Journal: International journal of oncology

Article Title: Inhibition of basal cell carcinoma cells by cold atmospheric plasma‑activated solution and differential gene expression analysis.

doi: 10.3892/ijo.2020.5009

Figure Lengend Snippet: Figure 9. After 3, 6 and 12 h treatment with PAS, the expression of cas3/cle‑cas3 and cas9/cle‑cas9 in TE354T cells is analyzed by western blotting. Changes in cas3/cle‑cas3 and cas9/cle‑cas9 protein expression in TE354T cells after PAS treatment by (A) western blotting and (B) subsequent densitometry. **P<0.01 vs. respective con. (C) Changes in the ratio of cle‑cas3/cas3 and cle‑cas9/cas9 after PAS treatment. Data are presented as the mean ± SD of three independent experiments. **P<0.01 vs. respective 0 h. PAS, plasma‑activated solution; cas3, caspase 3; cle, cleaved; cas9, caspase 9; con, control; NS, not significant.

Article Snippet: CAP, cold atmospheric plasma; NS, not significant. antibodies used for Cas3, cleaved Cas3 and cleaved Cas9 were goat rabbit anti‐lgG (cat. no. ZB2301; OriGene Technologies, Inc.); the secondary antibodies used for Cas9 were goat mouse anti‐IgG (cat. no. ZB2305; OriGene Technologies, Inc.).

Techniques: Expressing, Western Blot, Control

Journal: Cellular and Molecular Gastroenterology and Hepatology

Article Title: A β-Catenin-TCF-Sensitive Locus Control Region Mediates GUCY2C Ligand Loss in Colorectal Cancer

doi: 10.1016/j.jcmgh.2021.12.014

Figure Lengend Snippet: Cas9 deletion confirms a super-enhancer necessary for β-catenin-TCF-sensitive locus control. DLD1(DNTCF) cells expressing Cas9 and untargeted (clone #1) or GUCA2A-locus-targeted gRNAs harbor biallelic deletions encompassing the GUCA2A promoter (clone #2), DNase site #6 (clone #3), DNase site #7 (clone #4), or both DNase sites (clone #5). Expression of ( A ) GUCA2A protein, ( B ) GUCA2A mRNA, and ( C ) GUCA2B mRNA was quantified with (+) or without (–) 1 μg/mL DOX for 24 hours. ( D ) Hypothetical model of GUCA2A and GUCA2B promoter positioning relative to a Pol II-rich super-enhancer region upstream of GUCA2A, consisting of multiple DNase sites. ( E ) ChIP-pcr in HT29(APC) cells treated with (+) or without (–) 300 μM zinc for 24 hours reveals enrichment of TCF at the promoter of a Wnt target gene, SP5, with a TCF-specific antibody, but not with control IgG. In contrast, TCF was not detected at sites within 6kb of the GUCA2A TSS: DNase site #5 (GUCA2A promoter), site #6, site #7, or site #8. ( F ) GUCA2A mRNA expression in HT29(APC) cells stably expressing an untargeted (Ctr) or TCF-targeted shRNA, and treated with (+) or without (–) 300 μM zinc for 24 hours. GUCA2A mRNA expression is retained despite TCF knockdown, illustrated by Western blot. ( A–C ) Bars represent the average of ( A ) 2 or ( B , C ) 3 independent experiments ± SEM, and data are presented relative to noninduced cells. Significance was determined by 2-way analysis of variance with matched analysis for independent experiments on log2-transformed results. ( E ) Bars represent the mean ± SD of 3 IPs, and data are presented relative to input DNA. Significance was determined by 2-way analysis of variance. ( F ) Data points represent the average of 3 wells of cells from a single experiment, with the mean of 2 independent experiments indicated. Significance was determined by 2-way analysis of variance with matched analysis for independent experiments on log2-transformed results. Data are presented relative to noninduced cells receiving control shRNA. ∗ P < .05; ∗∗ P < .01; ∗∗∗ P < .001; ∗∗∗∗ P < .0001.

