Genome Editing Comes of Age
45
CRISPR, rAAV and the new landscape of molecular cell biology Genome Editing Comes of Age Normal human karyotype HeLa cell karyotype www.horizondiscovery.com
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Transcript of Genome Editing Comes of Age
CRISPR, rAAV and the new landscape of molecular cell biology
Genome Editing Comes of Age
Normal human karyotype
HeLa cell karyotype
www.horizondiscovery.com
Genome Editing Comes of Age
Jan Hryca, MSc (Leipzig University)• Business Development - Europe• [email protected]• +44 1223 20 47 42
Chris Thorne, PhD (Liverpool University)• Gene Editing Community Specialist• [email protected]• +44 1223 204799
Genome Editing Comes of Age
Horizon Discovery - Experts in Genome Editing • Integrated products and services built on genome editing applicable across the drug
development continuum• Deep roots in academia (with many existing colaborations through CoE program)
Our Mission
To translate the human genome and accelerate the discovery of personalised medicines
Our Approach
Leveraging genome editing across to speed the translation of genetic observations into clinical outcomes
Genome Editing Comes of Age
Gene Targeting Techniques – an overview
Genome Editing Tools• rAAV• CRISPR/Cas9
Key Considerations for Gene Editing
Genome Editing at scale• High through Knock-out Cell Line Generation• Genome Wide sgRNA Synthetic Lethality Screening
Genome Editing Comes of Age
Gene targeting techniques – an overview
ApproachGain of
functionLoss of
functionEndogenous expression
Long-term stability
Off-target integrations
Time vs. Cost
Transient over-expression Yes No No No No Low
Stable over-expression Yes No No Yes*Random
integrationMed/Low
Transient RNAi No Yes No No No Low
Stable RNAi No Yes No Yes*Random
integrationMed/Low
Dominant negative over-expression
No Yes No No** No Low
* Assuming viral promoter methylation does not occur
** Commonly transient vectors
Gene targeting techniques – an overview
ApproachGain of
functionLoss of
functionEndogenous expression
Long-term stability
Off-target integrations
Time vs. Cost
Transient over-expression Yes No No No No Low
Stable over-expression Yes No No Yes*Random
integrationMed/Low
Transient RNAi No Yes No No No Low
Stable RNAi No Yes No Yes*Random
integrationMed/Low
Dominant negative over-expression
No Yes No No** No Low
Nuclease-Based Genome Editing
Yes Yes Yes Yes Varies Low-High
rAAV-Based Genome Editing Yes Yes Yes Yes Controlled Med
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Endogenous targeting of PIK3CA
Large growth induction phenotype
Transforming alone
Milder growth induction phenotype
Non-transforming alone
Di Nicolantonio et al., PNAS, Dec. 2008Isakoff et al., Cancer Research, Jan 2006
9
Endogenous targeting of KRAS
Konishi et al (2007) - Endogenous knock-in of KRAS G12V does not transform cells, unlike KRAS G12V
overexpression
KRAS G12V overexpression is not a physiological model
The Opportunity: Translating Genetic Information into Personalised Medicines
Genome Editing: The Right Tool For The Right Outcome
Genome Editing: The Right Tool For The Right Outcome
rAAV
• High precision / low thru-put
• Any locus, wide cell tropism
• Well validated, KI focus
Zinc Fingers
• Med precision / med thru-put
• Good genome coverage
• Well validated / KO Focus
CRISPR
• New but high potential
• Capable of multi-gene targeting
• Simple RNA-directed cleavage
• Combinable with AAV
Great for knock-outs Great for heterozygous
knock-ins
rAAV: How Does It Work?
Nature Genetics 18, 325 - 330 (1998)
AAV = Adeno Associated Virus (ssDNA)
Crispr (cr) RNA + trans-activating (tra) crRNA combined = single guide (sg) RNA
CRISPR/Cas9: How Does It Work?
