A Basic Introduction In The CRISPR/Cas9 Genome Editing .

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cgec.erasmusmc.nlA basic introduction in the CRISPR/Cas9 genomeediting techniqueEmma de PaterCGECCancer Genome Editing Center

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 lab2

RS1RS2RS3Clustered Regularly Interspaced Short Palindromic Repeats3

RS1RS2crRNARS3tracrRNA4

Cas95

Cas96

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 lab7

CRISPR/Cas9 as a tool for biomedical research8

Genome editing options for CRISPR/Cas9 Generation of: Mutations (large) deletions Integrations (reporters, tags) Activation/repression of transcriptionDNADelete Gene functionIntroduce new gene/sequence9

Non homologous end joining (NHEJ)10

Homology directed repair (HDR)11

What to think of when you design your experiment Cas9 delivery Off target effects Repairable cell Editing efficiency

Generating a patiënt specific mutationDNADelete Gene functionIntroduce new gene/sequence**PuroLoxPLoxP13

Off target effects14

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 lab15

How to make CRISPR/Cas9 more specific?16

Kleinstiver, Nature, 2016 (HF-Cas9)Slaymaker, Science, 2015 (eSpCas9)17

Base editingKomor et al., Nature, 2016 (Cytidine deaminase) C TGaudelli et al., Nature in press (deoxyadenosine deaminase) A G18

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!19

Modification of gene expression Activation (CRISPRa) Repression (CRISPRi)20

How it works in the labVisit cgec.erasmusmc.nl for a detailed protocol 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 GFPcontrol vector Pick and screen clones by PCR/sequence verification21

22

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 exp

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