Journal: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 1. gRNA Design for Cpf1, Cas9, and Cas9-EQR Systems and Dual-Fluorescence Reporter Plasmids for Evaluation of Editing Efficacy and Selectivity (A) Design of single-guide RNAs (sgRNAs) for Cpf1, Cas9, and Cas9-EQR systems targeting mutant BRAF allele, with a hypothesis that they cannot bind and cleave wild-type BRAF allele. Two gRNAs (gRNA-1 and gRNA-2) were designed for both AsCpf1 and LbCpf1. gRNA-3 was used for spCas9. Mutant BRAF forms a PAM of spCas9 EQR variant, which is absent from the wild-type BRAF, and thus gRNA-4 was designed for Cas9-EQR. (B) Schematic illustration of the GFP-RFP reporter plasmids for evaluation of the editing efficacy and selectivity. The reporter vector contains a CMV promoter and sequences encoding EGFP and RFP, which are separated by multiple cloning sites. gRNA binding regions and PAM motifs were inserted between EcoRI and XhoI sites, and this insertion caused RFP coding region to be shifted out of frame. Target cleavage and subsequent non-homologous end-joining repair can lead to sequence indels/frameshifts and bring the downstream RFP back in frame.

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Fluorescence, Mutagenesis, Variant Assay, Plasmid Preparation, Cloning, Binding Assay, Non-Homologous End Joining, Sequencing

Journal: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 2. Evaluation of Editing Efficacy and Selectivity in HEK293T Cells (A) Transient transfection of GFP-vehicle-RFP control, GFP-WT-RFP reporter, and GFP-Mut-RFP reporter in HEK293T cells for 48 hr. (B–E) Evaluation of the editing efficacy and selectivity of the indicated CRISPR systems. All gRNAs were designed for targeting mutant BRAF allele. HEK293T cells constitutively expressing AsCpf1, LbCpf1, Cas9, or Cas9-EQR were established respectively by lentiviral transduction and puromycin selection. Images were acquired 60 hr after co-transfection with indicated GFP-RFP reporter vectors and gRNAs. (F) Statistical analysis of editing efficiency and selectivity by FACS. Error bars represent SD from experiments performed in triplicates. One-way ANOVA: *p < 0.05, **p < 0.01. WT, wild-type; Mut, mutant. The scale bar represents 200 mm.

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Transfection, Control, CRISPR, Mutagenesis, Expressing, Transduction, Selection, Cotransfection

Journal: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 3. Representative Sequencing Results of Corresponding DNA Samples in HEK293T Cells Sixty hours after co-transfection as described above, genome DNA was extracted and subjected to PCR amplification. PCR products were sequenced directly. All gRNAs were designed for targeting mutant BRAF allele as indicated. (A, C, and E) Sequencing results of gene editing of wild-type allele. (B, D, and F) Results of mutant allele. Regions with continuous overlapping peaks, caused by cleavage and subsequent non-homologous end-joining repair, are indicated with dashed boxes. Alignment to the mutant BRAF regions inserted in EGFP-Mut-RFP reporter is shown in (F).

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Sequencing, Cotransfection, Mutagenesis, Non-Homologous End Joining

Journal: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 4. Evaluation of Editing Efficacy and Selectivity in Cpf1-gRNA-Treated A375, a Melanoma Cell Line with a Homozygous BRAF V600E Mutation (A) Schematic illustration of the lentiviral vectors used for gRNA expressing and fluorescently labeling. (B) Highly efficient transduction of A375 stable cells with indicated lentiviral vectors. Images were acquired on day 2 following transduction. (C) Cpf1 stable cells were transducted with vehicle or gRNA-expression lentiviral vectors. Images were acquired on day 7 following transduction. A375 cells constitutively expressing AsCpf1 or LbCpf1 were established respectively by lentiviral transduction and puromycin selection. (D) Statistical analysis of cell viability by trypan blue exclusion test. Error bars represent SD from experiments performed in triplicate. One-way ANOVA analysis: NS, not significant; **p < 0.01. (E) Expression status of BRAF, ERK1/2, and pERK1/2 in A375 stable cells following Cpf1-gRNA treatment for 72 hr by western blotting. AsCpf1 positive stable A375 cells transducted with lenti-mCherry-vehicle were set as control group. GAPDH serves as a loading control. AsgRNA, gRNA for AsCpf1; LbgRNA, gRNA for LbCpf1. The scale bar represents 100 mm.

