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CRISPR/Cas9 The new frontier of genome
engineering
03.06.2015
Jennifer A. Doudna & Emmanuelle Charpentier, Science 346, 2014
DOI: 10.1126/science.1258096
Wikipedia
Genome editing is a technique where DNA is
inserted, replaced or removed from a genome
using artificially engineered nucleases
Genome Editing
Target gene mutation
Knockout gene
Study gene function
Create transgenic organism
Synthetic biology
3
Genome Editing
Target gene mutation
Knockout gene
Study gene function
Create transgenic organism
Synthetic biology
Gene therapy
3
2 phases
Create a Double-Stranded Break (DSB)
Let the cell repair mechanisms fix it
Meganucleases
ZFNs
TALENS
CRISPR/Cas9
4
2 phases
Create a Double-Stranded Break (DSB)
Let the cell repair mechanisms fix it
Meganucleases
ZFNs
TALENS
CRISPR/Cas9
Non-Homogolous End Joining (NHEJ)
Homology Direct Repair (HDR) 4
DSB repair mechanisms
Mechanisms of which pathway is taken is not fully understood
Techniques exists to induce one or another
DNA templates
5
How bacteria prevent DNA invasion from viruses
CRISPR
Protospacer
7 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
CRISPR = Clustered Regularly Interspaced Short Palindromic
Repeats CRISPR
Protospacer
Protospacer: Invading DNA from viruses, phages, …
7 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
CRISPR = Clustered Regularly Interspaced Short Palindromic
Repeats CRISPR
Protospacer
Protospacer: Invading DNA from viruses, phages, …
7 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
tracRNA: transactivating CRISPR RNA
tracRNA
tracRNA
8 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
tracRNA: transactivating CRISPR RNA
tracRNA
tracRNA
8 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
tracRNA: transactivating CRISPR RNA
tracRNA
tracRNA
8 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
9 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
9 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
tracRNA will
activate nuclease
Cas9
9 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
Cas9 will search the
matching foreign DNA
to create DSB and
promote degradation
tracRNA will
activate nuclease
Cas9
9 Sander et al, Nature Biotechnology, 32, 347-355, 2014
How bacteria prevent DNA invasion from viruses
9 Sander et al, Nature Biotechnology, 32, 347-355, 2014
New foreign DNA is added to CRISPR regions
Cas9 mechanism
PAM
PAM motif (‘NGG’)
mandatory to cleave DNA
2 cleavage domains:
HNH and RuvC-like
RuvC
HNH
10
A simplified system
Fusion of crRNA and tracrRNA to a single gRNA
(20 bp)
12 Sander et al, Nature Biotechnology, 32, 347-355, 2014
A simplified system
Fusion of crRNA and tracrRNA to a single gRNA
(20 bp)
12 Sander et al, Nature Biotechnology, 32, 347-355, 2014
A simplified system
Fusion of crRNA and tracrRNA to a single gRNA
2-component system
(20 bp)
12 Sander et al, Nature Biotechnology, 32, 347-355, 2014
CRISPR/Cas 9 engineering tool
DNA cleavage is based on RNA/DNA pattern and not
anymore on Protein/DNA
13
CRISPR/Cas 9 engineering tool
DNA cleavage is based on RNA/DNA pattern and not
anymore on Protein/DNA
Change require only in the 20’ first nucleotides of the
gRNA (former crRNA)
13
CRISPR/Cas 9 engineering tool
DNA cleavage is based on RNA/DNA pattern and not
anymore on Protein/DNA
Change require only in the 20’ first nucleotides of the
gRNA (former crRNA)
Possibility of targeting multiple DNA sequences at once
13
CRISPR/Cas 9 engineering tool
DNA cleavage is based on RNA/DNA pattern and not
anymore on Protein/DNA
Change require only in the 20’ first nucleotides of the
gRNA (former crRNA)
Much more easier to target DNA sequence
Possibility of targeting multiple DNA sequences at once
13
Some limitations: off-target
Off-target: tolerance of Cas9 to mismatches in the RNA
guide sequence.
14
Some limitations: off-target
Limited by PAM motif
Off-target: tolerance of Cas9 to mismatches in the RNA
guide sequence.
