Journal: Scientific Reports

Article Title: Efficient viral delivery of Cas9 into human safe harbor

doi: 10.1038/s41598-020-78450-8

Figure Lengend Snippet: Schematic diagram of the four-step integration strategy of Cas9 into the AAVS1 locus. (1) The first step is to generate a cell line expressing Cas9v1 by random integration of the recombinant lentivirus in the host genome. (2) Using the Cas9v1 nuclease activity, the N-terminal 1.9 kb fragment of Cas9v2 was introduced into the AAVS1 locus by AAV vector #1 with the sgRNA1-targeting AAVS1 site and the homology arms flanking the Cas9 N-terminal 1.9 kb fragment. (3) full-length Cas9v2 was reconstituted in the AAVS1 locus using HDR, with AAV vector #2 providing the 2.3 kb Cas9v2 C-terminal fragment, as well as a 0.3 kb region overlapping the 1.9 kb N-terminal fragment; (4) lentivirus, containing Cas9v1, was removed from the genome using AAV vector #3, which contained sgRNA targeting long terminal repeats (LTR) in the lentiviral vector. The schematic structures of wild-type Cas9v2, Cas9v2 nickase (Cas9nikase), and dead Cas9v2 (dCas9) made by the same procedures as wild-type Cas9v2 are shown in brackets. The sgRNAs targeting AAVS1, the 3′-end of the Cas9v1 N-terminal 1.9 kb fragment and the LTR of the lentivirus are shown as sg1, sg2, and sg3, respectively. AAVS1, adeno-associated virus integration site 1 (intron1 of the PPP1R12C, protein phosphatase 1 regulatory subunit 12C); EX, exon; Cas9v1, S. pyogenes-derived Cas9; Cas9v2, human codon-optimized Cas9; VP64, Herpes simplex virus transcriptional activator domain (TA).

Article Snippet: Specific proteins were detected using an anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc-tag (9B11) mouse monoclonal antibody (Cell Signaling Technology #2276S), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich #F1804), or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) and were used at 1:500–3000 dilution.

Techniques: Expressing, Recombinant, Activity Assay, Plasmid Preparation, Derivative Assay

Journal: Scientific Reports

Article Title: Efficient viral delivery of Cas9 into human safe harbor

doi: 10.1038/s41598-020-78450-8

Figure Lengend Snippet: AAV vector #1 construct for introduction of N-terminal 1.9 kb Cas9v2 into the AAVS1 locus. ( A ) AAV vector #1 is designed to introduce the N-terminal Cas9v2 into the AAVS1 locus as a double-cut donor. The vector sequence is shown in Supplementary Table . T1 is the AAVS1 target site including the protospacer adjacent motif (PAM). The T1 sequence was synthesized and added adjacent to each homology arm to cut out the donor DNA from the AAV vector genome. U6, U6 RNA polymerase III promoter; sg1, AAVS1-specific sgRNA1; LA and RA, left and right homology arms; SA, splicing acceptor; T2A and P2A: Thosea asigna virus and porcine teschovirus-1 2A self-cleaving peptides; Puro, puromycin resistance gene; pA, a polyadenylation signal sequence; ITR, inverted terminal repeat. ( B ) Genome structure after integration of the donor by HDR. Primers P1–P2 for PCR were placed outside of the homology arms. Primers P1–P3 and P4–P2 were used to detect the N-terminal Cas9 and the Puro genes, respectively. The PCR primer sequences are listed in Supplementary Table .

Article Snippet: Specific proteins were detected using an anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc-tag (9B11) mouse monoclonal antibody (Cell Signaling Technology #2276S), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich #F1804), or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) and were used at 1:500–3000 dilution.