Article Snippet: Blots were blocked for 1 hour in phosphate-buffered saline (PBS) containing 0.1% Tween 20 and 10% milk, then incubated overnight at 4°C with primary antibodies at the indicated dilutions targeting GAPDH (#2118, 1:5000), APC (#2504, 1:1000), β-catenin (#8480, 1:1000), TCF (#2569, 1:1000), Histone H3 (#4499, 1:1000), or Cas9 (#19526, 1:1000) from Cell Signaling Technology (Danvers, MA), or GUCA2A (#HPA018215, 1:250) from Sigma-Aldrich.

Techniques: Control, Expressing, Stable Transfection, shRNA, Knockdown, Western Blot, Transformation Assay

Journal: bioRxiv

Article Title: Evolution of CRISPR-associated Endonucleases as Inferred from Resurrected Proteins

doi: 10.1101/2022.03.30.485982

Figure Lengend Snippet: (a) Phylogenetic chronogram of Cas9 endonucleases. Fifty-nine sequences were chosen from two phyla, Firmicutes and Actinobacteria, with two classes, Bacilli and Clostridia, belonging to Firmicutes. Identification codes of all sequence can be found in the Supplementary Information. Divergence times were estimated using Bayesian inference and information from the Time Tree of Life. Internal nodes from Firmicutes Common Ancestor (FCA), Bacilli Common Ancestor (BCA), Streptococci Common Ancestor (SCA), Pyogenic Common Ancestor (PCA) and Pyogenes-Dysgalactiae Common Ancestor (PDCA) were selected for testing. (b) Superposition of structural prediction of FCA Cas9 using AlphaFold2 (blue) with x-Ray structure of SpCas9 with guide RNA and target DNA (PDB:4oo8, red). (c) Isolated HNH domains of SpCas9 (red) and FCA anCas (blue) from whole structure coordinates. (d) Superposition of structural prediction of PDCA anCas with guide RNA and target DNA using AlphaFold2 (purple) with x-Ray structure of SpCas9 (PDB:4oo8, red). (e) pLDDT values for all anCas as estimated from AlphaFold2 prediction.

Article Snippet: Anti-Cas9 rabbit antibody (Rockland, 600-401-GK0) was diluted 1:25000 in 1% BSA blocking solution and plates were incubated for 2 hours at room temperature.

Techniques: Sequencing, Structural Proteomics, Isolation

Journal: bioRxiv

Article Title: Evolution of CRISPR-associated Endonucleases as Inferred from Resurrected Proteins

doi: 10.1101/2022.03.30.485982

Figure Lengend Snippet: (a) In vitro cleavage assay on a supercoiled DNA substrate of anCas and SpCas9 using sgRNAs from different species. FCA, BCA anCas and SpCas9 are shown. (b) Quantification of in vitro cleavage for all anCas and SpCas9 using the different sgRNAs. (c) In vitro cleavage assay on an 85 nt ssDNA fragment at different incubation times for FCA, BCA anCas and SpCas9. (d) In vitro cleavage assay on a 60 nt ssRNA at different incubation times for FCA, BCA anCas and SpCas9. (e) Quantification of fraction cleavage of ssDNA at different times and exponential fits for determination of kinetics parameters. In both (c) and (d), the control lane is the same for the three proteins. (f) Quantification of fraction cleavage of ssRNA at different times and exponential fits for determination of kinetics parameters. All kinetics parameters are summarized in . (g) Results from ELISA test of Anti-Cas9 rabbit antibody against SpCas9, FCA anCas, BCA anCas and BSA, used as control. Results are reported as average and S.D from three independent experiments.

Article Snippet: Anti-Cas9 rabbit antibody (Rockland, 600-401-GK0) was diluted 1:25000 in 1% BSA blocking solution and plates were incubated for 2 hours at room temperature.

