CRISPR/Cas9 Gene Editing
Discover how to speed up your workflow with Guide-itTM!
Cornelia Hampe, PhD
Technical Support Specialist & Product Manager
Takara Bio Europe
Outline
• Introduction to CRISPR/Cas9 Gene Editing
– What is CRISPR/Cas9?
– How can CRISPR/Cas9 be exploited for genome editing?
• CRISPR/Cas9 in 5 easy steps using Guide-itTM
1. Choose your target
2. Design your sgRNA
3. Test sgRNA efficacy in vitro
4. Deliver Cas9/sgRNA to your cells
5. Monitor Indels
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2016
CRISPR/Cas9 prokaryotic immune system
Clustered
Regularly
Interspaced
Short
Palindromic
Repeats
Adapted from Bhaya et al. Annu Rev Genet. 2011;45:273-97.
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Milestones in the development of CRISPR/Cas9
Cell 157, June 5, 2014
http://zlab.mit.edu/assets/reprints/Hsu_PD_Cell_2014.pdf
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Applications of Genome Engineering
Cell 157, June 5, 2014
http://zlab.mit.edu/assets/reprints/Hsu_PD_Cell_2014.pdf
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Back to basics…
• Cas9 is an endonuclease creating double-strand DNA
breaks (DSB)
• DSB are dangerous DNA lesions!
Cells have evolved 2 repair pathways:
1.
2.
Non-homologous end joining (NHEJ)
• Rapid, flexible, but error prone
Homologous Recombination (HR)
• Slower, restricted to late S and G2
but precise
Adapted from Bee et al. 2013 PLoS One. 2013 Jul 11;8(7)
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Repairing double-strand breaks
Double-strand break
NHEJ
HR
Repair template
X
Error-Prone:
Creates small insertions/deletions
(Indels)
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X
Error-free:
Perfect repair
How can we exploit this process for genome
engineering?
My Favorite Gene
Artificially create a double-strand break
NHEJ
HR
Repair template
GFP
X
X
Knock-Out
Knock-In
GFP
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Genome engineering using ZFNs/TALENs
NHEJ
HR
ZFN = Zinc Finger
Nuclease
TALEN = Transcription
activator–like effector
nuclease
Adapted from Sanjana et al., Nat Protoc. 2012 Jan 5;7(1):171-92.
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Genome engineering using ZFNs/TALENs
Every time you want to target a different sequence,
you have to re-engineer the protein
 Not easy, only a few labs are experts
Adapted from Sanjana et al., Nat Protoc. 2012 Jan 5;7(1):171-92.
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Another great idea ……
What if there was a nuclease…
Nuclease
…that is targeted to a specific genome sequence by RNA
Nuclease
UA G C C G C U
You could change its target specificity really easily and at
low cost - simply change the RNA!
Cas9
UA G C C G C U
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CRISPR/Cas9
guide RNA (sgRNA)
CRISPR/Cas9 Principle
Target specific
sequence
Fixed
sequence
PAM
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Indels
Outline
• Introduction to CRISPR/Cas9 Gene Editing
– What is CRISPR/Cas9?
– How can CRISPR/Cas9 be exploited for genome editing?