Techniques: Plasmid Preparation, Construct, Introduce, Sequencing, Synthesized

Journal: Scientific Reports

Article Title: Efficient viral delivery of Cas9 into human safe harbor

doi: 10.1038/s41598-020-78450-8

Figure Lengend Snippet: PCR and immunoblot to confirm N-terminal Cas9v2 genomic integration and expression. ( A ) Genomic DNA from parent HEK293T-Cas9v1-Myc cells (Cas9v1#2) and the 293 T-Cas9v1-Myc cells expressing the 1.9 kb N-terminal Cas9v2 after AAV vector #1 infection and puromycin selection (+ v2-NT #1–#15) was analyzed by PCR with primers P1–P2 (4-min extension at 68 °C). The 4.5 kb and 1.5 kb PCR products are from the N-terminal Cas9v2-integrated and non-integrated original alleles, respectively. The Cas9v2 NT #8 clone had 4.5 kb and 4.2 kb PCR products. ( B ) Further analysis of the Cas9v2-integrated clones with primers P1–P3 (1.2 kb for Cas9v2). ( C ) Further analysis of the Cas9v2-integrated clones with primers P4–P2 (1.1 kb for Puro). D. The lysates obtained from the cells used for ( B , C ) were analyzed by immunoblot using an antibody against Cas9 that recognizes both Cas9s, after 6% SDS-PAGE. The arrows indicate the Cas9v1-Myc (160 kDa) and the 1.9 kb N-terminal Cas9v2 (70 kDa).

Article Snippet: Specific proteins were detected using an anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc-tag (9B11) mouse monoclonal antibody (Cell Signaling Technology #2276S), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich #F1804), or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) and were used at 1:500–3000 dilution.

Techniques: Western Blot, Expressing, Plasmid Preparation, Infection, Selection, Clone Assay, SDS Page

Journal: Scientific Reports

Article Title: Efficient viral delivery of Cas9 into human safe harbor

doi: 10.1038/s41598-020-78450-8

Figure Lengend Snippet: PCR and immunoblot to confirm the reconstitution of full-length Cas9v2-FLAG in AAVS1 locus. ( A ) PCR analysis of genomic DNA prepared from the HEK293T-Cas9v1-Myc + Cas9v2-NT #4 cells and the HEK293T-Cas9v1-Myc cells expressing full-length Cas9v2-FLAG (+ v2-CT-FLAG #1–#9), after AAV vector #2 infection and blasticidin selection. The DNA was amplified by primers P5–P6 and the 1.2 kb PCR products indicating the integration of donor elements (Bsd gene) were detected in all clones. ( B – D ) Lysates obtained from the cells used in ( A ) were analyzed by immunoblot with an antibody against Cas9 that recognizes both Cas9s, after 6% SDS-PAGE. The 70-kDa band indicates the 1.9 kb N-terminal Cas9v2 ( B ). The 160-kDa band was confirmed with the specific tag antibodies to contain the reconstituted full-length Cas9v2-FLAG ( C ) and Cas9v1-Myc ( D ).

Article Snippet: Specific proteins were detected using an anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc-tag (9B11) mouse monoclonal antibody (Cell Signaling Technology #2276S), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich #F1804), or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) and were used at 1:500–3000 dilution.

Techniques: Western Blot, Expressing, Plasmid Preparation, Infection, Selection, Amplification, Clone Assay, SDS Page