AGCTGGGATCAACTATAGCG CGG
gRNA target sequence PAM
CRISPR/Cas9: How Does It Work?
Cas9 Wild type Cas9 Nickase (Cas9n)
Induces double strand break Only “nicks” a single strand
Only requires single gRNARequires two guide RNAs for reasonable
activity
Concerns about off-target specificity Reduced likelihood of off-target events
High efficiency of cleavage Especially good for random indels (= KO)
Guide efficiency dictated by efficiency of the weakest gRNA
Nishimasu et al Cell
CRISPR/Cas9: How Does It Work?
Hsu et al. Cell. 2014
CRISPR/Cas9: What can you do?
Genome Editing: As Simple As…
... HOWEVER …
Cell Line
Screen for clones
Engineered cells!
Genome Editing Vector
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Normal human karyotype
HeLa cell karyotype
Gene copy number Effect of modification on growth
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Transfection/electroporation Single-cell dilution
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Sequence source Off-target potential Guide proximity Wild-type Cas9 or mutant nickase
Ran et al Cell 2014
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Number of gRNAs gRNA activity measurement
NTCas9 wt
only4uncut 1 52 3
gRNA
200
300
400
500
100
600
+ve
700
200
300
400
500
100
600700
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Donor sequence modifications Modification effects on expression or splicing Size and type of donor (AAV, oligo, plasmid) Selection based strategies
Cas9 Cut Site
Genomic Sequence
Donor Sequence containing mutation
Technology Development at Horizon: Combining rAAV + CRISPR
Can we combine technologies for improved efficiency?
Tested using a reporter cell-line harbouring an inactivating mutation in GFP
Correction donor-vector supplied either as dsPlasmid, ssDNA oligos, or ssDNA rAAV
rAAV = the most efficient donor vector (50 fold)
% G
ree
n c
ells
(FA
Cs)
Technology Development at Horizon: Systematic improvements
Donor lengths: sODNs ranging from 50-200nt, with single phosthothioate modifications at both outer nucleotides
100nt ssODN is optimal
4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0
0 .0
0 .5
1 .0
1 .5
H R e f f ic ie n c y u s in g s s O D N s o f d i f fe r e n t le n g th s
O lig o le n g th (N T )
Eff
icie
nc
y (
%)
4 0 6 0 8 0 1 0 0 1 2 0 1 4 0 1 6 0 1 8 0 2 0 0
0
5
1 0
1 5
T ra n s fe c tio n e ff ic ie n c y u s in g 1 0 p m o l s s O D N
O lig o le n g th (N T )
Tra
ns
fec
tio
n %
(R
FP
)
Size Oligo Sequence
50 C*ACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCC*C
70 T*CCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTG*C
90 T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A
110 A*CAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG*C
130 T*TTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC*G
150 G*TATCTGGTATTTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCA*A
170 T*AAGCCTGCAGTATCTGGTATTTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAA*C
200 A*AATGTCTTTATAAATAAGCCTGCAGTATCTGGTATTTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA*C
GFP Mutation, PAM mutation
Technology Development at Horizon: Systematic improvements
Donor modifications: number and position of phosphothioate medications
Only 3’ PTO modifications required for ssODNs tested
Oligo Sequence
None TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA