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Mutagenesis, Expressing, Labeling, Transduction, Selection, Western Blot, Control

Journal: Molecular therapy. Nucleic acids

Article Title: Targeted Disruption of V600E-Mutant BRAF Gene by CRISPR-Cpf1.

doi: 10.1016/j.omtn.2017.05.009

Figure Lengend Snippet: Figure 5. Mutant Sequences Induced at the Cpf1-gRNA-1 Target Site in A375 Cells Genome DNA was extracted after lentiviral transduction of A375 stable cells as described above. All gRNAs were designed for targeting mutant BRAF allele as indicated. Data are representative of three independent experiments. Mutant sequence induced by AsCpf1-gRNA-1 and LbCpf1-gRNA-1 are shown on the left and right, respectively. (A–D) PCR products harboring target region were sequenced directly 72 hr after transduction. Regions with continuous overlapping peaks, caused by cleavage and subsequent non-homologous end-joining repair, are indicated with dashed boxes. (E–J) PCR products harboring target region were subjected to TA cloning 7 days after transduction. In total, 60 clones from three independent samples (20 clones per independent sample) were analyzed by sequencing. (E and F) Representative mutant sequences are shown. The deleted bases are marked with dashes, and the inserted or substituted bases are shown in red. Mutation types and the occurrence of each sequence are indicated to the left of the alignment. Unmodified reads are marked as “WT.” (G and H) Size distribution of deletions identified at the target site. (I and J) Mutation profiling of the sequenced clones.

Article Snippet: After blocking with 5% skimmed milk, membrane was incubated with the following primary antibodies at 4 C overnight: mouse monoclonal antibody to BRAF (Zen BioScience, 1:2000, 200532-4E1), mouse monoclonal antibody to MEK1 (Zen BioScience, 1:1,000, 200424), rabbit monoclonal antibody to ERK1/2 (Cell Signaling Technology, 1:1,000, 4695S), rabbit monoclonal antibody to pERK1/2 (Cell Signaling Technology, 1:2,000, 4370S), and rabbit polyclonal antibody to GADPH (BOSTER, 1:400, BA2913).

Techniques: Mutagenesis, Transduction, Sequencing, Non-Homologous End Joining, TA Cloning, Clone Assay

Journal: Human molecular genetics

Article Title: Activated Braf induces esophageal dilation and gastric epithelial hyperplasia in mice.

doi: 10.1093/hmg/ddx354

Figure Lengend Snippet: Figure 3. Delayed esophageal musculature in BrafQ241R/þ mice. (A) Sections of the LES from 10-week-old Brafþ/þ and BrafQ241R/þ mice were stained with antibodies

Article Snippet: Immunohistochemistry was performed with the Histofine simple stain kit (Nichirei Bio Sciences, Tokyo, Japan) and the antibodies to the following proteins (with catalog numbers in parentheses): BRAF (9433) from Cell Signaling; a-SMA (M0851) and SA (M0874) from DAKO; PGP9.5 (AB1761-I) from Millipore; Ki67 (418071) from Nichirei Bio Sciences; and Keratin 5 (905501) from BioLegend.

Techniques: Staining

Journal: Human molecular genetics

Article Title: Activated Braf induces esophageal dilation and gastric epithelial hyperplasia in mice.

doi: 10.1093/hmg/ddx354

Figure Lengend Snippet: Figure 5. Increased expression of cell adhesion- and epithelium development-associated genes in the forestomach in BrafQ241R/þ mice. (A) Longitudinal forestomach sections from Brafþ/þ and BrafQ241R/þ mice at P 1, 7, 10 and 14 were stained with an antibody against S100. (B) Longitudinal forestomach sections from 10-week-old

Article Snippet: Immunohistochemistry was performed with the Histofine simple stain kit (Nichirei Bio Sciences, Tokyo, Japan) and the antibodies to the following proteins (with catalog numbers in parentheses): BRAF (9433) from Cell Signaling; a-SMA (M0851) and SA (M0874) from DAKO; PGP9.5 (AB1761-I) from Millipore; Ki67 (418071) from Nichirei Bio Sciences; and Keratin 5 (905501) from BioLegend.

Techniques: Expressing, Staining