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: Frontiers in Plant Science

Article Title: Biocircuits in plants and eukaryotic algae

doi: 10.3389/fpls.2022.982959

Figure Lengend Snippet: Synthetic biology tools for plants and algae.

Article Snippet: , , MoClo CRISPR/Cas toolkit for Plants ( https://www.addgene.org/kits/nekrasov-moclo-plant-crispr/ ) , Kit collection with 95 plasmids for transformation and expression of CRISPR/Cas nucleases, base editors, gRNA backbones, and promoters in plants. , .

Techniques: Algae, Transformation Assay, Construct, Translocation Assay, CRISPR, Expressing, Functional Assay, Sequencing, In Silico, Software, Bacteria, Gene Expression

Journal: Cancer cell

Article Title: Stress Activated NRF2-MDM2 Cascade Controls Neoplastic Progression in Pancreas

doi: 10.1016/j.ccell.2017.10.011

Figure Lengend Snippet: (A) Effects of p53, p62 and NRF2 overexpression, NRF2 silencing and sulforaphane on Mdm2 promoter activity in transfected KPC cells (n = 3). (B) Chromatin immunoprecipitation assays probing NRF2 and small MAF protein recruitment to the MDM2 promoter in WT and NRF2 ablated MIA PaCa-2 (n = 3) cells. (C) Sphere formation of control or MDM2-ablated MIA PaCa-2 and Capan-2 cells with or without NICD1 overexpression. (D) SA-β-gal staining and γ-H2AX IB of cells as in C. (E) Sphere formation of control and p62-ablated MIA PaCa-2 and Capan-2 cells with or without MDM2 CRISPR-mediated activation vector (n = 3). (F) SA-β-gal staining and γ-H2AX IB of cells as in E. (G) Sphere formation of control and NRF2-ablated MIA PaCa-2 and Capan-2 cells with or without MDM2 CRISPR-mediated activation vector (n = 3). (H) SA-β-gal staining and γ-H2AX IB of cells as in G. (I) A scheme explaining how p62 accumulation, operating via the NRF2-MDM2 module leads to dedifferentiation and cell cycle progression in preneoplastic lesions in pancreas. Results are mean ± SEM; *, p < 0.05; **, p < 0.01; ***, p < 0.001 by Student’s t test. See also Figure S7.

Article Snippet: Transfections of MIA PaCa-2, Capan-2 cells, KC and KPC cell lines with CRISPR/Cas9 KO, HDR or CRISPR activation plasmids listed in the key resources table were performed with UltraCruz® Transfection Reagent (Santa Cruz, sc-395739) according to the manufacturer’s instructions.

Techniques: Over Expression, Activity Assay, Transfection, Chromatin Immunoprecipitation, Control, Staining, CRISPR, Activation Assay, Plasmid Preparation

Journal: Nature cell biology

Article Title: RNA-targeting CRISPR systems from metagenomic discovery to transcriptomic engineering

doi: 10.1038/s41556-019-0454-7

Figure Lengend Snippet: a, Cas proteins, including RCas, are discovered in metagenomic sequencing of bacteria and archaea using an established bioinformatic pipeline. Starting with a CRISPR array search seed, nearby putative Cas effectors are filtered on the basis of various criteria, including co-correlation with CRISPR arrays, size, and predicted protein domains. After grouping based on homology, distinct groups of effectors are selected for experimental validation follow-up. Researchers deduce the targeting substrate and mechanism through biochemical incubation of substrate nucleic acids with purified CRISPR ribonucleoprotein complexes and by observing resultant cleavage and binding. Genetic interrogation of targeting rules can be achieved through a spacer depletion screen assay consisting of a CRISPR library with spacers targeting an antibiotic resistance gene. Bacterial co-transformation via plasmids containing (i) the spacer library and Cas effector and (ii) the antibiotic resistance gene, followed by computational analysis of depleted spacers in the sequenced surviving population, will yield any targeting-dependent sequence requirements vis-à-vis the spacer. b, Type II and VI systems, encapsulating Cas9 and Cas13, respectively, represent the most commonly used RCas systems. Cas9 binds to a tracrRNA and crRNA (often combined into a single sgRNA) to target RNA. Depending on the Cas9 variant, a PAMmer oligonucleotide comprising a PAM sequence may be required for RNA binding10 and an additional domain (such as PIN domain endonuclease) may be required for RNA cleavage47. Cas13 binds to a crRNA for effective RNA targeting and subsequent indiscriminate cleavage. c, Prokaryotic Argonaute (pAgo) systems resemble CRISPR-Cas systems in their ability to target nucleic acids with a programmable guide. They exist as part of a broader RNA-targeting ecosystem that includes antisense oligonucleotides (ASOs), RNA interference (RNAi), CRISPR-Cas-inspired RNA targeting systems (CIRTS), viral coat proteins (VCPs), zinc fingers, Pumilio and FBF homology proteins (PUFs), and pentatricopeptide repeat proteins (PPRs), among others.

Article Snippet: Competing interests A.A.S. declares inventorship on the following published patents, applied for by the Broad Institute of MIT and Harvard and the Massachusetts Institute of Technology: WO2018035250A1 on methods for bioinformatic discovery of class 2 CRISPR-Cas systems; WO2017070605 on systems, methods, and compositions for targeting nucleic acids with type VI-B CRISPR-Cas systems.

Techniques: Sequencing, Bacteria, CRISPR, Biomarker Discovery, Incubation, Purification, Binding Assay, Transformation Assay, Variant Assay, Zinc-Fingers

Journal: iScience

Article Title: FOXO1 stimulates tip cell-enriched gene expression in endothelial cells

doi: 10.1016/j.isci.2024.109161

Figure Lengend Snippet:

Article Snippet: ANGPT2 enhancer region-targeting CRISPR-Cas9 & gRNAs #1 (#1-5’; 5’-ACCTCTGAC TGAGGCACGTT-3’, #1-3’; 5’-TGAAGTGT TAGGGCGCCTTT-3’) lentivirus , VectorBuilder , VB230331.

Techniques: Virus, CRISPR, Control, Recombinant, Modification, Saline, Chromatin Immunoprecipitation, Library Quantification, Biomarker Discovery, Selection, Plasmid Preparation, Software