crispr design webpage tool  (Benchling Inc)

Journal: Journal of Cell Science

Article Title: Kinesin-1 transports morphologically distinct intracellular virions during vaccinia infection

doi: 10.1242/jcs.260175

Figure Lengend Snippet: Quantifying the number of kinesin-1 complexes on IMVs and IEVs. (A) Schematic of the intracellular TagGFP2-tagged 60-mer nanocage. Each subunit of the nanocage (grey) is fused with TagGFP2 (green) and FKBP (blue), although this is shown only for one subunit for clarity. FRB (pink) is targeted to the plasma membrane by its palmitoylation and myristoylation sequence and dimerises with FKBP in the presence of the rapamycin analogue AP21967. PDB structures used: 5KP9 , 2Y0G and 4DRI . (B) Representative average-intensity projection images of transiently expressed TagGFP2-tagged nanocages in HeLa cells treated with 500 nM AP21967. Scale bar: 2 µm. (C) Quantification of fluorescence intensities of TagGFP2-tagged 24-, 60-, 120- and 180-mer nanocages. Error bars represent mean±s.d. Linear line of regression is fitted. n =51–114 measurements per nanocage from three independent experiments. (D) Immunoblot analysis of total cell lysates from HeLa wild-type (WT) or TagGFP2–KIF5B CRISPR knock-in (KI) cells using the indicated antibodies. (E) Representative images from time-lapse movie showing the association of kinesin-1 (green) with IMVs (magenta) during moving (red arrowheads) and stationary (blue arrowheads) phases in the HeLa TagGFP2–KIF5B knock-in cell line at 7.5 h post infection with the ΔB5 RFP–A3 virus (see Movie 8 ). Time in seconds is indicated in each image. Scale bar: 2 µm. The graph on the right shows quantification of the TagGFP2–KIF5B:RFP–A3 fluorescence intensity ratio on IMV particles during moving and stationary phases. n =11 virions from two independent experiments. (F) Representative average-intensity projections of endogenously expressed TagGFP2–KIF5B (green) on IEVs or IMVs (magenta) in HeLa TagGFP2–KIF5B knock-in cells 7.5 h post infection with ΔB5 RFP–A3 (left) or WR B5-RFP (right). Scale bar: 2 µm. (G) The left graph shows the mean background-subtracted fluorescence intensity of TagGFP2–KIF5B together with the calculated number of molecules on IMVs and IEVs, superimposed (dotted red lines) on the nanocage calibration plot from C. The table below shows the summary of the readout. SuperPlot (right) showing the number of kinesin-1 complexes associated with IMVs or IEVs from three independent experiments in which 84 and 121 virions were analysed for IMVs and IEVs, respectively. Bars represent mean±s.e.m. Two-tailed unpaired ­Student's t -test was used to determine statistical significance. ** P ≤0.01. (H) SuperPlots showing the background-subtracted antibody intensity signals of KIF5B associated with IMVs (left graph) or IEVs (right graph) in HeLa wild-type (WT) or tagGFP2–KIF5B knock-in (KI) cells. The fold difference between the mean number of KIF5B associated with virions in WT or KI cells is shown. The table summarises the mean number of kinesin-1 complexes associated with IMVs or IEVs in HeLa WT or KI cells after correcting for low levels of tagGFP2–KIF5B expression in the latter. a.u., arbitrary units.

Article Snippet: The guide RNA (gRNA) for KIF5B was designed using a CRISPR design webpage tool ( https://www.benchling.com/ ).

Techniques: Clinical Proteomics, Membrane, Sequencing, Fluorescence, Western Blot, CRISPR, Knock-In, Infection, Virus, Two Tailed Test, Expressing