Journal: Biological & pharmaceutical bulletin

Article Title: Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

doi: 10.1248/bpb.b16-00542

Figure Lengend Snippet: Fig. 1. Enzymatic Activity of Cas9 Was Confirmed by in Vitro Plasmid Cleavage Assay

Article Snippet: The membranes were blocked in Blocking One (Nacalai Tesque, Kyoto, Japan) and then incubated with diluted Guide-itTM Cas9 Polyclonal Antibody (TaKaRa Bio USA, Mountain View, CA, U.S.A.).

Techniques: Activity Assay, In Vitro, Plasmid Preparation, Cleavage Assay

Journal: Biological & pharmaceutical bulletin

Article Title: Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

doi: 10.1248/bpb.b16-00542

Figure Lengend Snippet: Fig. 2. Cas9 Was Digested in Simulated Gastric Fluid (SGF) for the Indicated Periods (min)

Article Snippet: The membranes were blocked in Blocking One (Nacalai Tesque, Kyoto, Japan) and then incubated with diluted Guide-itTM Cas9 Polyclonal Antibody (TaKaRa Bio USA, Mountain View, CA, U.S.A.).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

doi: 10.1248/bpb.b16-00542

Figure Lengend Snippet: Fig. 3. Cas9 Was Digested in Simulated Gastric Fluid for the Indicated Periods (min), Which Were Longer Than Those in Fig. 2

Article Snippet: The membranes were blocked in Blocking One (Nacalai Tesque, Kyoto, Japan) and then incubated with diluted Guide-itTM Cas9 Polyclonal Antibody (TaKaRa Bio USA, Mountain View, CA, U.S.A.).

Techniques:

Journal: Biological & pharmaceutical bulletin

Article Title: Cas9 in Genetically Modified Food Is Unlikely to Cause Food Allergy.

doi: 10.1248/bpb.b16-00542

Figure Lengend Snippet: Fig. 4. Thermal Stability of Cas9 Was Examined

Article Snippet: The membranes were blocked in Blocking One (Nacalai Tesque, Kyoto, Japan) and then incubated with diluted Guide-itTM Cas9 Polyclonal Antibody (TaKaRa Bio USA, Mountain View, CA, U.S.A.).

Techniques:

Journal: Cell Communication and Signaling : CCS

Article Title: Targeting the orphan nuclear receptor NR2F6 in T cells primes tumors for immune checkpoint therapy

doi: 10.1186/s12964-019-0454-z

Figure Lengend Snippet: CRISPR/Cas9 mediated gene knockout in primary mouse T cells. a Schematic overview of CRISPR/Cas9 mediated gene knockout in isolated CD4 + T cells from Cas9 transgenic mice. b 10 days post-treatment, flow cytometry assays were performed to measure the loss of CD44 or CD69 in CD4 + Cas9 transgenic T cells targeted with sgRNAs against CD44 or CD69. c Efficient gene deletion achieved in primary T cells cells following treatment with different sgRNAs. Knockout efficiencies were calculated based on surface marker expression in comparison to NTC treated cells (CD44 KO: d6 p = 0.0002, d10 p = 0.0009, d13 p = 0.0026, CD69 KO: d6 p = 0.0003, d10 p = 0.0009, d13 p = 0.0062). d FACS plots and e quantification of CD4 + T cells with NTC or Nr2f6 CRISPR/Cas9 mediated knockout on day 10, re-stimulated with PdBU/Ionomycin for 4 h showing enhanced IFNγ cytokine production with Nr2f6 loss compared to NTC control cells ( p = 0.0429). NTC, non-targeting control, sgRNA, single guide RNA, Cas9 Tg, Cas9 transgenic. The above experiments are repeated at least two times with similar results. Error bars represent the mean ± SEM

Article Snippet: The efficiency of sgRNA cleavage was tested on gDNA from wild-type thymocytes with the Guide-itTM sgRNA Screening Kit from Takara (632639).

Techniques: CRISPR, Gene Knockout, Isolation, Transgenic Assay, Flow Cytometry, Knock-Out, Marker, Expressing, Comparison, Control

Journal: Cell Communication and Signaling : CCS

Article Title: Targeting the orphan nuclear receptor NR2F6 in T cells primes tumors for immune checkpoint therapy

doi: 10.1186/s12964-019-0454-z

Figure Lengend Snippet: sgRNA target sequence

Article Snippet: The efficiency of sgRNA cleavage was tested on gDNA from wild-type thymocytes with the Guide-itTM sgRNA Screening Kit from Takara (632639).

Techniques: Sequencing

Journal: Cell Communication and Signaling : CCS

Article Title: Targeting the orphan nuclear receptor NR2F6 in T cells primes tumors for immune checkpoint therapy

doi: 10.1186/s12964-019-0454-z

Figure Lengend Snippet: Validation of sgRNAs-mediated knockout targeting NR2F6 in primary lymphocytes. a Schematic of the sgRNA-targeting sites within the Nr2f6 genomic locus. The sgRNA-targeting sequence in red, the protospacer-adjacent motif (PAM) sequence in bold grey and flanking primer pairs (bold, underlined, italics). b Detection of sgRNA Cas9-mediated cleavage of Nr2f6 by PCR with the Takara kit on wildtype thymocytes for sgRNA.Nr2f6.03 and sgRNA.Nr2f6.04 including the kit control and untreated cells without cleavage. c T7 cleavage assay on genomic DNA isolated from CD3 + Cas9 transgenic T cells using sgRNA.Nr2f6.04 or combining sg. RNA.Nr2f6.04 and sg. RNA.Nr2f6.05 (sgRNA.Nr2f6.4.5) including a non targeting control (NTC) sg. RNA. Results shown are derived from at least two independent experiments. Untr., untreated, NTC, non-targeting control

Article Snippet: The efficiency of sgRNA cleavage was tested on gDNA from wild-type thymocytes with the Guide-itTM sgRNA Screening Kit from Takara (632639).

Techniques: Biomarker Discovery, Knock-Out, Sequencing, Control, Cleavage Assay, Isolation, Transgenic Assay, Derivative Assay

Journal: Communications biology

Article Title: Severe cardiac and skeletal manifestations in DMD-edited microminipigs: an advanced surrogate for Duchenne muscular dystrophy.

doi: 10.1038/s42003-024-06222-5

Figure Lengend Snippet: Fig. 1 | CRISPR/Cas9-mediated generation of dystrophin-deficient microminipigs. a CRISPR/ Cas9-mediated gene-targeting strategy to generate dystrophin-deficient microminipigs. The target sequence and protospacer adjacent motif in the CRISPR/Cas9 genome editing system are indicated with a blue arrow and as PAM. The arrows in the diagram of the pig DMD gene indicate the positions of the promoters of the dystrophin isoforms. b Out- of-frame deletion of exon 23 of the microminipig DMD gene produced using the CRISPR/Cas9 gene- editing system. The numbers indicate the positions of the bases from the 5′ end of exon 23, which consists of 213 bases. c Three-month-old DMD- edited male microminipigs, F2-03 and F2-04. d, e A DMD-edited microminipig (F2-03) at 29 months of age. Oblique frontal standing view and supine view of anesthetised F2-03. The scale bar indicates 10 cm.

Article Snippet: Using a micro-injector (Narishige, Tokyo, Japan), 2–4 pL of a mixture of sgRNA (25 ng/μL) and Cas9 protein (25 ng/μL) (Guide-itTM Recombinant Cas9 Nuclease; Takara Bio) was injected into the cytoplasm of each embryo in PXM-HEPES medium under aerated conditions at 25–30 °C38.

Techniques: CRISPR, Sequencing, Produced