crispr/cas9 gene editing -...
TRANSCRIPT
CRISPR/Cas9 Gene EditingDiscover how to speed up your workflow with Guide-itTM!
Cornelia Hampe, PhD
Technical Support Specialist & Product Manager
Takara Bio Europe
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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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CRISPR/Cas9 prokaryotic immune system
3
Adapted from Bhaya et al. Annu Rev Genet. 2011;45:273-97.
Clustered
Regularly
Interspaced
Short
Palindromic
Repeats
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Milestones in the development of CRISPR/Cas9
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Cell 157, June 5, 2014
http://zlab.mit.edu/assets/reprints/Hsu_PD_Cell_2014.pdf
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Applications of Genome Engineering
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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!
Adapted from Bee et al. 2013 PLoS One. 2013 Jul 11;8(7)
Cells have evolved 2 repair pathways:
1. Non-homologous end joining (NHEJ)
• Rapid, flexible, but error prone
2. Homologous Recombination (HR)
• Slower, restricted to late S and G2
but precise
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Repairing double-strand breaks
Double-strand break
NHEJ
Error-Prone:
Creates small insertions/deletions
(Indels)
Repair templateX X
Error-free:
Perfect repair
HR
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How can we exploit this process for genome engineering?
NHEJ
Knock-Out
Repair templateX X
HR
GFP
GFP
Knock-In
Artificially create a double-strand break
My Favorite Gene
8
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Genome engineering using ZFNs/TALENs
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NHEJ HR
Adapted from Sanjana et al., Nat Protoc. 2012 Jan 5;7(1):171-92.
ZFN = Zinc Finger
Nuclease
TALEN = Transcription
activator–like effector
nuclease
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Genome engineering using ZFNs/TALENs
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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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What if there was a nuclease…
…that is targeted to a specific genome sequence by RNA
You could change its target specificity really easily and at low cost - simply change the RNA!
Another great idea ……
Nuclease
GGA CCU CU
Nuclease
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GGA CCU CU
Cas9
guide RNA (sgRNA)
CRISPR/Cas9
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CRISPR/Cas9 Principle
Target specific
sequence
Fixed
sequence
PAM
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Indels
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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
13
CRISPR/Cas9
Gene Editing
in 5 easy steps
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CRISPR/Cas9 Workflow
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Guide-it™ Complete sgRNA Screening System
• Guide-it™ sgRNA In Vitro Transcription Kit
• Guide-it™ sgRNA Screening Kit
Guide-it™ Mutation Detection Kit
Guide-it™ Genotype Confirmation Kit
Guide-it™ Indel Identification Kit
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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CRISPR/Cas9 Workflow
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• That’s the easiest bit…it’s your gene of interest
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CRISPR/Cas9 Workflow
17
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Design your single guide RNA
• There are online tools (e.g., http://crispr.mit.edu/ or https://chopchop.rc.fas.harvard.edu/)
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PAM
PAM
PAM
PAM
List of online tools for sgRNA design
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CRISPR/Cas9 Workflow
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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?
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• In vitro testing of guide RNAs enables you to pre-
evaluate 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?
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• Very good correlation between in vitro test and sgRNA efficiency in
cells (shown for CXCR-4 knockout)
1 2 3 4C -
0% ~55% ~42% ~8% ~71%
sgRNA3 didn’t work in vitro And in HeLa cells neither
% efficiency of cutting
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CRISPR/Cas9 Workflow
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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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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 GesicleProduction 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?
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293T cells are co-
transfected with target-
specific 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?
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Glycoprotein induces
gesicle formation
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How are Gesicles produced?
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iDimerize technology
incorporates Cas9-
sgRNA complex into
gesicles: Cas9 will
be associated to the
CherryPicker red
fluorescent protein.
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How are Gesicles produced?
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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?
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Gesicles applied to
target cells fuse and
release Cas9-sgRNA
for editing.
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Gesicle Production Workflow
NLS
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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
-ve Control +ve Control 6 hrs 24 hrs
% KO 99.91% 2.21% 41.21% 47.60%
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6h
24h
• iPS cells were treated with
gesicles according to the
standard protocol for either
6 hours our 24 hours
• Culture System:
Cellartis® DEF-CS™ 500
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Importance of an optimized sgRNAscaffold
We strongly recommend using the supplied pGuide-it-sgRNA1 Vector
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Dose-dependent efficiency
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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
39
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Reduced off-target effects
40
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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
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Guide-it™ Mutation Detection Kit
Guide-it™ Genotype Confirmation Kit
Guide-it™ Indel Identification Kit
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• Quickly determine if your gene engineering
experiment was successful, best for populations
TALENs, ZFNs
Cas9/CRISPRs
Cells with mutated gene
Cells with wild type gene
What is the Mutation Detection Kit for?
43
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Mutation Detection Kit Workflow
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Mutation Detection Kit vs Cel1 Assay
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• 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
47
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Genotype Confirmation Kit
48
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Genotype Confirmation Kit
49
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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
Western Blot
• Confirms functional knock-out
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
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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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