Journal: Scientific Reports

Article Title: Efficient viral delivery of Cas9 into human safe harbor

doi: 10.1038/s41598-020-78450-8

Figure Lengend Snippet: AAV vector #3 construct targeting the genome integrated lentivirus long terminal repeat (LTR). ( A , B ) AAV vector #3 targeting the LTR of the lentiviral vector (T3) was created by placing a pair of sgRNAs (sg3-1 and sg3-2) with 16 bp offset for DSB (double-strand break) by Cas9 nickase as well as wild-type Cas9 and placing a puromycin resistance gene between the ITRs. Primers P7–P8 for Cas9v1 are used to examine whether the lentivirus containing Cas9v1-Myc is removed by NHEJ with AAV vector #3 infection ( B ) The sgRNA and PCR primer sequences are shown in Supplementary Tables and , respectively. ( C – E ) PCR analysis of genomic DNA prepared from the HEK293T-Cas9v1-Myc + full-length Cas9v2-FLAG #7 cells, and the HEK293T-Cas9v1-Myc + Cas9v2-FLAG cells (sgLenti #1–#10) expressing sgRNA3 targeting the lentivirus LTR, after AAV #3 vector infection and puromycin selection. The DNA was amplified using primers P7–P8 (for Cas9v1) ( C ). The lysates from the cells that had no 1.2 kb PCR products in ( C ) were analyzed by immunoblot for Cas9v1-Myc against Myc-tag ( D ) and Cas9v2-FLAG with an antibody against FLAG-tag ( E ).

Article Snippet: Specific proteins were detected using an anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc-tag (9B11) mouse monoclonal antibody (Cell Signaling Technology #2276S), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich #F1804), or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) and were used at 1:500–3000 dilution.

Techniques: Plasmid Preparation, Construct, Infection, Expressing, Selection, Amplification, Western Blot, FLAG-tag

Journal: Scientific Reports

Article Title: Efficient viral delivery of Cas9 into human safe harbor

doi: 10.1038/s41598-020-78450-8

Figure Lengend Snippet: Schematic diagram of the four-step strategy using Lentiviral Cas9 (Nick)-Myc. (1) To reduce possible off-target effects, lentiviral Myc-tagged Cas9 nickase, termed Cas9 (Nick)-Myc (Supplementary Fig ), was used in the first step. (2) AAV#7 (Supplementary Fig ) was used to introduce DSB at the AAVS1 site via Cas9 (Nick)-Myc nickase. (3) AAV#8 (Supplementary Fig ) was used to reconstitute the full-length 4.2 kb Cas9v2 nickase-FLAG. If AAV#9 (Supplementary Fig ) was used instead of AAV#8, dead Cas9 (v2)-VP64 was produced, as shown in brackets. (4) Randomly integrated lentivirus, containing Cas9 (Nick)-Myc, was removed by AAV vector #3, with sgRNA3 targeting the long terminal repeat (LTR) of the lentivirus vector. sgRNAs targeting AAVS1, the 3′-end of the 1.9 kb Cas9v1 N-terminal fragment, and the LTR of the lentivirus are shown as sg7/1, sg8/2, and sg3, respectively.

Article Snippet: Specific proteins were detected using an anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc-tag (9B11) mouse monoclonal antibody (Cell Signaling Technology #2276S), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich #F1804), or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) and were used at 1:500–3000 dilution.

Techniques: Introduce, Produced, Plasmid Preparation

Journal: Scientific Reports

Article Title: Efficient viral delivery of Cas9 into human safe harbor

doi: 10.1038/s41598-020-78450-8

Figure Lengend Snippet: Sequential transduction procedure using AAV vectors. ( A ) According to the scheme shown in figure, 293 T-Cas9(Nick)-Myc cells (500/well) plated in a 24-well plate were incubated with 10,000 transduction units of AAV#7, which provided sgRNA targeting the AAVS1 site, and 1.9 kb Cas9v2 nickase N-terminal fragments (Supplementary Fig ), for 36 h. Following medium aspiration, cells were sequentially incubated with 10,000 transduction units of AAV#8 (Supplementary Fig ), for 24 h, to reconstitute full-length Cas9 (v2) nickase-FLAG. Cells were transferred to a 6-well plate, with blasticidin added two days after transfer. Colonies were collected after 10 days in the presence of blasticidin and analyzed using PCR and immunoblot. ( B ) 293 T-Cas9 (Nick)-Myc cells (500/well) were sequentially incubated with AAV#7 and AAV#9 (Supplementary Fig ) to reconstitute full-length dCas9 (v2)-VP64. Blasticidin-resistant cells were analyzed using PCR and immunoblot.