5' PTO T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA
3' PTO TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A
5+3 PTO T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A
Mut Flank TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACT*A*C*C*AGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA
Mut Flank + 5+3 PTO T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACT*A*C*C*AGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A
3x5' PTO T*G*A*TGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA
3x3' PTO TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA*C*C*A
3x5'+3' PTO T*G*A*TGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA
Mut Flank + 3x5'+3' PTO T*G*A*TGGTTCTTCCATCTTCCCACAGCTGGCCGACCACT*A*C*C*AGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA*C*C*A
GFP Mutation, PAM mutation
No
ne
5' P
TO
3' P
TO
5+3 P
TO
Mu
t F
lan
k
Mu
t F
lan
k +
5+3 P
TO
3x5' P
TO
3x3' P
TO
3x5'+
3' P
TO
Mu
t F
lan
k +
3x5'+
3' P
TO
0 .0
0 .5
1 .0
Ta
rg
eti
ng
fre
qu
en
cy
(G
FP
%)
H R e f f ic ie n c y u s in g s s O D N s w ith v a r y in g n u m b e r s a n d p o s ito n s o f
p h o s p h t io la t e p r o t e c t e d n u c le o t id e s
No
ne
5' P
TO
3' P
TO
5+3 P
TO
Mu
t F
lan
k
Mu
t F
lan
k +
5+3 P
TO
3x5' P
TO
3x3' P
TO
3x5+3 P
TO
Mu
t F
lan
k +
3x5+3 P
TO
0
5
1 0
1 5
2 0
2 5
Tra
ns
fec
tio
n %
(R
FP
)
T r a n s fe c t io n e f f ic ie n c y u s in g s s O D N s w ith v a r y in g n u m b e r s a n d p o s it o n s o f
p h o s p h t io la t e p r o t e c t e d n u c le o t id e s
No
ne
5' P
TO
3' P
TO
5+3 P
TO
Mu
t F
lan
k
Mu
t F
lan
k +
5+3 P
TO
3x5' P
TO
3x3' P
TO
3x5'+
3' P
TO
Mu
t F
lan
k +
3x5'+
3' P
TO
0 .0
0 .5
1 .0
Ta
rg
eti
ng
fre
qu
en
cy
(G
FP
%)
H R e f f ic ie n c y u s in g s s O D N s w ith v a r y in g n u m b e r s a n d p o s ito n s o f
p h o s p h t io la t e p r o t e c t e d n u c le o t id e s
No
ne
5' P
TO
3' P
TO
5+3 P
TO
Mu
t F
lan
k
Mu
t F
lan
k +
5+3 P
TO
3x5' P
TO
3x3' P
TO
3x5+3 P
TO
Mu
t F
lan
k +
3x5+3 P
TO
0
5
1 0
1 5
2 0
2 5
Tra
ns
fec
tio
n %
(R
FP
)
T r a n s fe c t io n e f f ic ie n c y u s in g s s O D N s w ith v a r y in g n u m b e r s a n d p o s it o n s o f
p h o s p h t io la t e p r o t e c t e d n u c le o t id e s
Introducing gUIDEBook™ - Coming soon!
Supports all Cas9 nuclease variants
Advanced tools for knock-in design
Comprehensive gRNA scoring • Off target• Activity
Full integration with annotated reference genomes
Flexible and easy to use
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Donor sequence modifications Modification effects on expression or splicing Size and type of donor (AAV, oligo, plasmid) Selection based strategies
(+/+)
(+/-)
(-/-)
(KI/-)
(KI/+)
(KI/KI)
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Number of cells to screen Screening strategy Modifications on different alleles Homozygous or heterozygous
modifications versus mixed cultures
% cells targeted
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Confirmatory genotyping strategies Off-target site analysis Modification expression Contamination
Heterozygous knock-in
Wild type
Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
How many copies?
Is it suitable?
What’s my goal? (Precision vs Efficiency)
Does my guide cut?
Have I minimised re-cutting?
How many clones to find a positive?
Is my engineering as expected?
