis, MN). Pamoic acid and dalbavancin were studied together in experiment 1, whereas carbenoxolone was studied in experiment 2. Means ± SDs (the groups of Cas9 + sg PCSK9 + saline and + 80 mg/kg pamoic acid in the 1 st and 2 nd treatment cycles of experiment 1, n = 12; the other groups, n = 6). Statistical analysis was performed using one-way ANOVA with Dunnett’s multiple comparisons test (for the samples from 1 st and 2 nd treatment cycles of experiment 1) or with Tukey’s multiple comparisons test (for the samples from the 3 rd treatment cycles of experiment 1 and the samples from experiment 2). Ns, no significance; * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: HEK293FT wt cells or HEK293FT cells stably expressing GFP were seeded at a volume of 1 ml with ~ 2 × 10 5 cells per well in 12-well plates for 1 day, and 1 μg of PX459- Spy Cas9 (Addgene, #62,988) together with 1 μg of pGL3-sg GFP or empty vector (Additional file 1: Table S2) were then transiently transfected into the cells using LipofectamineTM 3000 transfection reagent according to the manufacturer’s protocol (Thermal Fisher, L3000008).

Techniques: Injection, Control, Clinical Proteomics, Enzyme-linked Immunosorbent Assay, Saline

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: Three proposed modes of action of four newly identified leads in inhibiting the activity of Cas9. Mode of action: pamoic acid or carbenoxolone binds competitively with DNA substrates to the PAM binding site of Cas9 positioned by Arg1333 and Arg1335, and shows a various inhibitory potency to the cleavage of the supercoil plasmid or linear DNA substrate by Cas9, and thus leading a selectivity on the cleavage of different topology of DNA. Dalbavancin directly binds to sgRNA and forms the quaternary complexes of Cas9 and sgRNA. Epirubicin directly binds to the DNA substrate and forms the quaternary complexes of Cas9 and DNA

Article Snippet: HEK293FT wt cells or HEK293FT cells stably expressing GFP were seeded at a volume of 1 ml with ~ 2 × 10 5 cells per well in 12-well plates for 1 day, and 1 μg of PX459- Spy Cas9 (Addgene, #62,988) together with 1 μg of pGL3-sg GFP or empty vector (Additional file 1: Table S2) were then transiently transfected into the cells using LipofectamineTM 3000 transfection reagent according to the manufacturer’s protocol (Thermal Fisher, L3000008).

Techniques: Activity Assay, Binding Assay, Plasmid Preparation

Journal: Plant Communications

Article Title: Expanding plant genome-editing scope by an engineered iSpyMacCas9 system that targets A-rich PAM sequences

doi: 10.1016/j.xplc.2020.100101

Figure Lengend Snippet: Development of an iSpyMacCas9 plant genome-editing toolbox and its assessment in rice. (A) Protein domains of the hybrid SpyMacCas9 and iSpyMacCas9 nucleases, which were generated based on two codon-optimized SpyCas9 versions, pcoCas9 and zCas9. The z-iSpyMacCas9 entry clone was deposited into Addgene (no. 149553). (B) Genome-editing efficiency of four SpyMacCas9 systems at four target sites in rice protoplasts. Error bars represent the standard deviation of three independent experiments. (C) Genome-editing efficiency of z-iSpyMacCas9 at 32 target sites with NAAA, NAAT, NAAG, and NAAC PAMs in rice protoplasts.

Article Snippet: To prepare plant codon optimized (pco) and maize codon optimized (z) SpyMacCas9 attL1-attR5 Gateway-compatible entry clones pYPQ150-SpyMac and pYPQ166-SpyMac, Arabidopsis thaliana and maize codon-optimized PI domains from Streptococcus macacae ( ) were synthesized (IDT gBlocks). pYPQ150 (Addgene no. 69301) ( ) containing pco-SpyCas9 was enzymatically digested with XmnI (NEB, catalog no. R0194∗) and AatlI (NEB, catalog no. R0117∗). pYPQ166 (Addgene no. 109328) ( ) containing z-SpyCas9 was enzymatically digested with MscI (NEB, catalog no. R0534∗) and SacI (NEB, catalog no. R3156∗).

