Journal: Molecular Biology of the Cell

Article Title: CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses

doi: 10.1091/mbc.E18-04-0214

Figure Lengend Snippet: Comparison of efficiency of crRNAs and sgRNAs for gene disruption. (A, B) Quantification of knockout phenotype for cells transfected with 15 nM synthetic crRNA or sgRNA targeting KIF11 (A) or CENPN (B), imaged at the indicated times posttransfection. Mitotic index indicates the percentage of total cells in mitosis. Cas9 expression was induced 18–20 h before transfection for KIF11 or simultaneously with RNA transfection for CENPN. Number of cells counted and statistical comparisons performed can be found in Supplemental Tables S1 and S2. (C–E) Titration of RNA guide concentrations for crRNA and sgRNA targeting KIF11 (C), CENPN (D), and RELA (E). Cells transfected with KIF11-targeting crRNA and sgRNA were treated as described above and imaged at 48 and 30 h posttransfection, respectively, to catch the peak knockout period for each synthetic guide. Cells transfected with CENPN or RELA RNA guides were treated as described in and analyzed at 60 or 72 h posttransfection, respectively. Quantification of the knockout phenotypes was done using immunofluorescence (KIF11/CENPN) and flow cytometry (RELA). Data are displayed as mean with error bars indicating SD.

Article Snippet: Doxycycline was refreshed every 24 h. For reverse transfection of cells on sgRNA arrays (prepared by Persomics; see below), cells were induced over night with 1 mg/l doxycycline hyclate before seeding on the array.

Techniques: Comparison, Disruption, Knock-Out, Transfection, Expressing, Titration, Immunofluorescence, Flow Cytometry

Journal: Molecular Biology of the Cell

Article Title: CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses

doi: 10.1091/mbc.E18-04-0214

Figure Lengend Snippet: Reverse transfection allows efficient delivery of sgRNAs for phenotypic analyses. Immunofluorescence images of HeLa cells that were fixed and imaged 72 h postseeding on printed sgRNA array with control spots or spots containing sgRNA targeting KIF11 (A) or RELA (B). Cas9 expression in these cells was induced 12–18 h before seeding. For the KIF11 sgRNA array, cells were stained for DNA (cyan) and microtubules (red). Dotted boxes (white) indicate magnified region (right). For the RELA sgRNA array, cells were stained for RELA (cyan) and microtubules (red). In all images, the dotted circles (orange) indicate region of printed spot. Scale bar = 100 µm.

Article Snippet: Doxycycline was refreshed every 24 h. For reverse transfection of cells on sgRNA arrays (prepared by Persomics; see below), cells were induced over night with 1 mg/l doxycycline hyclate before seeding on the array.

Techniques: Transfection, Immunofluorescence, Control, Expressing, Staining

Journal: Molecular Biology of the Cell

Article Title: CRISPR/Cas9-based gene targeting using synthetic guide RNAs enables robust cell biological analyses

doi: 10.1091/mbc.E18-04-0214

Figure Lengend Snippet: Reverse transfection allows real-time imaging of depletion phenotypes. Time-lapse stills of HeLa cells seeded on a printed sgRNA array with control spots (left) or spots containing an sgRNA targeting KIF11 (right). Spots were identified using a fluorescence dye-conjugated RNA (channel not shown). Cas9 expression in these cells was induced 12–18 h before seeding. Cells were stained for DNA and imaged in a heated microscope chamber at the indicated time postseeding. Dotted circles (orange) indicate the region of the printed spot. Red arrowheads highlight cells with monopolar spindles. Scale bar = 100 µm.

Article Snippet: Doxycycline was refreshed every 24 h. For reverse transfection of cells on sgRNA arrays (prepared by Persomics; see below), cells were induced over night with 1 mg/l doxycycline hyclate before seeding on the array.

Techniques: Transfection, Imaging, Control, Fluorescence, Expressing, Staining, Microscopy