making genome edits in mammalian cells
TRANSCRIPT
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HORIZON DISCOVERY
Making Genome Edits In Mammalian Cells
Chris Thorne, PhD | Commercial Marketing Manager
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Contents
1. Quick recap
2. Introducing haploid genetics
3. Observations from over 1000 knockout experiments
4. Genome editing options beyond knockouts• Knock-ins, genomic deletions, translocations, gene tagging
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The Opportunity: Genome Editing
Genome editing is the most robust and biologically relevant method for studying how genes and mutations function in driving disease
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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 1Frameshift mutation
Exon 1
Most frequently CRISPR-Cas9 is used to make either knockouts (via NHEJ mediated gene disruption) or knockins (via HDR)
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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?
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The Challenge? Polyploid cells…e.g. Disruption of the MAPK3 gene in the A375 cell line (copy number = 3)
1
2
3
Validation of frameshift disruptions in polyploid cells is a significant bottleneck
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Kotecki et al. (1999) in Exp Cell ResCarette et al. (2009) in Science
KBM-7 is a human cell line that is haploid for all chromosomes but chromosome 8.
Thijn BrummelkampNKI/CeMM
The Solution? Haploid cells...
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Genotyping analysis in haploid cells
Exon 1 Exon 2 Exon 3
PCR with custom primers
Sanger sequencing of PCR product
Mutation masked by second copy
Mutation leads to knockout
Diploid Haploid
Both editing and validation is more efficient in haploid cells
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(Near-) Haploid Human Cell Lines
KBM-7Near-haploid (diploid chr8, chr15)Isolated from CML patientMyeloid lineageSuspension cells
HAP1Near-haploid (chr15)Derived from KBM-7Fibroblast likeAdherent cells
eHAPFully haploidDerived from HAP1Patent EP 13194940.6
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Haploid
High efficiency
Unambiguous genotyping
Diploid
Defined copy number
KnockoutsDiploid/haploid: >2fold
Defined mutationsDiploid/haploid: >10fold
Knowledge base
RNA sequencingPredict suitability as cellular model
Essentiality datasetPredict success ratefor knockouts
Advantages of haploid cells for genome editing
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Customer
Design
ProductionQualitycontrol
Packaging
Shipment
On-demand knockoutsfor any human genein 10 weeks
Production pipeline
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Knockout cell line collections
Gene CollectionsKinases, Bromodomain genes, Deubiquitinases, Ubiquitin E2 ligases, HDACs, Caspases, Rab GTPases
Pathway CollectionsSialylation, mTOR signaling, TNF- signaling, Autophagy, Epigenetics, DNA damage responses
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1500 gene targeting experiments later…
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Editing efficiency in human cells
0-10 10-20 20-30 30-40 40-50 50-60 60-70 70-80 80-90 90-100
0
20
40
60
80
100
120
140
160
180
200
Editing Efficiency (in %)
# of
gRN
A pr
ojec
ts
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Cas9-induced mutational pattern
PAM-20 -18 -16 -14 -12 -10 -8 -6 -4 -2 0 2 4 6 8 10 12 14 16 18 20
0
200
400
600
800
1000
1200
1400
1600
1800
2000
Peak at position -3
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Cas9-induced mutational patternDeletions Insertions
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Assessment of off-target editing in clonal cell lines
Off-target sites
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Hap1 Gene Targeting – what we‘ve learned
CRISPR/Cas9 is highly efficient Mutations cluster at PAM -3
Deletions are favored over insertionsOff-target editing represents a minor issue
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So what can we do?
