2 6 b r 48 3 7 1  (ATCC)

Journal: ACS synthetic biology

Article Title: The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells

doi: 10.1021/acssynbio.5b00299

Figure Lengend Snippet: Editing efficiency at different Cas9/sgRNA target sites detected by SURVEYOR assays. (a) Diagram of the SURVEYOR assay. INDELS: Insertions and deletions; WT: wildtype (b) Map of all sgRNAs tested in this study. Arrows show Cas9/sgRNA binding sites. (c) SURVEYOR results from a screen to identify sgRNAs with detectable editing rates at the UAS-TK-luciferase reporter in Luc14 cells.

Article Snippet: Plasmid DNA Construction In order to determine transfection efficiencies using flow cytometry, we modified the vectors pX330-U6-Chimeric_BB-CBh-hSpCas9 4 (a gift from Feng Zhang, Addgene plasmid #42230) and pX330A_d-Cas9–1 × 4 [Nakagawa 2015] (a gift from Takashi Yamamoto, Addgene plasmid #63598) using the T2A peptide skipping sequence to express EGFP from the same mRNA transcript as the Cas9 protein.

Techniques: Binding Assay, Luciferase

Journal: ACS synthetic biology

Article Title: The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells

doi: 10.1021/acssynbio.5b00299

Figure Lengend Snippet: Cas9 editing efficiencies at target sites in unsilenced, partially silenced, and fully silenced chromatin states. (a) A map of the Cas9/sgRNA-expressing plasmid. Cas9 and EGFP expression are both driven by the CBh promoter. The T2A signal allows EGFP to be translated as a separate peptide to avoid interference of Cas9 function. (b) Table of average frequencies of EGFP-expressing cells (transfection efficiencies) as determined by flow cytometry of triplicate samples for transfected Luc14, GAL4EED, and GAL4EED cells treated with doxycycline. (c) Mean editing frequencies normalized to transfection efficiency in Luc14, GAL4EED, or GAL4EED + dox cells for Cas9 targeted to sites sg046, sg055, sg032, sg034, sg054, sg031, sg025, sg044, and sg048. *Indicates significantly reduced editing efficiencies at fully silenced chromatin compared to unsilenced chromatin (p < 0.025 for 3 biological replicates). Editing frequencies for target sites sg046, sg055, sg032, and sg054 for both GAL4EED or GAL4EED + dox cell types were below detection limits. Error bars indicate s.d. for n = 3 biological replicates. (d) Summary of the data in (c). Cas9 target sites sg046, sg032, and sg054 show a reduction in editing efficiency in both the partially and fully silenced states compared to the unsilenced states (red arrows). Cas9 target sites sg034 and sg044 show a reduction in editing efficiency in the fully silence states compared to the unsilenced states (yellow arrows).

Article Snippet: Plasmid DNA Construction In order to determine transfection efficiencies using flow cytometry, we modified the vectors pX330-U6-Chimeric_BB-CBh-hSpCas9 4 (a gift from Feng Zhang, Addgene plasmid #42230) and pX330A_d-Cas9–1 × 4 [Nakagawa 2015] (a gift from Takashi Yamamoto, Addgene plasmid #63598) using the T2A peptide skipping sequence to express EGFP from the same mRNA transcript as the Cas9 protein.

Techniques: Expressing, Plasmid Preparation, Transfection, Flow Cytometry

Journal: ACS synthetic biology

Article Title: The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells

doi: 10.1021/acssynbio.5b00299

Figure Lengend Snippet: Frequencies of mutations in genomic DNA from Cas9/sg034-treated Luc14 or GAL4EED + dox cells. (a) Each row shows the mutated sequence aligned to the wild type sequence (WT). Bar graphs show the percentage of library clones that correspond to the sequence in the same row on the left. (b) The heat map shows the number of times each nucleotide position was affected by a deletion that arose from nonhomologous end joining (NHEJ) repair.

Article Snippet: Plasmid DNA Construction In order to determine transfection efficiencies using flow cytometry, we modified the vectors pX330-U6-Chimeric_BB-CBh-hSpCas9 4 (a gift from Feng Zhang, Addgene plasmid #42230) and pX330A_d-Cas9–1 × 4 [Nakagawa 2015] (a gift from Takashi Yamamoto, Addgene plasmid #63598) using the T2A peptide skipping sequence to express EGFP from the same mRNA transcript as the Cas9 protein.

