Genome Editing Comes Of Age
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Transcript of Genome Editing Comes Of Age
HORIZON DISCOVERY
Genome Editing Comes of AgeCRISPR, rAAV and the new landscape of molecular cell biology
Chris Thorne | Gene Editing Specialist
2
Horizon Discovery
Powering Genomic Research and Translational Medicine,
…from Sequence to Treatment
5
Overview
• The case for Genome Editing
• 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
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6
The Opportunity:
The challenge has shifted from obtaining genomic information to understanding what it means
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Gene function analysis - Patient-derived cell lines
Human cell lines contain pre-existing mutations
are derived directly from human tumors
Immense genetic diversity
However Lack of wild type controls
Availability of rare mutation models
Cell line diversity makes it very hard link observations to specific genetics
(Domke et al Nat. Comms 2013)
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Gene function analysis - RNAi
Problems with RNAi can result in false positives or negatives
Loss of function analysis using RNAi is
inexpensive and widely applicable
Incomplete knockdown
However Lack of reproducibility
Off-target effects
Brass et al.Science
273 genes
König et al.
Cell
213 genes
Zhou
et a
l.
Cell H
ost M
icrob
e
300
gene
s
Total overlap only 3 genes
Shalem et al Science 2014 HIV Host Factors
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Gene function analysis - Overexpression
Overexpression of oncogenes can over represent their role in disease biology
Gain of function analysis using
overexpression is inexpensive and widely
applicable
Result may be artefact of overexpression
HoweverDifficult to achieve long-
term overexpression
• Large growth induction phenotype• Transforming alone
• Milder growth induction phenotype• Non-transforming alone
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Gene function analysis – Gene Editing
Gene editing represents the most biologically relevant means to explore gene function in cells
Genetically defined mutant cell line
Matched isogenic wild type control
Complete loss of function possible
Endogenous gain of function possible
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Genome Editing: The Right Tool For The Right Outcome
Horizon is ‘technology agnostic’, using the right technology to
generate virtually any genomic modification
in a cell
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The Right Tool For The Right Outcome
rAAV• High precision / low thru-put• Any locus, wide cell tropism• Well validated, KI focus• Exclusive to HD
Zinc Fingers • Med precision / med thru-put• Good genome coverage• Well validated / KO Focus• Licensed from Sigma
CRISPR • New but high potential• Capable of multi-gene targeting• Simple RNA-directed cleavage• Combinable with AAV• Extensive IP position
Great for knock-outsGreat for heterozygous knock-ins
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rAAV: How Does It Work?
Nature Genetics 18, 325 - 330 (1998)
AAV = Adeno Associated Virus (ssDNA)
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Crispr (cr) RNA + trans-activating (tra) crRNA combined = single guide (sg) RNA
CRISPR/Cas9: How Does It Work?
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Cas9 Wild type Cas9 Nickase (Cas9n)
Induces double strand break Only “nicks” a single strand
Only requires single gRNA Requires 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
CRISPR/Cas9: How Does It Work?
Nishimasu et al Cell
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Genome Editing: As Simple As…
... HOWEVER …
Cell Line
Screen for clones
Engineered cells!