14
Some limitations: off-target
Limited by PAM motif
Depend of mismatchs locations, lengths, compositions
Off-target: tolerance of Cas9 to mismatches in the RNA
guide sequence.
14
Some limitations: off-target
Limited by PAM motif
Depend of mismatchs locations, lengths, compositions
Off-target: tolerance of Cas9 to mismatches in the RNA
guide sequence.
Difficult to predict 14
Variants of the Cas9 systems: CRISPRi
Repress multiple target genes with reversibility
dCas9
16
No cleavage
domain
Variants of the Cas9 systems: CRISPRi
Repress multiple target genes with reversibility
dCas9
Fuse Cas9 with activator/repressor/fluorescent domains 16
No cleavage
domain
CRISPR/Cas9 is the new ‘graphene’ hype
Jinek et al, Science 337, 2012
Now
> 1000 publications
Dozens of organisms
tested
18
CRISPR/Cas9 is the new ‘graphene’ hype
Jinek et al, Science 337, 2012
Now
> 1000 publications Patent War
Dozens of organisms
tested
18
CRISPR/Cas9 is the new ‘graphene’ hype
Jinek et al, Science 337, 2012
Now
> 1000 publications
Several start-ups
created
Patent War
Dozens of organisms
tested
18
Dynamic Imaging of genomic loci
Chen et al., Cell, 2013, Dynamic imaging of genomic loci in living human cells
by an optimized CRISPR/Cas system 21
Dynamic Imaging of genomic loci
Chen et al., Cell, 2013, Dynamic imaging of genomic loci in living human cells
by an optimized CRISPR/Cas system
Attached a GFP to a nuclease-deficient Cas9 (dCas9)
21
Dynamic Imaging of genomic loci
Chen et al., Cell, 2013, Dynamic imaging of genomic loci in living human cells
by an optimized CRISPR/Cas system
Attached a GFP to a nuclease-deficient Cas9 (dCas9)
21
Dynamic Imaging of genomic loci
Chen et al., Cell, 2013, Dynamic imaging of genomic loci in living human cells
by an optimized CRISPR/Cas system
Attached a GFP to a nuclease-deficient Cas9 (dCas9)
21
First monkeys with customized mutations born
Niu et al., Cell, 2014, doi:
10.1016/j.cell.2014.01.027
22
First monkeys with customized mutations born
Niu et al., Cell, 2014, doi:
10.1016/j.cell.2014.01.027
22
First monkeys with customized mutations born
CRISPR/Cas9 targeting of multiple
genes in monkey embryos
Ppar-g and Rag1 double mutation in
monkeys in one step
Niu et al., Cell, 2014, doi:
10.1016/j.cell.2014.01.027
22
Genetically modify human embryos
Liang et al., Protein Cell, 2015, CRISPR/Cas9-mediated
gene editing in human tripronuclear zygotes
23
Genetically modify human embryos
Liang et al., Protein Cell, 2015, CRISPR/Cas9-mediated
gene editing in human tripronuclear zygotes
Tried to mutate the human β-globin (HBB) gene in ‘non-viable’
embryos (β-thalassaemia)
23
Genetically modify human embryos
Liang et al., Protein Cell, 2015, CRISPR/Cas9-mediated
gene editing in human tripronuclear zygotes
Tried to mutate the human β-globin (HBB) gene in ‘non-viable’
embryos (β-thalassaemia)
7 of 86 embryos were successfully mutated
Much more higher rates of off-targeting
23
Genetically modify human embryos
Liang et al., Protein Cell, 2015, CRISPR/Cas9-mediated
gene editing in human tripronuclear zygotes
Tried to mutate the human β-globin (HBB) gene in ‘non-viable’
embryos (β-thalassaemia)
7 of 86 embryos were successfully mutated
Much more higher rates of off-targeting
Raise huge ethical concerns…
23
Most powerful & easiest tool to genome editing
Limitations due to off-targeting
Works on any DNA (bacteria, mouse, rise, humans…)
Most powerful & easiest tool to genome editing
Limitations due to off-targeting
Works on any DNA (bacteria, mouse, rise, humans…)
Many applications with the different variants
Raise ethical questions: “How can we use this powerful tool
in such a way as to ensure maximum benefit while
minimizing risks?”
Raise ethical questions: “How can we use this powerful tool
in such a way as to ensure maximum benefit while
minimizing risks?”