Article Snippet: Specific proteins were detected using an anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc-tag (9B11) mouse monoclonal antibody (Cell Signaling Technology #2276S), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich #F1804), or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) and were used at 1:500–3000 dilution.

Techniques: Transduction, Incubation, Western Blot

Journal: Scientific Reports

Article Title: Efficient viral delivery of Cas9 into human safe harbor

doi: 10.1038/s41598-020-78450-8

Figure Lengend Snippet: PCR and immunoblot analysis to confirm the reconstitution of full-length Cas9v2nickase-FLAG in the AAVS1 locus. ( A , B ) PCR analysis of genomic DNA prepared from HEK293T, HEK293T-Cas9(Nick)-Myc, and HEK293T-Cas9(Nick)-Myc + v2n-NT + CT-FLAG #1–#6 cells, after sequential infections of AAV vectors #7 and #8, as in Fig. A. v2n-NT, 1.9 kb Cas9v2 nickase-NT; CT-FLAG, 2.3 kb Cas9v2-CT-FLAG. Primers P1–P3 (Cas9v2), and P4–P2 (Puro), were used to examine the integration efficiency of the reconstituted full-length Cas9v2nickase-FLAG into the AAVS1 locus at the 5′- and 3′-regions. ( C – E ) Lysates obtained from the same cells used for the PCR analysis were analyzed using immunoblot, with the antibodies indicated. Arrows indicate reconstituted full-length Cas9v2nickase-FLAG (160 kDa), lentiviral Cas9 (Nick)-Myc (160 kDa), and β-actin (45 kDa).

Article Snippet: Specific proteins were detected using an anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc-tag (9B11) mouse monoclonal antibody (Cell Signaling Technology #2276S), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich #F1804), or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) and were used at 1:500–3000 dilution.

Techniques: Western Blot

Journal: Scientific Reports

Article Title: Efficient viral delivery of Cas9 into human safe harbor

doi: 10.1038/s41598-020-78450-8

Figure Lengend Snippet: PCR and immunoblot analysis to confirm the reconstitution of full-length dCas9v2-VP64 in the AAVS1 locus. ( A , B ) PCR analysis of genomic DNA prepared from HEK293T, HEK293T-Cas9(Nick)-Myc, and HEK293T-Cas9(Nick)-Myc + v2n-NT + dCT-VP64 #1–#6 cells, after sequential infections of AAV vectors #7 and #9, as in Fig. B. v2n-NT, 1.9 kb Cas9v2 nickase-NT; dCT-VP64, 2.3 kb dCas9v2-CT-VP64. Primers P1–P3 (Cas9v2), and P4–P2 (Puro), were used to examine the integration efficiency of the reconstituted full-length dCas9v2-VP64 into the AAVS1 locus at the 5′- and 3′-regions. ( C – E ) Lysates obtained from the same cells used for the PCR analysis were analyzed using immunoblot, with the antibodies indicated. Arrows indicate reconstituted full-length dCas9v2-VP64 (170 kDa), lentiviral Cas9 (Nick)-Myc (160 kDa), and β-actin (45 kDa).

Article Snippet: Specific proteins were detected using an anti-Cas9 polyclonal antibody (Takara Bio #632607), anti-Myc-tag (9B11) mouse monoclonal antibody (Cell Signaling Technology #2276S), anti-FLAG M2 monoclonal antibody (Sigma-Aldrich #F1804), or anti-β-actin mouse monoclonal antibody (Santa-Cruz #SC-47778) and were used at 1:500–3000 dilution.

