review of crispr/cas9

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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

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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

3

Genome Editing

Genome Editing

Target gene mutation

Knockout gene

Study gene function

3

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

4

2 phases

Create a Double-Stranded Break (DSB)

4

2 phases

Create a Double-Stranded Break (DSB)

Meganucleases

ZFNs

TALENS

4

2 phases

Create a Double-Stranded Break (DSB)

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

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

5

DSB repair mechanisms

5

DSB repair mechanisms

5

Join directly the DNA ends

Prone to errors

DSB repair mechanisms

5

DNA templates

Use a template DNA

Error free

DSB repair mechanisms

Mechanisms of which pathway is taken is not fully understood

DNA templates

5

DSB repair mechanisms

Mechanisms of which pathway is taken is not fully understood

Techniques exists to induce one or another

DNA templates

5

CRISPR/Cas9

A clever immune system

How bacteria prevent DNA invasion from viruses

7

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

10

Cas9 mechanism

PAM

PAM motif (‘NGG’)

mandatory to cleave DNA

10

Cas9 mechanism

PAM

PAM motif (‘NGG’)

mandatory to cleave DNA

2 cleavage domains:

HNH and RuvC-like

RuvC

HNH

10

From an immune system to

an engineered technique

A simplified system

12

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

13

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

14

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

15

Variants of the Cas9 systems: nickase

15

Variants of the Cas9 systems: nickase

Only one strand of the DNA will be cut

15

Variants of the Cas9 systems: CRISPRi

16

Variants of the Cas9 systems: CRISPRi

dCas9

16

No cleavage

domain

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

The Revolution

CRISPR/Cas9 is the new ‘graphene’ hype

Jinek et al, Science 337, 2012

18

CRISPR/Cas9 is the new ‘graphene’ hype

Jinek et al, Science 337, 2012

Now 18

CRISPR/Cas9 is the new ‘graphene’ hype

Jinek et al, Science 337, 2012

Now

> 1000 publications

18

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

Some Applications

Examples of cell types and organisms modified

20

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

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

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

23

Genetically modify human embryos

23

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

Conclusion

Most powerful & easiest tool to genome editing

Most powerful & easiest tool to genome editing

Limitations due to off-targeting

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?”

Thank you