Techniques: In Vitro, Cleavage Assay, Incubation, Control, Enzyme-linked Immunosorbent Assay

Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 1. Designing and development of the CRISPR-Cas9-based plant transformation vector for the incorporation of the desired mutations in the wild CcEPSPS gene where A. pCAMBIA1300-based NICTK-2_pCRISPR-Cas9 binary vector harboring the Cas9 expression cassette highlighted with NLS: nuclear localization signal, promoter sequence and restriction sites essential for cloning. B. The schematic representation depicting the standardized pipeline for the selection of the sgRNAs with high efficiency and no off-target activity and cloning of the selected sgRNAs in the NICTK-2_pCRISPR-Cas9 binary vector to develop complete CcEPSPS_NICTK- 2_pCRISPR-Cas9 plant transformation vector. C. Represents the sgRNA cassette where the effective AtU6-29 and AtU3b promoters drive the expression of the selected sequences (pink; highlighted PAM with red) of crRNA with scaffolds and terminator. D. Represents the sequence of the two selected sgRNAs confining the target region of the CcEPSPS gene. E. Represents the CcEPSPS donor repair template harboring the desired mutations in the target region ie., 182G > A (green), 183T > I (red), and 187P > S (blue) which will be utilized for the homology directed repair mechanism and development of mutated CcEPSPS. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: CRISPR, Transformation Assay, Plasmid Preparation, Expressing, Sequencing, Cloning, Selection, Activity Assay

Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 2. A. Representative images of stages in tissue culture of Pigeonpea (i) Seeds (seed coat removed) of pigeonpea on germination media (GM) (ii) Seed germination (iii) Excised embryonic axis (EA) explant on callus induction medium (CIM) (iv) Induction of callus on CIM (v) Initiation of multiple shooting from induced callus on the regeneration medium (REM) (vi) Regenerated shoots on the elongation media (EM) (vii) Rhizogenesis on rooting medium (RM) (viii) Rep resents the in vitro regenerated plants under hardening. B. The schematic representation of standardized protocol for biolistic transformation of the callus with 0.6 μm microcarrier (gold particle) coated with CcEPSPS_NICTK-2_pCRISPR-Cas9 plant transformation vector and donor template. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: In Vitro, Transformation Assay, Plasmid Preparation

Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 3. Molecular characterization of putative positive T0 and T1 edited plants. A. Illustrate the amplification with Cas9 specific primer in putative positive T0 plants showing Cas9 integration. B. Amplification with EPSPS target site overlapping primer set1 of the Cas9 positive T0 plants showing desired mutation. C. Illustrate the amplification with EPSPS target site overlapping primer set2 of the Cas9 positive T0 plants showing desired mutation. D. Amplification of a portion spanning the target site of the EPSPS gene from putative positive T0 plants utilized for sequencing. E. Illustrate the amplification with Cas9 specific primers of putative positive T1 plants showing Cas9 integration. F. Amplification of a portion spanning the target site of the EPSPS gene from positive T1 plants. Where, M- 1 kb DNA ladder, NTC- no template control, WT-wild type, PC- positive control, and WC- water control. G. Representative Southern blot analysis of established T1 transformants showing the single copy number of Cas9 gene in screened transgenic pigeonpea plants. These observations were obtained by the digestion of genomic DNA from eight T1 plants using restriction enzyme EcoRV and Cutsmart 2 buffer obtained from NEB. Lane M 100 ng DNA molecular weight marker III, Digoxigenin labeled, lane S1-8 20 μg genomic DNA, EcoRV, lane WT 20 μg genomic DNA of untransformed plant, EcoRV, lane PC 5 μg positive control plasmid DNA. H. Representative Western blot analysis of established T1 transformants showing Cas9 protein expression in T1 edited pigeonpea plants. These observations were obtained by the total protein extraction from eight edited plants using Cas9 specific primary antibody. Where in S1-8 lane, 40–45 μg/ml of plant extracts were loaded and for positive control (PC) 1ug/ul Cas9 protein was loaded. The marker used in the lane M was of size 10–180 KDa.

