This document provides an overview of genome editing techniques such as CRISPR/Cas9 and rAAV and considerations for their use. It discusses how CRISPR/Cas9 and rAAV work to edit genomes and compares their advantages. Key factors for CRISPR gene editing are discussed such as gRNA design, donor design, and screening/validation approaches. The document also summarizes research optimizing CRISPR gene editing through improvements like testing different donor lengths and modifications. The goal is to translate genetic information into personalized medicines by leveraging tools like CRISPR and rAAV.

1. CRISPR, rAAV and the new landscape of molecular cell biology
Genome Editing Comes of Age
Normal human karyotype
HeLa cell karyotype
www.horizondiscovery.com

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 204799

3. 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

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. 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

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
Nuclease-Based Genome
Editing
Yes Yes Yes Yes Varies Low-High
rAAV-Based Genome Editing Yes Yes Yes Yes Controlled Med

8. 8
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. 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

10. The Opportunity: Translating Genetic Information into Personalised Medicines

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

12. 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

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

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

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

16. 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?

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

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

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

20. 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

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

22. 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

23. Ran et al Cell 2014

24. 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

25. 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

26. 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)

27. 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

28. 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
N
o
n
e
5
'P
T
O
3
'P
T
O5
+
3
P
T
O
M
u
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F
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+
5
+
3
P
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F
la
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+
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x
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'+
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'P
T
O
0 .0
0 .5
1 .0
Targetingfrequency(GFP%)
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
2 5
)
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
N
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+
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+
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u
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F
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+
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x
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'+
3
'P
T
O
0 .0
0 .5
Targetingfrequency(GFP
N
o
n
e
5
'P
T
O
3
'P
T
O5
+
3
P
T
O
M
u
t
F
la
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k
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u
t
F
la
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k
+
5
+
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T
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x
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'P
T
O3
x
3
'P
T
O
3
x
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+
3
P
T
O
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u
t
F
la
n
k
+
3
x
5
+
3
P
T
O
0
5
1 0
1 5
2 0
2 5
Transfection%(RFP)
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

29. 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

30. 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)

31. 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

32. 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

33. 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?

34. 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

35. 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

36. 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

37. 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

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

39. 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. 4
Genome Wide sgRNA Screening
Shalem et al Science 2014

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
4
Synthetic lethal target ID via sgRNA screening

42. 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

43. 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

44. 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: 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

45. 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