Bo Huang: Seminar 02/12/15

Life inside the Cell: CRISPR GFP and Imagenomics


  • Joined UCSF in 2009. Recently tenured
  • Packard fellow and New innovator award (well deserved)


  • studying cars. Conserved gene driving.
  • one knockout not distinguish method.
  • physical biology approach — direct observation.
    • can see both camps are right – reduce friction and provide driving force.

Projects overview

  • centrosome
  • GPCR signaling
  • the genome.

Genome imaging

  • 100 bp is 30 nm, routine for super-resolution

Bo: what we want

  • sequence flexibility
    • label all DNA or all histone, hard to interpret
    • e.g. Wu lab, Cremer lab, Bo lab FISH studies
    • not what Bo’s been going after
  • minimal perturbation, live cell

Sequence speicfic live labeling

  • replication blocks with EdU incorporation?
    • replication blocks not robust
    • cell doesn’t like replication blocking
  • LacO/TetO insertions?
  • 4 papers recently came out in 2013/2014 using TALE-GFP on repetitive sequences in fixed cells
  • Qi Weisseman Lim 2013 CRISPRi (inteference — regulator by Activator / repressor targeting).
  • collaboration with GFP imaging. with Elizabeth Blackburn (telomeres), Weissman and Qi.
  • even leaky expression of GFP on tetR3G is on the edge of too much GFP. (even though this is a special low leaky version of Tet-On).
  • always get nucleolose strong signal. For 3 months no little dots aroudn the cell.
  • get 1 cell in an ‘extreme’ condition. Need extreme lvels of virus delivery (of guide RNA) to get signal.
  • try stem extension (natural squence is longer). 4Us in a row maybe bad. Try AU enginered flip.
  • combine all these together get much better signal.
  • brightness of telomere spots vary. Different telomere lengths?
  • can reduce and increase telomere length and observe change in brightness.
  • do colocalize the telomere binding proteins (in fixed cells) with GFP.
  • other genes? Go after genes and introns with tandem repeats. MUNC4 gene.
  • send cells for sequencing, validate that chromosome number has gone trisomy.
  • design 73 guide RNAs into a pool of viruses (total viral titer 2x previous)
  • 36 guide RNAs sufficient, 26 to 16 doesn’t work.
  • all 3 copies tend to stay on same side of cell (don’t really need live imaging to do this)
  • confined diffusion movie.
  • G2 cells 20 pairs of spots
  • have to dial down laser intensity a lot to take movie
  • Supernova tag (SUnTag) from Vale lab – attach repetitive epitope to protein, get 24 GFPs to attach to this.
  • use split-GFP (used in GRASP and super-res CALM).
  • reduce background to increase contrast.
  • can make YFP or CFP mutation on the split GFP11 component. Photo-activatable GFP.
  • tandem GFP-11s on target protein. add GFP 1-10 to increase signal with high contrast.


  • perturbation?
    • doesn’t block transcription (does in E coli?)
    • doesn’t block replication
    • local unwiding of DNA
    • nucleosome will have to be moved out of the way. Can’t co-occupy
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