Techniques: Western Blot

Journal: Frontiers in Oncology

Article Title: Suppression of EGFR/PKC-δ/NF-κB Signaling Associated With Imipramine-Inhibited Progression of Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2021.735183

Figure Lengend Snippet: The induction of apoptosis and cytotoxicity of imipramine in NSCLC cells. (A) The morphology of CL1-5-F4 cells after 100 and 150 μM imipramine treatment for 48 hr is displayed. (B) Cell viability of CL1-5-F4, A549, and NCI460 cells after 0-200 μM imipramine treatment for 48 hr are displayed. (C) The Annexin-V/PI staining pattern and quantification results of CL1-5-F4, A549, and NCI460 cells after 100 and 150 μM imipramine treatment for 48 hr are displayed. (D) Quantification results Annexin-V positive percentage after Z-VAD combined with 100 and 150 μM imipramine in CL1-5-F4 cells are showed. (E, G, H) Staining pattern and quantification results of cleaved caspase-3, cell cycle and cleaved PARP-1 in CL1-5-F4 cells after 100 and 150 μM imipramine treatment for 48 hr are presented. (F) IF staining images and quantification results after 100 and 150 μM imipramine treatment in CL1-5-F4 cells are displayed. (I) The protein expression of cleaved caspase-3 and cleaved-PARP-1 are displayed. (J) One represented comet tail movement from each treatment group is displayed. H 2 O 2 is represented as the positive control. ( ** p < 0.01 vs . 0 μM imipramine; ## and $$ p < 0.01 vs . imipramine alone).

Article Snippet: Primary antibodies against Matrix metalloproteinase-9 (MMP-9) (AB19016, Millipore), vascular endothelial growth factor (VEGF) (ab1316, Abcam, Cambridge, UK), EGFR (Try 1068) (#2234, Cell signaling, Danvers, MA, USA), EGFR (E-AB-63555, Elabscience, Houston, TX, USA), PKC-δ (Thr507) (E-AB-20968, Elabscience), PKC-δ (E-AB-14675, Elabscience), NF-κB p65 (Ser536) (E-AB-70335, Elabscience), NF-κB p65 (E-AB-22066, Elabscience), cell leukemia-1 (MCL-1) (BV-438, BioVision), cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) (D16A8, Cell signaling), X-linked inhibitor of apoptosis protein (XIAP) (PA5-29253, Thermo Fisher Scientific), Fas (E-AB-40063, Elabscience), Fas ligand (FasL) (E-AB-31410, Elabscience), cleaved caspase-3 (E-AB-30004, Elabscience), cleaved caspase-8 (E-AB-22107, Elabscience), cleaved caspase-9 (#9505, Cell Signaling Technology), PARP-1 (#9532, Cell Signaling Technology) and β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, Texas, Waltam, MA, USA) for Western blotting were purchased from different companies as listed.

Techniques: Staining, Expressing, Positive Control

Journal: Frontiers in Oncology

Article Title: Suppression of EGFR/PKC-δ/NF-κB Signaling Associated With Imipramine-Inhibited Progression of Non-Small Cell Lung Cancer

doi: 10.3389/fonc.2021.735183

Figure Lengend Snippet: The inhibition of EGFR/PKC-δ/NF-κB proteins phosphorylation and induction of apoptosis-related proteins by imipramine in CL1-5-F4/ NF-κB-luc2 bearing tumor. (A–C) The protein expression from IHC of EGFR (Try 1068), PKC-δ (Thr507), NF-κB (Ser536), cleaved caspase-3, -8, -9, MMP-9, XIAP, MCL-1 and their quantification bar chart are presented. (D, E) The tumor ex vivo Western blotting from each mice of cleaved caspase-3, -8, -9 and PARP-1 is presented. ( ** p < 0.01 vs . vehicle; scale bar =100 μm).