• CRISPR/Cas9 in 5 easy steps using Guide-itTM
1. Choose your target
2. Design your sgRNA
3. Test sgRNA efficacy in vitro
4. Deliver Cas9/sgRNA to your cells
5. Monitor Indels
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2016
CRISPR/Cas9
Gene Editing
in 5 easy steps
CRISPR/Cas9 Workflow
Guide-it™ Complete sgRNA Screening System
• Guide-it™ sgRNA In Vitro Transcription Kit
• Guide-it™ sgRNA Screening Kit
Guide-it™ CRISPR/Cas9 Systems (Green or Red)
AAVpro® CRISPR/Cas9 Systems
Xfect RNA Transfection Reagent
Guide-it™ CRISPR/Cas9 Gesicle Production System
Guide-it™ Mutation Detection Kit
Guide-it™ Genotype Confirmation Kit
Guide-it™ Indel Identification Kit
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CRISPR/Cas9 Workflow
• That’s the easiest bit…it’s your
gene of interest
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CRISPR/Cas9 Workflow
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2016
Design your single guide RNA
• There are online tools (e.g., http://crispr.mit.edu/ or
https://chopchop.rc.fas.harvard.edu/)
PAM
PAM
PAM
List of online tools for sgRNA design
PAM
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CRISPR/Cas9 Workflow
Guide-it™ Complete sgRNA Screening System
• Guide-it™ sgRNA In Vitro Transcription Kit
• Guide-it™ sgRNA Screening Kit
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Test efficacy of your sgRNA in vitro
• Guide-it™ Complete sgRNA Screening System
– Guide-it™ sgRNA In Vitro Transcription Kit
– Guide-it™ sgRNA Screening Kit
• All the components you need to:
– Create several sgRNAs by in vitro transcription
– Amplify target DNA to test-cleave
• Cleave your target in vitro using sgRNA and recombinant
Cas9
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sgRNA Screening Kit Workflow
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Why should I test my sgRNA in vitro?
• In vitro testing of guide RNAs enables you to preevaluate the sgRNA efficiency for your target gene
Do not waste time with guide RNAs that don’t work.
Choose and work with the best guide RNA
Compare efficiency of cleavage of potential off-target sites
No cloning steps required with Screening Kit  streamlined
protocol, save time
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Do sgRNAs that work best in vitro
also work best in cells?
• Very good correlation between in vitro test and sgRNA efficiency in
cells (shown for CXCR-4 knockout)
C-
0%
1
~55%
2
~42%
3
4
~8%
~71%
% efficiency of cutting
sgRNA3 didn’t work in vitro
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And in HeLa cells neither
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CRISPR/Cas9 Workflow
Guide-it™ CRISPR/Cas9 Systems (Green or Red)
AAVpro® CRISPR/Cas9 Systems
Xfect RNA Transfection Reagent
Guide-it™ CRISPR/Cas9 Gesicle Production System
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2016
What are gesicles?
• Cell-derived nanovesicles that are
made by a producer cell line after
overexpression of a specific glycoprotein
• Used for delivering proteins (or other
macromolecular cargoes)
• Cargoes can be delivered to a similar
range of cells as would be expected for
VSV-G pseudotyped lentivirus
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What’s the CRISPR/Cas9 Gesicle
Production System ?
• A complete system to produce your own Gesicles to deliver
active Cas9 protein together with a target specific sgRNA
for genome editing experiments.
• What is inside of CRISPR/Cas9 gesicles?
– Gesicles contain both:
• Active Cas9 protein
• Single guide RNA (sgRNA) specific to a target gene
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How are Gesicles produced?
293T cells are cotransfected with targetspecific sgRNA plasmid
and gesicle packaging
mix
Packaging mix contains:
• Xfect Transfection Reagent
• Nanovesicle-inducing
glycoprotein
• Cas9 endonuclease
• CherryPicker RFP
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How are Gesicles produced?
Glycoprotein induces
gesicle formation
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How are Gesicles produced?
iDimerize technology
incorporates Cas9sgRNA complex into
gesicles: Cas9 will
be associated to the
CherryPicker red
fluorescent protein.
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How are Gesicles produced?
Loaded gesicles
pinch off and are
collected from media.
Gesicles can be used
immediately or stored
at -70˚C for over a
year.
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How are Gesicles produced?
Gesicles applied to
target cells fuse and
release Cas9-sgRNA
for editing.