Techniques: Sequencing, Clone Assay

Journal: ACS synthetic biology

Article Title: The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells

doi: 10.1021/acssynbio.5b00299

Figure Lengend Snippet: Chromatin mapping data show differential enrichment of H3K27me3 and dCas9 at luciferase in the open, partially closed, and closed chromatin states. (a) Cross-linked, sheared chromatin was prepared from Luc14 (unsilenced), GAL4EED (partially silenced), or GAL4EED + dox (fully silenced) cells. An anti-H3K27me3 antibody was used to immunoprecipitate (IP) chromatin from untreated cells. An anti-FLAG antibody was used to IP chromatin from dCas9_gRNA-transfected cells. Quantitative PCR (qPCR) was used to measure IP’ed DNA. Primers (described in Methods) and amplicon sizes are shown in the illustrated maps. A primer pair located at the constitutively active GAPDH promoter was used as a negative control to determine off-target binding. (b) Mean H3K27me3 enrichments for Luc14 (unsilenced), GAL4EED (partially silenced), and GAL4EED + dox (fully silenced) cells at four sites spanning the Cas9/sgRNA target sites. Enrichment is shown as percentages of input minus IgG mock IP. (c) Enrichments of IP DNA for dCas9 are shown for three gRNA target sites, sg032, sg034, and sg031 in three chromatin states, unsilenced, partially silenced, and fully silenced. Enrichments are shown as percentages of input minus background (IgG mock IP and GAPDH) (see Methods). In (b) and (c), error bars indicate s.d. for n = 3 replicate IPs from a single chromatin preparation. A one-tailed t test was done to compare Gal4EED or Gal4EED + dox to Luc14 (*p < 0.5, **p < 0.01).

Article Snippet: Plasmid DNA Construction In order to determine transfection efficiencies using flow cytometry, we modified the vectors pX330-U6-Chimeric_BB-CBh-hSpCas9 4 (a gift from Feng Zhang, Addgene plasmid #42230) and pX330A_d-Cas9–1 × 4 [Nakagawa 2015] (a gift from Takashi Yamamoto, Addgene plasmid #63598) using the T2A peptide skipping sequence to express EGFP from the same mRNA transcript as the Cas9 protein.

Techniques: Luciferase, Transfection, Real-time Polymerase Chain Reaction, Amplification, Negative Control, Binding Assay, One-tailed Test

Journal: ACS synthetic biology

Article Title: The Impact of Chromatin Dynamics on Cas9-Mediated Genome Editing in Human Cells

doi: 10.1021/acssynbio.5b00299

Figure Lengend Snippet: Dynamic regulatory states impact Cas9-mediated editing at the luciferase transgene. (a) Illustration of the luciferase transgene in the basal expression state and in different, artificially regulated states. (b) Background-subtracted Luciferase expression levels per cell were measured 96 h after dox treatment (GAL4EED + dox), immediately before transfection with Gal4-p65 plasmid DNA (+p65), or mock-transfection (vehicle only). Luciferase expression was measured in siRNA-treated cells 336 h after transfection. a.u.: arbitrary units. (c) Editing efficiency for Cas9/sg034 was determined by SURVEYOR assays. Editing was reduced in the hyperactive expression state (Luc14 + p65) compared to the Luc14 basal state (*p = 0.018) and the GAL4EED partially repressed state (*p = 0.004). Reversal of the closed state via siRNA treatment (GAL4EED + dox + siRNA) was accompanied by an increase in luciferase expression and editing efficiency (p = 0.045) compared to the fully repressed state. Editing efficiencies for Luc14, GAL4EED, and GAL4EED + dox shown here are the same data shown in Figure 3c.

Article Snippet: Plasmid DNA Construction In order to determine transfection efficiencies using flow cytometry, we modified the vectors pX330-U6-Chimeric_BB-CBh-hSpCas9 4 (a gift from Feng Zhang, Addgene plasmid #42230) and pX330A_d-Cas9–1 × 4 [Nakagawa 2015] (a gift from Takashi Yamamoto, Addgene plasmid #63598) using the T2A peptide skipping sequence to express EGFP from the same mRNA transcript as the Cas9 protein.

Techniques: Luciferase, Expressing, Transfection, Plasmid Preparation