Article Snippet: Primary antibodies against Matrix metalloproteinase-9 (MMP-9) (AB19016, Millipore), vascular endothelial growth factor (VEGF) (ab1316, Abcam, Cambridge, UK), EGFR (Try 1068) (#2234, Cell signaling, Danvers, MA, USA), EGFR (E-AB-63555, Elabscience, Houston, TX, USA), PKC-δ (Thr507) (E-AB-20968, Elabscience), PKC-δ (E-AB-14675, Elabscience), NF-κB p65 (Ser536) (E-AB-70335, Elabscience), NF-κB p65 (E-AB-22066, Elabscience), cell leukemia-1 (MCL-1) (BV-438, BioVision), cellular FLICE (FADD-like IL-1β-converting enzyme)-inhibitory protein (cFLIP) (D16A8, Cell signaling), X-linked inhibitor of apoptosis protein (XIAP) (PA5-29253, Thermo Fisher Scientific), Fas (E-AB-40063, Elabscience), Fas ligand (FasL) (E-AB-31410, Elabscience), cleaved caspase-3 (E-AB-30004, Elabscience), cleaved caspase-8 (E-AB-22107, Elabscience), cleaved caspase-9 (#9505, Cell Signaling Technology), PARP-1 (#9532, Cell Signaling Technology) and β-actin (sc-47778, Santa Cruz Biotechnology, Dallas, Texas, Waltam, MA, USA) for Western blotting were purchased from different companies as listed.

Techniques: Inhibition, Expressing, Ex Vivo, Western Blot

Journal: The Journal of Biological Chemistry

Article Title: Recharacterization of the mammalian cytosolic type 2 ( R )-β-hydroxybutyrate dehydrogenase as 4-oxo- l -proline reductase (EC 1.1.1.104)

doi: 10.1016/j.jbc.2022.101708

Figure Lengend Snippet: Western blot analysis of BDH2 expression in four Bdh2 -deficient HEK293T clonal cell lines ( Bdh2 -KO) and WT HEK293T cells (WT). The KO clonal cell lines (A12, A15, B9, and B13) were generated by the CRISPR–Cas9 gene-inactivation procedure. The Western blot analysis was carried out using 40 μg of the cell lysate protein, a primary rabbit antibody against the human BDH2 (catalog no.: PA5-44760; Invitrogen), and a horseradish peroxidase–conjugated goat anti-rabbit secondary antibody. The secondary antibody was detected by measuring enhanced chemiluminescence. The presence of a nonspecific signal (≈30 kDa) is in agreement with the specification of the primary antibody. BDH2, type 2 ( R )-β-hydroxybutyrate dehydrogenase; HEK293T, human embryonic kidney 293T cell line.

Article Snippet: This vector encodes an enhanced specificity Cas9 enzyme (a gift from Andrea Németh; Addgene; plasmid no.: 101039) ( ).

Techniques: Western Blot, Expressing, Generated, CRISPR

Journal: Developmental cell

Article Title: Cancer Cells Upregulate NRF2 Signaling to Adapt to Autophagy Inhibition

doi: 10.1016/j.devcel.2019.07.010

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Specifically, 200 ng of recombinant Cas9 (PNAbio) was incubated for 10 minutes with 5 ng of each guide RNA (two that target GFP and two that target the gene of interest).

Techniques: Recombinant, Protease Inhibitor, CRISPR, Transfection, Isolation, SYBR Green Assay, Activity Assay, Flow Cytometry, shRNA, Plasmid Preparation

Journal: Hernia

Article Title: What is the evidence for the use of biologic or biosynthetic meshes in abdominal wall reconstruction?

doi: 10.1007/s10029-018-1735-y

Figure Lengend Snippet: CST and biologic mesh

Article Snippet: Clemens [ ] , 2013 , Plast Reconstr Surg , 106/120 CST , 21 ± 9.9 months , 36.6% overall (44.9% PADM vs. 25.5% BADM), surgical complications 29.2%/21.6% , 2.9%/3.9% , 69/120 PADM (98.6% CST) 51/120 BADM (74.5% CST) , , Both BADM and PADM are associated with similar rates of postoperative surgical complications and appear to result in similar outcomes. PADM may be prone to intraoperative device failure.

Techniques: Infection, Preserving, Hood

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