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: Amplification, Mutagenesis, Sequencing, Control, Positive Control, Southern Blot, Transgenic Assay, Molecular Weight, Marker, Labeling, Plasmid Preparation, Western Blot, Expressing, Protein Extraction

Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 5. The molecular characterization of T2 edited plants. A. Illustrate the amplification with Cas9 specific primers of T2 edited plants showing Cas9 integration (T2.1

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: Amplification

Journal: Plant physiology and biochemistry : PPB

Article Title: Combating aggressive weeds: Reinforcing herbicide resistance in pigeonpea (Cajanus cajan L.) through genome editing.

doi: 10.1016/j.plaphy.2025.109550

Figure Lengend Snippet: Fig. 6. Physiological analysis of WT, TC and edited T2 plants under glyphosate treatment. A. Seed germination analysis: seeds of WT and T2 generation edited seeds were germinated on germination medium supplemented with gradually increasing concentration of glyphosate (0–50 mM) in a glass jar under controlled tissue culture conditions. Additionally, the effect of 4 mM of glyphosate on the growth of edited (T2 generation) and WT seeds displayed that all the edited seeds germinated while WT seeds did not germinate. Data was recorded after two week of incubation. B. Glyphosate spray analysis: 42-days-old T2 plants and C. 28-days-old T2 plants were sprayed with 6 ml/L commercial glyphosate (Roundup: 41.0% w/v; Monsanto Inc., Montreal, QC, Canada) under controlled greenhouse conditions (RH = 85%; Temp. = 28±2 ◦C). The data was recorded after one week. D. Post glyphosate treatment the edited plants were allowed to grow till maturity and the recovered Cas9- free T2 plants displayed optimum growth. E. Weed competition assay: the WT and edited plants seeds were germinated along with associated weeds on a vermiculite tray and they were sprayed with 6 ml/L Roundup solution and after one week only edited plants survived.

Article Snippet: The blocked blot was probed with 1:1000 dilution of Rabbit-raised anti-Cas9 primary antibody (specific to Cas9; procured from Rockland, USA) followed by incubation with 1:20000 dilution ratio of Goat anti-Rabbit HRP conjugated secondary antibody for 1H at RT.

Techniques: Concentration Assay, Incubation, Competitive Binding Assay

Journal: Pathogens and Disease

Article Title: Nonspecific toxicities of Streptococcus pyogenes and Staphylococcus aureus dCas9 in Chlamydia trachomatis

doi: 10.1093/femspd/ftaa005

Figure Lengend Snippet: Structures of pTRL2(Δgfp) (A), pTRL2-Sp-dCas9 (B), pTRL2-Sp-dCas9 (C) and pTRL2-metG (D). Abbreviations: Sp, S. pyogenesis; Sa, S. aureus; dCas9, deactivated Cas9; MetG, methionine-tRNA ligase; Pgp, chlamydial plasmid-encoded protein; PdnaK, dnaK promoter; mKate, a monomeric basic far-red fluorescence protein; gRNA, guide RNA; PdefA, defA promoter; AmpR, penicillin/ampicillin-resistance gene; tetR, tet repressor; ColE1, E. coli replication origin E1; PtetA, tetracycline/anhydrotetracycline (ATC)-activated promoter. pL2 ori, CtL2 plasmid replication origin.

Article Snippet: The fixed cells were sequentially reacted with a mouse monoclonal anti-Sa-Cas9 primary antibody (Boster Biological Technology, catalog # {"type":"entrez-nucleotide","attrs":{"text":"M30929","term_id":"159088","term_text":"M30929"}} M30929 –3) and an FITC-conjugated anti-mouse IgG secondary antibody (Millipore Sigma, catalog # F9137).

Techniques: Plasmid Preparation, Fluorescence