Exon 8 Exon 9 NanoLuc polyA
Exon 1 Exon 3
Translocations and Fusions
Gene taggingChromosomal deletions
Chr 1 Chr 19
Point mutations
Exon 8 Exon 9
*
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Exon 1 Exon 2 Exon 3
Exon Exon 2 Exon 31
Cas9-induceddouble-strand break
Exon 2 Exon 3
Homology-directed repair (precise)
Exon 1
Exon 1
Introduction of point mutations by homology-directed repair
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Point mutation in EGFR L858R
Targeting Efficiency ~8%
AACGTACTGGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCTGGCCAAACTGAsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyLeuAlaLysLeu
AACGTACTAGTGAAAACACCGCAGCATGTCAAGATCACAGATTTTGGGCGGGCCAAACTGAsnValLeuValLysThrProGlnHisValLysIleThrAspPheGlyArgAlaLysLeu
Clone 5
Wild-type
SpeI
Positive
contro
l
Clone 1
Clone 2
Clone 3
Clone 4
Clone 5
Clone 6
Clone 7
Clone 8
Clone 9
Clone 10
Clone 11
Clone 12
Clone 13
Clone 14
Clone 15
Clone 16
Clone 17
Clone 18
Clone 19
Clone 20
Clone 21
Clone 22
Clone 23
PCR +SpeI
Inclusion of a restriction site knockin allows rapid screening
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Chromosomal deletions
HAP1 cells are disomic for a fragment from chromosome 15
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Strategy for CRISPR/Cas-mediated excision of chr15 fragment
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Deletion of chr15 fragment is detectable by PCR
400 clones screened
5 positive clones identified
~1% targeting efficiencyEssletzbichler et al Genome Research 2014
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Single cell clones that carry the deletion can be isolated
SKY staining of clone E9
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Translocations / Chromosomal Fusions
Chin J Cancer. 2013 Nov;32(11):594-603
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Interchromosomal translocation leads to CD74-ROS1 fusion
Chr 5
Chr 6
Chr 5
Chr 6
ROS1-CD74
CD74-ROS1
Translocation
CD74
ROS1
ex6 ex7
Chr 5
Chr 6
Simultaneous cleavage with Cas9
ex33 ex34
ex7
ex6
Screen for fusion by PCR
ex33
ex34
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PCR screening identifies two clones with CD74-ROS1 fusion
CD74-ROS1
ROS1-CD74
A10
E4
E4
A10
~1% Clones Tested are positive for
fusion
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Validation of both DNA and RNA
Analysis of CD74-ROS1 break point in chr6 of genomic DNA
CTTACGCATACTGCTGACAGTTAAATTTAGTTGAAG-GCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAACTTACGCATACTGCTGACAGTTAAATTTAGTTGAAG-GCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAACTTACGCATACTGCTGACAGTTAAATTTAGTTGAAGTGCCTGGGGCCTCAGTTTCTGCATCAGATTCATAGAA
Predicted1C21G13
ROS1 CD74
CCTGAAGTAGAAGGTCAAAGGGCCACCCTCACAGGCTGGATTACTTAATCCCTCTCTGAAATACCCACAATCCTGAAGTAGAAGGTCAAAGGGCCACCCTCACAGGCTGGATTACTTAATCCCTCTCTGAAATACCCACAATCCTGAAGTAGAAGGTCAAAGGGCCACCCTC------TGGATTACTTAATCCCTCTCTGAAATACCCACAAT
Predicted1C21G13
CD74 ROS1
HCT116
eHAP1C2
1G13wate
rHCT116
eHAP1C2
1G13wate
rHCT116
eHAP1C2
1G13wate
rHCT116
eHAP1C2
1G13wate
r
CD74-ROS1 ROS1-CD74 CD74 ROS1
CD74 exon 6 ROS1 exon 34
Analysis of expression of CD74-ROS1 fusion transcript
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So where next?
Exon 8 Exon 9 NanoLuc polyA
Exon 1 Exon 3
Translocations and Fusions
Gene taggingChromosomal deletions
Chr 1 Chr 19
Point mutations
Exon 8 Exon 9
*
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Gene Tagging - The conventional approach
Gene tagging by homology-directed repair
Exon 7 Exon 8 Exon 9
polyANanoLuc
Exon 7 Exon 8 Exon 9
Homology-directed repair
polyANanoLuc
Exon 9
Genome
Homology donor
Two major shortcomings: a. Low overall efficiencyb. Requires the synthesis of gene-specific donor templates
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Gene tagging by non-homologous end joining
Developed further by Thijn Brummelkamp (NKI) and Daniel Lackner (Horizon)
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Gene tagging by non-homologous end joining
Based on generic donor cassettes flanked by tia11 guide RNA recognition sites
polyANanoLuc tia11tia11
tia11 gRNAU6
Cas9
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Gene tagging by non-homologous end joining
Based on generic donor cassettes flanked by tia11 guide RNA recognition sites
Exon 7 Exon 8 Exon 9 polyANanoLuctia11
Exon 7 Exon 8 Exon 9
Generic donor cassettes
Non-homologous end joining (imprecise)
polyANanoLuc
tia11
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Genotyping on pools of cells after transfection
gRNA 2655
---2656
--- 2657
2658
--- 2659
2660
2661
--- 2662
--- ---2663
2665
2664
2666
2667
--- 669
---
ID1 MX2 IRF9 STAT1 TAP2 CCL2 IL9
13 out of 14 pools show integration of reporter cassette in right orientation
Exon 9 polyANanoLuc
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Single clones bearing reporter constructs
Gene ID gRNA ID # clones # PCR-positive clones
Integration confirmed by sequencing
Editing Efficiency
ID1 2655 24 3 2 8%
ID1 2656 24 5 5 21%
IRF9 2659 24 1 1 4%IRF9 2660 24 1 0 N/A
TAP2 2663 24 0 0 N/A
TAP1 2664 24 0 0 N/A
CCL2 2665 24 0 0 N/A
CCL2 2666 24 1 1 4%
IL6 996 24 3 3 13%
BUT... only one clone contained an in-frame cassette integration!
