SLIDE 1 A basic introduction in the CRISPR/Cas9 genome editing technique
Emma de Pater CGEC
Cancer Genome Editing Center
cgec.erasmusmc.nl
SLIDE 2 CRISPR/Cas9
- CRISPR/Cas9 – The immune system of bacteria
- CRISPR/Cas9 – as a biomedical tool
- What to think of when you design your experiment
- Cas9 Variants
- CRISPR in the lab
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Clustered Regularly Interspaced Short Palindromic Repeats R R R S1 S2 S3
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SLIDE 4
R R R S1 S2 S3
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crRNA tracrRNA
SLIDE 5
Cas9
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SLIDE 6
Cas9
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SLIDE 7 CRISPR/Cas9
- CRISPR/Cas9 – The immune system of bacteria
- CRISPR/Cas9 – as a biomedical tool
- What to think of when you design your experiment
- Cas9 Variants
- CRISPR in the lab
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SLIDE 8
CRISPR/Cas9 as a tool for biomedical research
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SLIDE 9 Genome editing options for CRISPR/Cas9
- Generation of:
- Mutations
- (large) deletions
- Integrations (reporters, tags)
- Activation/repression of transcription
Delete Gene function Introduce new gene/sequence DNA
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SLIDE 10
Non homologous end joining (NHEJ)
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SLIDE 11
Homology directed repair (HDR)
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SLIDE 12 What to think of when you design your experiment
- Cas9 delivery
- Off target effects
- Repairable cell
- Editing efficiency
SLIDE 13 Generating a patiënt specific mutation
Delete Gene function DNA Introduce new gene/sequence *
Puro
LoxP LoxP
*
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SLIDE 14
Off target effects
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SLIDE 15 CRISPR/Cas9
- CRISPR/Cas9 – The immune system of bacteria
- CRISPR/Cas9 – as a biomedical tool
- What to think of when you design your experiment
- Cas9 Variants
- CRISPR in the lab
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How to make CRISPR/Cas9 more specific?
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SLIDE 17
Slaymaker, Science, 2015 (eSpCas9)
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Kleinstiver, Nature, 2016 (HF-Cas9)
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Base editing
18 Komor et al., Nature, 2016 (Cytidine deaminase) C>T Gaudelli et al., Nature in press (deoxyadenosine deaminase) A > G
SLIDE 19 How to deal with off-target effects
- Check your clone with NGS
- Redesign your guide
- HF or eCas9
- Nickase Cas9
- Use multiple clones or multiple guides
- Use a hit and run method (ribonuclearprotein transfection)
- Backcross your mouse line
- When Cas9 is loaded, fewer off-targets!
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- Modification of gene expression
- Activation (CRISPRa)
- Repression (CRISPRi)
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SLIDE 21 How it works in the lab
- Make sure your target sequence is what you think
- www.ensembl.org (and sequence verify)
- Design your guide (GG-18N-NGG)
- crispr.mit.edu/
- chopchop.rc.fas.harvard.edu/
- Clone your guide into proper Cas9 expression vector
- Transfect your cells
- Cas9 is large, make sure you get ~90% efficiency with a GFP
control vector
- Pick and screen clones by PCR/sequence verification
Visit cgec.erasmusmc.nl for a detailed protocol
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