Genome Editing Vector
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Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
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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
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Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
Transfection/electroporation Single-cell dilution
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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
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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
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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
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Technology Development at Horizon: Systematic improvementsDonor lengths: sODNs ranging from 50-200nt, with single phosthothioate modifications at both outer nucleotides
100nt ssODN is optimal
40 60 80 100 120 140 160 180 2000.0
0.5
1.0
1.5
HR efficiency using ssODNs of different lengths
Oligo length (NT)
Effi
cien
cy (
%)
40 60 80 100 120 140 160 180 2000
5
10
15
Transfection efficiency using 10 pmol ssODN
Oligo length (NT)
Tra
nsfe
ctio
n %
(R
FP
)
Size Oligo Sequence50 C*ACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCC*C70 T*CCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTG*C90 T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A
110 A*CAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAG*C130 T*TTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCC*G150 G*TATCTGGTATTTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCA*A170 T*AAGCCTGCAGTATCTGGTATTTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAA*C200 A*AATGTCTTTATAAATAAGCCTGCAGTATCTGGTATTTTTGCTCTACAGTTATGTTGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCACTACCTGAGCACCCAGTCCGCCCTGAGCAAAGACCCCAACGAGAAGCGCGATCA*C
GFP Mutation, PAM mutation
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Technology Development at Horizon: Systematic improvementsDonor modifications: number and position of phosphothioate medications
Only 3’ PTO modifications required for ssODNs tested
Oligo Sequence
None TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA5' PTO T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA3' PTO TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A
5+3 PTO T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*AMut Flank TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACT*A*C*C*AGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA
Mut Flank + 5+3 PTO T*GATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACT*A*C*C*AGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACC*A3x5' PTO T*G*A*TGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCA3x3' PTO TGATGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA*C*C*A
3x5'+3' PTO T*G*A*TGGTTCTTCCATCTTCCCACAGCTGGCCGACCACTACCAGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAACCAMut Flank + 3x5'+3' PTO T*G*A*TGGTTCTTCCATCTTCCCACAGCTGGCCGACCACT*A*C*C*AGCAGAACACACCCATCGGCGACGGCCCCGTGCTGCTGCCCGACAA*C*C*A
GFP Mutation, PAM mutation
None
5' P
TO
3' P
TO
5+3
PTO
Mut F
lank
Mut F
lank
+ 5+
3 PTO
3x5'
PTO
3x3'
PTO
3x5'
+3' P
TO
Mut F
lank
+ 3x
5'+3
' PTO
0.0
0.5
1.0
Ta
rge
tin
g f
req
ue
nc
y (
GF
P%
)
HR efficiency using ssODNs with varying numbers and positons ofphosphtiolate protected nucleotides
None
5' P
TO
3' P
TO
5+3
PTO
Mut F
lank
Mut F
lank
+ 5+
3 PTO
3x5'
PTO
3x3'
PTO
3x5+
3 PTO
Mut F
lank
+ 3x5
+3 P
TO
0
5
10
15
20
25
Tra
nsfe
ctio
n %
(R
FP
)
Transfection efficiency using ssODNs with varying numbers and positons ofphosphtiolate protected nucleotides
None
5' P
TO
3' P
TO
5+3
PTO
Mut F
lank
Mut F
lank
+ 5+
3 PTO
3x5'
PTO
3x3'
PTO
3x5'
+3' P
TO
Mut F
lank
+ 3x
5'+3
' PTO
0.0
0.5
1.0
Ta
rge
tin
g f
req
ue
nc
y (
GF
P%
)
HR efficiency using ssODNs with varying numbers and positons ofphosphtiolate protected nucleotides
None
5' P
TO
3' P
TO
5+3
PTO
Mut F
lank
Mut F
lank
+ 5+
3 PTO
3x5'
PTO
3x3'
PTO
3x5+
3 PTO
Mut F
lank
+ 3x5
+3 P
TO
0
5
10
15
20
25
Tra
nsfe
ctio
n %
(R
FP
)
Transfection efficiency using ssODNs with varying numbers and positons ofphosphtiolate protected nucleotides
28
Introducing gUIDEBook™
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
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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)
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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
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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
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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?
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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
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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
35
Wildtype TCCTTTGCGGAGAGCTGCAAGCCGGTGCAGCAG ||||||||||| ||||||||||||||Knockout TCCTTTGCGGA--------AGCCGGTGCAGCAG
Wildtype SerPheAlaGluSerCysLysProValGlnGlnKnockout 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
36
Customer
Design
ProductionQualitycontrol
Packaging
Shipment
Custom knockoutsfor any human gene
in 10 weeks
High throughput knock-out cell line generation
37
Exon 7 Exon 8 Exon 9
Exon 8
Cas9-induceddouble-strand break
Non-homologous end joining (imprecise)
Exon 9Exon 7
Exon 9Exon 8Exon 7
Insertion of DNA cassettes by NHEJ
39
Genome Wide sgRNA Screening
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
41
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
Synthetic lethal target ID via sgRNA screening
41
42
Ready-made knock-out X-MAN® cell linesX-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
43
Genome Editing Tools and Services
• Wild type and nickase• Separate or all-in one vectors
• gRNA design and validation service• Pre-validated guides available
• Custom donor design and synthesis• Multiple formats inc. rAAV available
• >1000 ready-modified cell lines• Custom cell line generation service
• Viral encapsulation of rAAV donor• Project design support
Your Horizon Contact:
t + 44 (0)1223 655580f + 44 (0)1223 655581e [email protected] www.horizondiscovery.comHorizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Chris ThorneGene Editing [email protected] +44 1223 204 742
Useful Resources
From Horizon
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