Introduction and key considerations around gene-editing using CRISPR and rAAV. With an overview of our knock-out library using the haploid cell line HAP1

1. CRISPR, rAAV and the new landscape of molecular cell biology
Genome Editing Comes of Age

2. Genome Editing Comes of Age
 Jan Hryca, MSc
 Leipzig University
 Business Development - Europe
 J.hryca@horizondiscovery.com
 +44 1223 20 47 42
 Chris Thorne, PhD
 Liverpool University
 Gene Editing Community Specialist
 c.thorne@horizondiscovery.com
 +44 1223 20 47 99

3. Genome Editing Comes of Age
 Who is Horizon?
 strong link to Academia
 What does Horizon do?
 Our Mission
• Translational Genomics
• Targeting Genetic Diseases
• Personalized Medicine

4. 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

5. Genome Editing Comes of Age

6. Genome Editing Comes of Age

7. Gene targeting techniques – an overview
Approach
Gain of
function
Loss of
function
Endogenous
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
integration
Med/Low
Transient RNAi No Yes No No No Low
Stable RNAi No Yes No Yes*
Random
integration
Med/Low
Dominant negative over-
expression
No Yes No No** No Low
* Assuming viral promoter methylation does not occur
** Commonly transient vectors

8. Gene targeting techniques – an overview
Approach
Gain of
function
Loss of
function
Endogenous
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
integration
Med/Low
Transient RNAi No Yes No No No Low
Stable RNAi No Yes No Yes*
Random
integration
Med/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

9. 9
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

10. 1
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

11. The Opportunity: Translating Genetic Information into Personalised Medicines

12. Genome Editing: The Right Tool For The Right Outcome

13. 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

14. rAAV: How Does It Work?
Nature Genetics 18, 325 - 330 (1998)
AAV = Adeno Associated Virus (ssDNA)

15. Crispr (cr) RNA + trans-activating (tra) crRNA combined = single guide (sg) RNA
CRISPR/Cas9: How Does It Work?

16. AGCTGGGATCAACTATAGCG CGG
gRNA target sequence PAM
CRISPR/Cas9: How Does It Work?

17. 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
Nishimasu et al Cell
CRISPR/Cas9: How Does It Work?

18. Hsu et al. Cell. 2014
CRISPR/Cas9: What can you do?

19. Genome Editing: As Simple As…
... HOWEVER …
Cell Line
Screen for clones
Engineered cells!
Genome Editing Vector

20. Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation

21. 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

22. Key Considerations For CRISPR Gene Editing
Gene Target Specifics
Cell Line
gRNA Design
gRNA Activity
Donor Design
Screening
Validation
 Transfection/electroporation
 Single-cell dilution

23. 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

24. Ran et al Cell 2014

25. 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
NT
Cas9
wt
only
4uncut
1 52 3
gRNA
200
300
400
500
100
600
+ve
700
200
300
400
500
100
600
700

26. 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

27. 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)
%Greencells(FACs)

28. 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 ffic ie n c y u s in g s s O D N s o f d iffe r e n t le n g th s
O lig o le n g th (N T )
Efficiency(%)
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 ffic ie n c y u s in g 1 0 p m o l s s O D N
O ligo length (N T )
Transfection%(RFP)
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

29. Technology Development at Horizon: Systematic improvements
Donor modifications: number and position of phosphothioate medications
Only 3’ PTO modifications required for ssODNs tested. Flanking the mutation with PTO groups does not
offer any additional benefit.
N
o
n
e5
'P
T
O
3
'P
T
O5
+
3
P
T
O
M
u
t
F
la
n
k
M
u
t
F
la
n
k
+
5
+
3
P
T
O3
x
5
'P
T
O3
x
3
'P
T
O
3
x
5
'+
3
'P
T
O
M
u
t
F
la
n
k
+
3
x
5
'+
3
'P
T
O
0 .0
0 .5
1 .0
Transfection%(RFP)
H R e ffic 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
N
o
n
e5
'P
T
O
3
'P
T
O5
+
3
P
T
O
M
u
t
F
la
n
k
M
u
t
F
la
n
k
+
5
+
3
P
T
O3
x
5
'P
T
O3
x
3
'P
T
O
3
x
5
+
3
P
T
O
M
u
t
F
la
n
k
+
3
x
5
+
3
P
T
O
0
5
1 0
1 5
2 0
2 5
Efficiency(%)
T r a n s fe c tio n e ffic 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
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

30. 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

31. 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)

32. 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

33. 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

34. 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?

35. 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

36. 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

37. 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

38. 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

39. Customer
Design
ProductionQuality
control
Packaging
Shipment
Custom knockouts
for any human gene
in 10 weeks
High throughput knock-out cell line generation

40. 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

41. 4
Genome Wide sgRNA Screening
Shalem et al Science 2014

42. 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
4
Synthetic lethal target ID via sgRNA screening

43. 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/
Bromodomain
40 genes
Autophagy
15 genes
mTOR pathway
50 genes
Kinases
350 genes
HATs/HDACs
15 genes
DNA damage
50 genes
RAB GTPases
15 genes
Deubiquitinases
80 genes

44. 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

45. Your Horizon Contact:
Horizon Discovery Ltd, 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: info@horizondiscovery.com Web:
www.horizondiscovery.com
Jan Hryca
Business Development - Europe
J.hryca@horizondiscovery.com
+44 1223 204 742
Chris Thorne PhD
Gene Editing Specialist
c.thorne@horizondiscovery.com
+44 1223 204 799

46. 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

47. CRISPR and rAAV Intellectual property
We have invested heavily with the intent to ensure that our customers have the broadest IP
coverage available to benefit from CRISPR gene editing now and in the future.
We bring to our customers the widest breadth of IP available from any commercial source:
 Our licenses allow for provision of products and services using CRISPR technology to third parties for
use in their internal research, in both an academic or commercial environment, excluding use in
plants.
 We have entered into license agreements with the Broad Institute, ERS Genomics, and Harvard
University to access intellectual property (IP) relating to the CRISPR/Cas9 gene editing system.
 Horizon has a worldwide exclusive patent position on the use of rAAV gene editing using homologous
recombination. As such, we are the only company able to offer hybrid rAAV/CRISPR systems that
draw from the best aspects of both approaches for far superior gene editing efficiencies.

48. Overcoming the Limitations of ES Cell Targeting
 Time-consuming process (12-18 months)
 Limited in strain
 Not widely adapted to other animal species
Microinjection
Founders born
Founders identified
Week
F2
F1 born
ZFN validation
Target
4 months
with 100%
germline
transmission
15
23
34
12
8
9
0
Step
breeding
Microinjection
Founders born
Founders identified
Week
F2
F1 born
sgRNA validation
Target
2 months
with 100%
germline
transmissi
on
9
17
28
6
2
3
0
Step
breeding
Historically….

49. Physiological In Vitro Models – Genetically Engineered Rats
15 Weeks 26 Weeks 32 Weeks
Pink1 and DJ-1 KO rats have a 50% reduction of dopaminergic neurons in the substantia nigra