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...
Caco-2 cell permeability assay for drug absorption
The key considerations of crispr genome editing
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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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
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
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
Editor's Notes
Pleasure to be here to today to tell you more about Horizon and our suite of technologies based around a core expertise in human genome editing and how we are applying this to better understand the human genome, find new validated targets and support targeted drug discovery with predictive, genetically-defined, in vitro models that accurately represent target patient groups.
Generally speaking when targeting genes of interest two DNA repair pathways are used to mediate the majority of genomic modifications we want to make.
The first of these is NHEJ
HR
For
For
For
For
For
Knockouts
Doench et al – actually position not as important as you might predict
BUT if you’re aware of splice variants and alternative start codons design your guides accordingly
Knockouts
Doench et al – actually position not as important as you might predict
BUT if you’re aware of splice variants and alternative start codons design your guides accordingly
Knockouts
Doench et al – actually position not as important as you might predict
BUT if you’re aware of splice variants and alternative start codons design your guides accordingly
Knockouts
Doench et al – actually position not as important as you might predict
BUT if you’re aware of splice variants and alternative start codons design your guides accordingly