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Sequencing of individual clones
>2655-13 AACCCCCGGGGGCCGAGGGCTGCCGGTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC
>2655-17 AACCCCCGGGGGCCGAGGGCTGCCGGTCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC
>2656-07 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC
>2656-10 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC
>2656-11 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC
>2656-15 CCGGTCCGGGCTCCGCTCAGCACCCTCATCCAGGGGCAGCGGATCCATGGTCTTCACACTC
>669-14 CTGACCCAACCACAAATGCCAGCCTGCTTCCAGGGGCAGCGGATCCATGGTCTTCACACTC
>2659-08 CAGATGGAGCAGGCCTTTGCCCGATACTTCCAGGGGCAGCGGATCCATGGTCTTCACACTC
>2666-10 CAGAAGTGGGTTCAGGATTCCATGGACCTCCAGGGGCAGCGGATCCATGGTCTTCACACTC
>669-24 ACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGCAGCGGATCCATGGTCTTCACACTC
>669-12 ACCACCCCTGACCCAACCACAAATGCCAGCCTGCTGCAGCGGATCCATGGTCTTCACACTC
>2656-24 CGGTCCGGGCTCCGCTCAGCACCCTCAATCCAGGGGCAGCGGATCCATGGTCTTCACACTC
Genomic Sequence Cassette Sequence
Precise cleavageLigationNo indels
Imprecise cleavageLigationIndels
Insertion is much more precise than originally predicted
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Assessing off-target integration of reporter cassette
HAP1 NanoLuc cell lines contain single integration events (as assessed by droplet digital PCR)
Hap1ID1-NanoLuc
HAP2DACT1-NanoLuc
HCT116HK2-NanoLuc
HAP1wt
NanoLuc copy number:
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A DACT1-NanoLuc reporter line
DACT1 expression is up-regulated in response to stimulation with Activin A
0
500
1000
1500
0
2000
4000
6000
8000
10000
Rela
tive
luni
nesc
ence
DACT1-NanoLuc levels
Rela
tive
luni
nesc
ence
Activin A(ng/ml)
0 10 10050 0 10 10050
4 h stimulation 24 h stimulation
Daniel Lackner
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The combination of CRISPR and a haploid background lends itself to both simple and complex genomic modifications
Modification Targeting Efficiency in Hap1Knockout >40%Point Mutation ~8%Chromosomal Deletion ~1%Chromosomal Translocation ~1%NHEJ Ligation Gene Tagging Up to 21%
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So lets wrap up!
Yes, CRISPR-Cas9 genome editing can be…
Easy to design
Efficient
Widely applicable
Flexible
…so how can Horizon help?
But…
× Not every cell line is easy to target
× Not every guide is active
× Genome editing is labour intensive and will not always be successful
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Already available…• Knockouts for >1,500 human genes• Verified by Sanger sequencing• Two independent clones per gene available• Can be supplied with gRNA used in generation• Supplied with wild type control line
$990 per cell line (Academic pricing)
How can Horizon advance your research?
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How can Horizon advance your research?
Haploid Genome Editing On Demand
• Hap1 cell line background
• Rapid, cost effective knockouts and genomic deletions
• Custom modifications also available (knockins, translocations, tags)
Custom Cell Line Engineering Service
• Your cell line
• Your choice of modification
• Fully custom service
iPSC Gene Editing Service
• Knockouts, knockins, mutation corrections
• You supply the iPSCs
• Custom modifications in 12-18 weeks
In vivo Genome Editing
• Many mice and rat knockout models already available
• Microinjection ready guide RNAs
• Custom in vivo genome editing service also available
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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
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 Thorne, PhDCommercial Marketing [email protected] +44 1223 204 799
Follow me on LinkedIn: cmcthorne