Journal: Journal of Genetic Engineering & Biotechnology

Article Title: Multiplex CRISPR/Cas9 gene-editing platform in oil palm targeting mutations in EgFAD2 and EgPAT genes

doi: 10.1186/s43141-022-00459-5

Figure Lengend Snippet: In vitro assay to evaluate cleavage efficiency by the Guide-it sgRNA screening kit. ( a ) PCR products from the amplification of EgFAD2 (lane 1: sgRNA1, lane 2: sgRNA2) and EgPAT (lane 3: sgRNA3, lane 4: sgRNA4) sgRNA templates at 130 bp for in vitro transcription. ( b ) PCR products are amplified from oil palm genomic DNA containing sgRNA target sites; the PCR templates of candidate sgRNAs are then in vitro combined with a recombinant Cas9 for a fragment cleavage site ( c ). EgFAD2-T1 (lane 1), EgFAD2-T2 (lane 2), EgPAT-T1 (lane 3), and EgPAT-T2 (lane 4) represent the PCR fragments with 921 bp, 986 bp, 903 bp, and 978 bp length, respectively. Cleavage fragments were present for all sgRNAs; they were cleaved into two fragments by the sgRNA/Cas9 complex: 503 bp and 395 bp for EgFAD2-T1; 559 bp and 404 bp for EgFAD2-T2; 531 bp and 349 bp for EgPAT-T1; and 287 bp and 668 bp for EgPAT-T2. Lane C is a control fragment that has a size of 614 bp and cleaved DNA of 350 bp and 264 bp. Lane W is a negative control, and lane M is a 1 kb plus DNA marker (Invitrogen)

Article Snippet: The sgRNAs designed for targeting the EgFAD2 and EgPAT genes were evaluated based on target cleavage efficiency using the Guide-it™ sgRNA In Vitro Transcription kit and the Guide-it™ Screening Systems Kit (Takara Bio Inc.) according to the manufacturer’s instructions.

Techniques: In Vitro, Amplification, Recombinant, Control, Negative Control, Marker

Journal: The Journal of Biological Chemistry

Article Title: Dicer Knockdown Inhibits Endothelial Cell Tumor Growth via MicroRNA 21a-3p Targeting of Nox-4 *

doi: 10.1074/jbc.M113.519264

Figure Lengend Snippet: MicroRNA biogenesis is essential for endothelial cell tumor formation. EOMA cell stable transfectants were generated using lentiviral shRNA delivery. A and B, targeted post-transcriptional gene silencing of dicer was confirmed by Western blot (A) and real time PCR (B). C and D, Matrigel angiogenesis assay was used to evaluate the functional effects of dicer knockdown in vitro. Cells were stained with calcein-AM (C), and the area within the formed tubes was quantitated by analyzing three high powered fields per well using AxioVision Rel 4.8 software (D). Matrigel tube formation was compromised in dicer knockdown EOMA cells. E, reduced tumor size in response to dicer knockdown. F, incidence of tumor formation in mice injected with EOMA cells transfected with controls shRNA or dicer shRNA. G, tumor volume as quantified using calipers (length × width × height). The results are means ± S.D. of at least three independent experiments. *, p < 0.05.

Article Snippet: In Vitro Angiogenesis Assay Four-well plates were coated with 100 μl of Matrigel® (Cultrex® Basement membrane extract reduced growth factor; R&D Systems, Minneapolis, MN) and let to solidify for 30 min at 37 °C.

Techniques: Generated, shRNA, Western Blot, Real-time Polymerase Chain Reaction, Angiogenesis Assay, Functional Assay, In Vitro, Staining, Software, Injection, Transfection

Journal: bioRxiv

Article Title: A Cost-Effective CRISPR/Cas9-Based Method for Sequencing the Functional Antibody Kappa Chain cDNA from Hybridomas Expressing Aberrant Kappa Chain mRNAs

doi: 10.1101/2023.06.08.544271

Figure Lengend Snippet: (A) shows the schematics of the proof of concept experiment. 4ug of the vector was first linearized using Seal (NEB) and rCutSmart buffer (NEB), the reaction is incubated for 1 hour at 37°C. The reaction then was run on a 1% agarose gel and purified. Next, the vector was digested using CRISPR/Cas9 (3μl 10X Cas9 buffer, l00dng DNA, 1 μl of 2μM sgRNA, 2μl of lμM Cas9, nucleases free water to bring up the total reaction volume to 30μl) the reaction was incubated at 37°C for 60 min followed by 10 minutes at 65°C. (B) shows the reaction products and appropriate controls run on a 1% agarose gel at 100V for 30 minutes.

Article Snippet: CRISPR/Cas9 digestion reaction was performed based on the following protocol: 3μl of the 10X CRISPR in vitro digestion buffer (20mM HEPES, 100mM NaCl, 5mM MgCl2, 0.1mM EDTA, pH 6.5), 1μl of 2μM sgRNA, 2μl of 1μM Cas9 protein, (Origene, TP790148), and 15ng of the cDNA (a molar ratio of 40:40:1) were added to a PCR tube and the volume was brought up to 30μl using nuclease free water.

Techniques: Plasmid Preparation, Incubation, Agarose Gel Electrophoresis, Purification, CRISPR

Journal: bioRxiv

Article Title: A Cost-Effective CRISPR/Cas9-Based Method for Sequencing the Functional Antibody Kappa Chain cDNA from Hybridomas Expressing Aberrant Kappa Chain mRNAs

doi: 10.1101/2023.06.08.544271

Figure Lengend Snippet: (A) shows the image of the CRISPR/Cas9 digested PCR product after the first digestion, the 560 bp band was purified and used as template for the second PCR. (B) shows the second PCR amplicon after the second CRISPR/Cas9 digestion. The 560 bp band was cloned and sequenced. (C) shows the sequencing results of the cloned fragment. This method decreased the percentage of the aberrant kappa chain transcripts by 88% in clone 262. (D) shows the comparison between method 1 versus method 2 in the percent reduction of the SP2/0 aberrant kappa chain sequences.

Article Snippet: CRISPR/Cas9 digestion reaction was performed based on the following protocol: 3μl of the 10X CRISPR in vitro digestion buffer (20mM HEPES, 100mM NaCl, 5mM MgCl2, 0.1mM EDTA, pH 6.5), 1μl of 2μM sgRNA, 2μl of 1μM Cas9 protein, (Origene, TP790148), and 15ng of the cDNA (a molar ratio of 40:40:1) were added to a PCR tube and the volume was brought up to 30μl using nuclease free water.

Techniques: CRISPR, Purification, Amplification, Clone Assay, Sequencing