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Gesicle Production Workflow
NLS
Gesicle Producer 293T Cell Line
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Characterization of Cas9 Gesicles
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Gesicle-mediated editing in a wide
range of cell types
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CD81 knockout in Human iPS cells
using gesicles
6h
• iPS cells were treated with
gesicles according to the
standard protocol for either
6 hours our 24 hours
24h
• Culture System:
Cellartis® DEF-CS™ 500
% KO
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-ve Control
+ve Control
6 hrs
24 hrs
99.91%
2.21%
41.21%
47.60%
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Importance of an optimized sgRNA
scaffold
We strongly recommend using the supplied pGuide-it-sgRNA1 Vector
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Dose-dependent efficiency
AcGFP1 knockout in
HT1080-AcGFP1 cells
measured by flow cytometry
6 days after gesicle delivery
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Reduced off-target effects
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Reduced off-target effects
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2016
Reduced off-target effects
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2016
Why use Gesicles for gene editing?
• Work in a broad range of cell types
• Cas9 protein delivery:
– No persistent expression, no risk of genomic integration 
decreased off-target effects
– Not limited by promoters or codon usage
• No toxicity
• Straightforward & complete kit for creating target-specific gesicles
– Make gesicles in producer cells & apply to target cells
– Collect and store gesicles at -70°C
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CRISPR/Cas9 Workflow
Guide-it™ Mutation Detection Kit
Guide-it™ Genotype Confirmation Kit
Guide-it™ Indel Identification Kit
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What is the Mutation Detection Kit for?
• Quickly determine if your gene engineering
experiment was successful, best for populations
Cells with wild type gene
TALENs, ZFNs
Cas9/CRISPRs
Cells with mutated gene
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Mutation Detection Kit Workflow
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Mutation Detection Kit vs Cel1 Assay
• Higher specificity: no smearing in contrast to Cel1 enzyme
• Higher sensitivity: detects a mutation present in less than
20% of genomic DNA pool
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Genotype Confirmation Kit
• Determine genotype of clones following genome editing.
• Simple protocol: PCR amplification of the target site and
in vitro cleavage with Cas9 and the sgRNA used for the
original CRISPR/Cas9 gene editing experiment.
• If indels are present at the target site, the original
sgRNA/Cas9 complex will be unable to cleave the site,
whereas wild-type alleles will be recognized and cleaved.
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Genotype Confirmation Kit
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Genotype Confirmation Kit
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Genotype Confirmation Kit
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Indel Identification Kit
• A complete cloning system for recovery of mutated
sequences so that you can sequence them and
determine the nature of the mutation
• Characterize variety of Indels that are present in the
cellular population
• Determine exact edits at sequence level in single
clones
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Indel Identification Kit Workflow
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Gene editing: different downstream analysis tools
Mutation Detection Kit
•
Quick evaluation of genome editing efficiency (mutation frequency),
best for populations
•
Whole protocol can be completed within 3.5 hours
•
Does not allow to see which mutations exactly occurred
Genotype Confirmation Kit
•
Determine genotype of clones following genome editing: identify WT,
monoallelic and biallelic mutations
•
Quick Screening, whole protocol can be completed within 1-2 days
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Gene editing: different downstream analysis tools
Indel Identification Kit
•
Evaluate genome editing efficiency in a cellular population
(mutation frequency)
•
Whole protocol takes 2-4 days (involves cloning & sequencing)
•
Characterize variety of Indels that are present in the cellular
population
•
Determine exact edits at sequence level in single clones or alleles
Western Blot
•
Confirms functional knock-out
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Summary
• CRISPR/Cas9 is making gene editing far simpler and cheaper compared
to other existing gene editing technologies (i.e. ZFNs, TALENs)
• Guide-it™ tools can help you throughout the whole workflow to:
– Produce sgRNAs and pre-evaluate sgRNA efficacy in vitro
– Efficiently deliver Cas9 and sgRNAs into mammalian cells / in vivo
– Quickly determine mutation frequency in a cellular population
– Confirm genotype of clones
– Determine exact edits in single clones/alleles by sequencing
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Visit our website: www.clontech.com/Guide-it
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