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.
- GPCR signaling
- the genome.
- 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.
- 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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