A basic introduction in the CRISPR/Cas9 genome editing ... Cancer Genome Editing Center cgec

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  • A basic introduction in the CRISPR/Cas9 genome

    editing technique

    Emma de Pater

    CGEC Cancer Genome Editing Center

    cgec.erasmusmc.nl

  • 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

    3

  • R R R S1 S2 S3

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    crRNA tracrRNA

  • Cas9

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  • Cas9

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  • 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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  • CRISPR/Cas9 as a tool for biomedical research

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  • 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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  • Non homologous end joining (NHEJ)

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  • Homology directed repair (HDR)

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  • What to think of when you design your experiment

     Cas9 delivery

     Off target effects

     Repairable cell

     Editing efficiency

  • Generating a patiënt specific mutation

    Delete Gene function

    DNA

    Introduce new gene/sequence

    *

    Puro

    LoxP LoxP

    *

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  • Off target effects

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  • 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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  • Slaymaker, Science, 2015 (eSpCas9) 17

    Kleinstiver, Nature, 2016 (HF-Cas9)

  • Base editing

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    Komor et al., Nature, 2016 (Cytidine deaminase) C>T

    Gaudelli et al., Nature in press (deoxyadenosine deaminase) A > G

  • 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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  • 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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    http://www.ensembl.org/ http://crispr.mit.edu/ https://chopchop.rc.fas.harvard.edu/

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