While CRISPR is simple to use, widely applicable and often highly efficient, there are a number of things to keep in mind to maximise experimental success. Here's what we recommend...

1. HORIZON DISCOVERY
The Key Considerations for CRISPR
Genome Editing
Chris Thorne, PhD | Commercial Marketing Manager

2. 2
Disclaimer
2
• This Presentation does not constitute or form any part of an offer to sell, or invitation to purchase or apply for or enter into any contract or make
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3. 3
CRISPR mediated genome editing
Exon 1 Exon 2 Exon 3
Exon Exon 2 Exon 31
CRISPR-induced
DNA double-strand break
Non-homologous
end joining
Exon 1
Homology-directed repair
Exon 2
Exon 2Exon 2Exon 1
Frameshift mutation
Exon 1
Knockout Knockin

4. 4
... HOWEVER …
Cell Line
Engineered cells!
Genome Editing Vector
Screen for clones
As simple as….

5. 5
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
There are a number of things to
keep in mind to maximise your
chances of success!

6. 6
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
• Transfection/electroporation
• Enrichment
• Single-cell dilution

7. 7
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
Normal human karyotype
HeLa cell karyotype
• Gene copy number
• Effect of modification on growth

8. 8
The Key Considerations For CRISPR Gene Editing
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
• Sequence source
• Off-target potential vs predicted activity
• Wild-type Cas9 or mutant nickase
https://www.deskgen.com/
guidebook/wizard.html

9. 9
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
• 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

10. 10
The Key Considerations For CRISPR Gene Editing
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
Knockouts
• Be aware of spice variants/alternative start
codons
• Functional domain targeting

11. 11
The Key Considerations For CRISPR Gene Editing
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
Doench et al

12. 12
The Key Considerations For CRISPR Gene Editing
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
Knockouts
• Be aware of spice variants/alternative start
codons
• Functional domain targeting
Knockins
• Cut site should be as close to site of knockin as
possible

13. 13
Ran et al Cell 2014

14. 14
The Key Considerations For CRISPR Gene Editing
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
Knockouts
• Be aware of spice variants/alternative start
codons
• Functional domain targeting
Knockins
• Cut site should be as close to site of knockin as
possible
Nickase
• Minimise offset
• D10A – 5’ overhang

15. 15
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
• Donor sequence modifications
• Effects on expression or splicing
• Size and type of donor (AAV, oligo, plasmid)
Cas9 Cut Site
Genomic
Sequence
Donor Sequence
containing mutation

16. 16
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
GF PCMV
Inactive GFP
P
P
P
RFP
DrugR
P
-ve
-ve DrugR
GFCMV
Active GFP
P
P
Homologous recombinationHomologous recombination
Fluorescent proteins
Test Donors:
Positive selection
Length of homology
Negative selection
Negative and positive
selection
Inactive FIRE-line
allele
Active FIRE-Line
allele
Our Testing Platform - Fluorescent Indicator of Recombination
Efficiency
Our Testing Platform - FIRELine

17. 17
ssODN Optimisation for CRISPR knock-ins
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 ligo length (N T)
Efficiency(%)
Length
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 tio la te p r o te c te d n u c le o tid e s
0 .5 1 2 4 8 1 6 3 2 6 4 1 2 8
0 .0
0 .5
1 .0
1 .5
H R e ffic ie n c y w ith v a r y in g c o n c e n tr a tio n o f o lig o d o n o r
[do no r] pm ol
Targetingfrequency(GFP%)
s
s
O
D
N
1
s
s
O
D
N
2
s
s
O
D
N
3
0
1
2
3
4
H R e ffic ie n c y u s in g o lig o s p u r fiie d b y d iffe r e n t m e th o d s
Targetingfrequency(GFP%)
D esalt
P A G E
H P LC
Modification Concentration
Purification

18. 18
ssODN Optimisation for CRISPR knock-ins
40 60 80 100 120 140 160 180 200
0.0
0.5
1.0
1.5
HR efficiency using ssODNs of different lengths
Oligo length (NT)
Efficiency(%)

