Life inside the Cell: CRISPR GFP and Imagenomics

Background

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

Intro

• 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.

Questions

• 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