Techniques: Generated, Standard Deviation

Journal: Molecular Systems Biology

Article Title: Unlocking CRISPR-Cas9 editing for widely diverse Dictyostelid species

doi: 10.1038/s44320-025-00180-8

Figure Lengend Snippet: ( A ) Schematic depiction of CRISPR-Cas9 editing with RNP complex consisting of Streptococcus pyogenes Cas9 (SpyCas9), CRISPR RNA (crRNA), and trans-activating crRNA (tracrRNA). Single-stranded donor oligo encodes for an insertion sequence as well as flanking homology arms mediating homology-directed repair (HDR). Knock-outs are produced by targeting amino acid 9 of mCherry CDS of D. discoideum AX2 act5 :: mCherry . The same target is used for figures hereafter, unless noted otherwise. ( B ) Knock-out efficiencies for 1 bp insertion (stop codon and frameshift mutation) and 14, 28, and 56 bp homology arms. ( C ) Knock-out efficiencies for 37 bp insertion (HA-tag sequence, frameshift mutation, and stop codon) with 14, 28, and 56 bp homology arms. ( D ) Knock-out efficiencies for 1, 4 (stop codon and frameshift mutation) or 37 bp insertion with 28 bp homology arms. Percentages in ( B – D ) show estimated maximum knock-out efficiencies and standard errors based on sigmoidal fits. As there can be considerable variation between cell batches (Appendix Figs. and ), the comparisons of knock-out efficiencies within each panel are based on a single batch of cells. The same applies to figure panels with sigmoidal fits hereafter. Knock-outs were generated without pre-annealing the gRNA complex. See Appendix Data for data, and Appendix File for ungated flow cytometry data.

Article Snippet: The identity of the SpyCas9 protein was verified by mass spectrometry, and the oligomerization state and absence of aggregates were checked by SEC-MALS and Refeyn mass photometry.

Techniques: CRISPR, Sequencing, Produced, Knock-Out, Mutagenesis, Generated, Flow Cytometry

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector.

doi: 10.1016/j.biopha.2022.114189

Figure Lengend Snippet: Fig. 4. External in vivo validation of SpyCas9 expressing SAd36.MBP vector in the Ai9-SauSpyCas9 mouse. 6–10 weeks old Ai9-SauSpyCas9 reporter mice were injected with either 6 × 1010 (two female (F) and two male (M)) or 1 × 1011 (two F and two M) virus particles (VPs) of SAd36.MBP.SpyCas9 adenoviral vectors in 160 μl of saline via the tail vein. As positive control (+), two mice (one F and one M) were injected with 6 × 1010 VPs SAd36.MBP.Cre, and as negative control (-), four mice (two F and two M) were injected with 160 μl saline solution. Tissues were harvested 7 days post administration. The frozen tissue sections were imaged on a Zeiss Axio Scan.Z1 scanner, using the 20X objective and Cy3 (for tdTomato, red signal) fluorescence filter. The external validation study was performed by re searchers at Baylor College of Medicine and Rice University (BCM-Rice) in the frame of Somatic Cell Genome Editing (SCGE) Consortium.

Article Snippet: The detect SpyCas9, we used mouse anti-SpyCas9 primary antibody (1:400, 14697, Cell Signaling Technology).

Techniques: In Vivo, Biomarker Discovery, Expressing, Plasmid Preparation, Injection, Virus, Saline, Positive Control, Negative Control, Fluorescence

Journal: Biomedicine & pharmacotherapy = Biomedecine & pharmacotherapie

Article Title: In vivo editing of the pan-endothelium by immunity evading simian adenoviral vector.

doi: 10.1016/j.biopha.2022.114189

Figure Lengend Snippet: Fig. 5. Quantification of tdTomato+ signal by flow cytometry analysis in the CD31+, CD102+ endothelial cells of multiple mouse organs. 6–10 weeks old Ai9- SauSpyCas9 reporter mice were iv injected either with 1 × 1011 VPs of the SAd36.MBP.Cre vector, SAd36.MBP.SpyCas9 or 160 μl saline (7–10 mice per group). Seven days later, lungs, spleen and adrenal glands were harvested, and the single cell suspensions were subjected for flow cytometry analysis by Attune NxT flow cytometer using YL-1 laser (ThermoFisher). Data from three replicate experiments were combined in each graphs. Mean tdTomato signals in CD31+ EC cells of the lungs, adrenal glands and the spleen, respectively: 3.85, 0.80, 0.71%. Mean tdTomato signals in CD102+ EC cells of the lungs, adrenal glands and the spleen, respectively: 3.81; 0.92; 0.56%.

Article Snippet: The detect SpyCas9, we used mouse anti-SpyCas9 primary antibody (1:400, 14697, Cell Signaling Technology).