19. 19
ssODN Optimisation for CRISPR knock-ins
0 .5 1 2 4 8 1 6 3 2 6 4 1 2 8
0 .0
0 .5
1 .0
1 .5
H R e ffic ie n c y w ith v a r y in g c o n c e n tr a tio n o f o lig o d o n o r
[do no r] pm ol
Targetingfrequency(GFP%)

20. 20
ssODN Optimisation for CRISPR knock-ins
s
s
O
D
N
1
s
s
O
D
N
2
s
s
O
D
N
3
0
1
2
3
4
H R e ffic ie n c y u s in g o lig o s p u r fiie d b y d iffe r e n t m e th o d s
Targetingfrequency(GFP%)
D esalt
P A G E
H P LC

21. 21
ssODN Optimisation for CRISPR knock-ins
HR efficiency using ssODNs with varying numbers
and positions of phosphorothioate protected
nucleotides
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
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

22. 22
ssODN Optimisation for CRISPR knock-ins
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 ligo length (N T)
Efficiency(%)
Length
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 tio la te p r o te c te d n u c le o tid e s
0 .5 1 2 4 8 1 6 3 2 6 4 1 2 8
0 .0
0 .5
1 .0
1 .5
H R e ffic ie n c y w ith v a r y in g c o n c e n tr a tio n o f o lig o d o n o r
[do no r] pm ol
Targetingfrequency(GFP%)
s
s
O
D
N
1
s
s
O
D
N
2
s
s
O
D
N
3
0
1
2
3
4
H R e ffic ie n c y u s in g o lig o s p u r fiie d b y d iffe r e n t m e th o d s
Targetingfrequency(GFP%)
D esalt
P A G E
H P LC
Modification Concentration
Purification
Conclusion: Length 90 nucleotides, at least 3’ end having a PTO, HPLC purified
Use ~20 pmol/ 600ng of ssODN (1.5x105 cells)

23. 23
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
• Donor sequence modifications
• Effects on expression or splicing
• Size and type of donor (AAV, oligo, plasmid)
(+/+)
(+/-)
(-/-)
(KI/-)
(KI/+)
(KI/KI)

24. 24
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
 Number of cells to screen
 Screening strategy
 Modifications on different alleles
 Homozygous or heterozygous
modifications versus mixed cultures
% cells targeted

25. 25
The Key Considerations For CRISPR Gene Editing
Targeting Efficiency
~8%
AACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTG
AsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyLeuAlaLysLeu
AACGTACTAGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCGGGCCAAACTG
AsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyArgAlaLysLeu
Clone 5
Wild-type
SpeI
PCR +
SpeI

26. 26
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
 Confirmatory genotyping strategies
 Off-target site analysis
 Modification expression
 Contamination
Heterozygous knock-in
Wild type

27. 27
Cell Line
Gene Target
Guide Choice
Guide Position
Donor Design
Screening
Validation
The Key Considerations For CRISPR Gene Editing
 Is it suitable?
 Is it essential/expressed/amplified?
 Specificity vs Efficiency
 Will depend on modification
 Donor design to maximise efficiency
 How many clones to find a positive?
 Is my engineering as expected?

28. 28
Next Webinar - CRISPR modified cell lines
What’s possible and how they will impact your research
Exon 8 Exon 9 NanoLuc polyA
Exon 1 Exon 3
Translocations and Fusions
Gene tagging
Chromosomal deletions
Chr 1 Chr 19
Point mutations
Exon 8 Exon 9
*

29. Your Horizon Contact:
t + 44 (0)1223 655580
f + 44 (0)1223 655581
e info@horizondiscovery.com
w www.horizondiscovery.com
Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Your Horizon Contact:
t + 44 (0)1223 655580
f + 44 (0)1223 655581
e info@horizondiscovery.com
w www.horizondiscovery.com
Horizon Discovery, 7100 Cambridge Research Park, Waterbeach, Cambridge, CB25 9TL, United Kingdom
Chris Thorne, PhD
Commercial Marketing Manager
c.thorne@horizondiscovery.com
+44 1223 204 799