Download The Copernican revolution of the biology

The Copernican Revolution of biology
The Copernican Revolution of biology
Iris Jonkers & John T. Nature Reviews Genetics 16, 2015; Lis, Lanctôt et al. Nature Reviews Genetics 8, 2007
Hopper, The nucleus and sub-nuclear domains, abcam
LADs= Lamina Associates Domains
LOCKs= Large Organized Chromatin K9
Modification
H3K9me2= Histone 3 Lysine 9 dimethylated
• Transcription
• DNA repair
• Splicing
• Gene Silencing
• Circadian rhythm
• Ecc.
Oscillating recruitment of CLOCK-controlled
genes
Repressed
compartment
Zhao et al, Mol Cell 2015
Nuclear envelope
What is our telescope?
What is our telescope?
We need a method to
visualize chromatin
fibers in the nucleus
and to monitor their
movements! mmm
3D DNA-FISH,
of course!!!!
I’m sorry, DNA-FISH is
not enough. With that
technique you can only
monitor transient event
in fixed cells.
…. Dammit!!!
And now???
Supervisor
PhD student
Reviewer
No problem, the
solution for you
is…
ChrMoTime
Chromatin
Movements
in real-time
… a CRISPR/dCas9
based method
ChrMoTime
Chromatin Movements
in real-time
Yheeee!!!!!
Thanks
ChroMoTime!
CRISPR/Cas9 technique
2014
CRISPR/Cas9 technique
Alex Reis et al., New England Biolabs, 2014
CRISPR/Cas9 technique
Simple, high efficient and versatile!
The new era of genetic engineering!!!
ChrMoTime:
Chromatin Movements in real-time
dCas9:
Nuclease-deficient Cas9
dCas9
gRNA
Alex Reis et al., New England Biolabs, 2014
ChrMoTime:
Chromatin Movements in real-time
Repressed
compartment
• Live imaging in real-time
• No fixed cells
• You only need the
sequence of your target
and ChrMoTime kit
• Simple protocol
• Fluorophores combination
• Commercial gRNAs
available
• Bio-informatics tools
Nuclear envelope
ChrMoTime: applied example
Visualization of
circadian chromatin movements in real-time
• Relative mobility of the core clockwork and PARP1 genes in
response to serum shock
• Sensitivity of circadian chromatin mobility to transcriptional
inhibitors
• Role of core clockwork in the recruitment and release of circadian
genes to and from the lamina
ChrMoTime:
Chromatin Movements in real-time
Not be in late and
not lost time!!!!
This is the era of
CRISPR/Cas9, this is the
moment of
ChrMoTime!!!
Your useful kit for real-time