Genome Editing Comes of Age
Gene Targeting Techniques – an overview
Genome Editing Tools• rAAV• CRISPR/Cas9
Key Considerations for Gene Editing
Genome Editing at scale• High through Knock-out Cell Line Generation• Genome Wide sgRNA Synthetic Lethality Screening
High throughput knock-out cell line generation
(Near) Haploid human cell lines
• Near-haploid (diploid for chr8, and chr15)• Isolated from CML patient• Myeloid lineage• Suspension cells
KBM-7
HAP1
• Near-haploid (diploid for chr15)• Derived from KBM-7• Fibroblast like• Adherent cells
Unambiguous genotyping
Defined copy number Knowledge base
RNA sequencing- Predict suitability
as cellular model
Essentiality dataset- Predict success rate
for knockouts
Haploid
High efficiency
Diploid
- Knockouts
- Defined mutations
High throughput knock-out cell line generation
Advantages of Haploid Cells
Wildtype TCCTTTGCGGAGAGCTGCAAGCCGGTGCAGCAG
||||||||||| ||||||||||||||
Knockout TCCTTTGCGGA--------AGCCGGTGCAGCAG
Wildtype SerPheAlaGluSerCysLysProValGlnGln
Knockout SerPheAlaGlu AlaGlyAlaAla
Exon 1
DNA sequencing
Exon 2
Cas9 cleavage
High throughput knock-out cell line generation
CRISPR/Cas9 allows rapid and high efficiency targeting
Customer
Design
ProductionQualitycontrol
Packaging
Shipment
Custom knockoutsfor any human gene
in 10 weeks
High throughput knock-out cell line generation
Lentivirally delivered sgRNA can drive efficient cleavage of target genomic
sequences for use in whole genome screens
Use massively-parallel next-gen sequencing to assess results
Possible addition/replacement to RNAi screens
Genome Wide sgRNA Screening
40
Genome Wide sgRNA Screening
Shalem et al Science 2014
We are combining CRISPR and isogenic cell lines to perform
CRISPR-based Synthetic Lethality Screens
sgRNA technology will be transformational for both Target ID and early-stage Validation
41
Synthetic lethal target ID via sgRNA screening
Ready-made knock-out X-MAN® cell lines
X-MAN® - gene X Mutant And Normal cell line
Advantages:• Genetically verified
• More than 3,000 available clones already available, in a variety of cell line backgrounds
• Quick and easy way to get first data on gene of interest
• Available with validated gRNAs to use with your own human cell line of choice.
More Information: www.horizon-genomics.com/
Bromodomain40 genes
Autophagy15 genes
mTOR pathway50 genes
Kinases350 genes
HATs/HDACs15 genes
DNA damage50 genes
RAB GTPases15 genes
Deubiquitinases80 genes
GENASSIST: CRISPR and rAAV enabled gene editing
Cas9 Vectors• Wild type and nickase• Separate or combined with guide
Guide RNA• Single or double guides• Available OTS for in-lab cloning• Custom guide generation available with validation
Donors• Available OTS for in-lab cloning• Plasmid or rAAV format• Custom donor generation available
Cell Lines• CRISPR-ready cell lines• 550+ OTS cell line menu available for further gene editing
Services• Viral encapsulation of rAAV donor• Project design support• On-going expert scientific support
Your Horizon Contact:
Horizon Discovery Group plc, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Tel: +44 (0) 1223 655 580 (Reception / Front desk) Fax: +44 (0) 1223 655 581 Email: [email protected] Web: www.horizondiscovery.com
Jan Hryca
Business Development - Europe
+44 1223 204 742
Chris Thorne PhD
Gene Editing Specialist
+44 1223 204 799
Useful Resources
From Horizon
Free gRNAs in Cas9 wild type vector – www.horizondiscovery.com/guidebook
Technical manuals for working with CRISPR - http://www.horizondiscovery.com/talk-to-us/technical-
manuals
In the Literature
Exploring the importance of offset and overhand for nickase -
http://www.cell.com/cell/abstract/S0092-8674(13)01015-5
sgRNA whole genome screening:• Shalem et al - http://www.sciencemag.org/content/343/6166/84.short
• Wang et al - http://www.sciencemag.org/content/343/6166/80.abstract
On the web
Feng Zhang on Game Changing Therapeutic Technology (Link to Feng’s Video)
Guide design - http://crispr.mit.edu/
CRISPR Google Group - https://groups.google.com/forum/#!forum/crispr