Techniques: Flow Cytometry, Injection, Plasmid Preparation, Saline

Journal: Nature communications

Article Title: Insights into the compact CRISPR-Cas9d system.

doi: 10.1038/s41467-025-57455-9

Figure Lengend Snippet: Fig. 3 | The hybrid REM of Cas9d comprising the REC domains and Stem 2 and stem 3 of the sgRNA. a Structural alignment of the Cas9d (RNA-coordinated target Engagement Module, REM) with the SpyCas9 REC domain. b Close-up view of the Cas9d REM, as highlighted by the ellipse in a. c Interaction interface of REC domain

Article Snippet: The gene encoding the full-length Cas9d (Sangon) was codon optimized for E.coli expression and assembled into a modified pET vector (2Bc-T, Addgene #37236) with a C-terminal thrombin-TwinStrepII-Histag usingGibson assembly (NewEnglandBiolabs, E2611L).Mutations in Cas9d and SpyCas9 (WT expression plasmid, Addgene #101199) were introduced using the QuickChange Mutagenesis kit (Takara, Cat# 638949) following the manufacturer’s instructions.

Techniques: Drug discovery

Journal: Science Advances

Article Title: A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9

doi: 10.1126/sciadv.abb4054

Figure Lengend Snippet: ( A ) Genetic circuit for linking PAM sequences to growth on d -xylose in E. coli . We call the resulting screen PAM-SEARCH. gRNA, guide RNA. ( B ) Schematic of the growth-based screen. NGS, next-generation sequencing. ( C ) Output of the screen performed with SpyCas9. The data are displayed as a nucleotide depletion plot, PAM wheel , and motif plot. Each dot represents a distinct 5-nt sequence within the library. See data file S1 for an interactive version of the PAM wheel based on the Krona plot. Results from a biological replicate of the screen can be found in fig. S3 and data file S2.

Article Snippet: The WT SpyCas9 gene (Addgene no. 42876) was used for plasmid clearance experiments and also as a backbone plasmid (CB339) for the high-fidelity variants.

Techniques: Next-Generation Sequencing, Sequencing

Journal: Science Advances

Article Title: A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9

doi: 10.1126/sciadv.abb4054

Figure Lengend Snippet: Assessing individual PAM sequences in E. coli ( A ) using the growth-based genetic circuit and measuring turbidity of the culture, ( B ) using the growth-based circuit with gfp replacing xylAB and measuring GFP fluorescence, ( C ) through direct transcriptional repression of gfp under the control of the lacZ promoter by measuring GFP fluorescence, and ( D ) through plasmid clearance by targeting the same construct from (B) with catalytically active SpyCas9. A nontargeting Sth1 sgRNA served as the nontargeting control. Assessing individual PAM sequences by targeting ( E ) gfp in U2OS-GFP cells and ( F ) genomic sites in HEK293T cells. Indels were measured with TIDE , CRISPResso analysis , and flow cytometry analysis. The NYGG data are the same in both (E) panels. Indel formation calculated by TIDE is in reference to a nontargeting control sample. Values are based on independent experiments starting from separate E. coli colonies or separate cell culture wells. Bars represent the mean of each set of triplicate or quadruplicate measurements. Statistical significance was calculated in comparison to the AGGTG sequence for (A) to (D) or the nontargeting sgRNA control in (E) using a two-tailed t test assuming equal variance with cutoffs of 0.05 (*) or 0.01 (**).

Article Snippet: The WT SpyCas9 gene (Addgene no. 42876) was used for plasmid clearance experiments and also as a backbone plasmid (CB339) for the high-fidelity variants.

Techniques: Fluorescence, Control, Plasmid Preparation, Construct, Flow Cytometry, Cell Culture, Comparison, Sequencing, Two Tailed Test

Journal: Science Advances

Article Title: A positive, growth-based PAM screen identifies noncanonical motifs recognized by the S. pyogenes Cas9

doi: 10.1126/sciadv.abb4054

Figure Lengend Snippet: ( A ) Mutations present in the four tested variants eSpyCas9(1.1), SpyCas9 HF1 , eSpyCas9(1.1) HF1 , and D1135E. ( B ) Assessing individual PAM sequences in E. coli through plasmid clearance. See for details. For SpyCas9 HF1 and eSpyCas9(1.1) HF1 , transformed cells were grown in liquid culture without selecting for the transformed plasmid between recovery from electroporation and plating on solid agar. See fig. S6 (C and D) for plasmid clearance with SpyCas9 HF1 and eSpyCas9(1.1) HF1 in the absence of outgrowth without selection. The data presented for WT Cas9 were collected separately from those in . Values represent measurements from independent experiments starting from separate E. coli colonies. Bars represent the mean of each set of triplicate or quadruplicate measurements. Statistical significance was calculated in comparison to the AGGTG sequence for each variant using a two-tailed t test assuming equal variance with a maximal value of 0.05 (*) or 0.01 (**).

Article Snippet: The WT SpyCas9 gene (Addgene no. 42876) was used for plasmid clearance experiments and also as a backbone plasmid (CB339) for the high-fidelity variants.

Techniques: Plasmid Preparation, Transformation Assay, Electroporation, Selection, Comparison, Sequencing, Variant Assay, Two Tailed Test