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Tristan FELIX, MSc. October 25th 2016
Dr. Miccio LabImagine Institute, Paris
Droplet Digital PCR: a tool to quantify CRISPR/Cas9 mediated genomic deletions
CRISPR Precision Genome Editing CongressBerlin 2016
In collaboration with:
2
Outline
CRISPR/Cas9 mediated genomic deletions
Molecular tools to detect and quantify genomic deletions- PCR- Cellular cloning- qPCR
ddPCR Experimental applications
Droplet Digital PCR (EvagreenTM)
3
Outline
CRISPR/Cas9 mediated genomic deletions
Molecular tools to detect and quantify genomic deletions- PCR- Cellular cloning- qPCR
ddPCR Experimental applications
Droplet Digital PCR (EvagreenTM)
CRISPR/Cas9 mediatedgenomic deletions
Potential applications:
- Study of regulatory elements (e.g. lncRNA genes, putative enhancers, silencers…)
- Therapeutic approaches (e.g exons containing stop codon or deletion of putative silencers…)
PUTATIVE REGULATORY ELEMENT
SILENCER
γ-globin
Nelson et al., Science, 2016.Long et al., Science, 2016.Duchenne Muscular Dystrophy: deletion of mutated Exon 23 carrying a stop codon (proof of concept on mdx mice).
Ye et al., PNAS, 2016.Beta-thalassemia and sickle cell disease: reproduction of HPFH5 (Hereditary Persistence of Fetal Hemoglobin) genomic deletion in HSPCs.
EXON 22 EXON 23 EXON 24 EXON 25
EXON 22 EXON 24 EXON 25
4
5
CRISPR/Cas9 mediatedgenomic deletions
Cell linesHSPCsiPSCs
• Evaluation of genome editing efficiency
Viral delivery:
DNA delivery:
Cas9 gRNA1gRNA2
gRNA1
gRNA2
RNA delivery:
CRISPR/Cas9 system delivery
• Impact on gene expression andfunctional rescue of the phenotype
Cas9 RNP delivery:
6
CRISPR/Cas9 mediatedgenomic deletions
Cell linesHSPCsiPSCs
• Evaluation of genome editing efficiency
Viral delivery:
DNA delivery:
Cas9 gRNA1gRNA2
gRNA1
gRNA2
RNA delivery:
CRISPR/Cas9 system delivery
• Impact on gene expression andfunctional rescue of the phenotype
Cas9 RNP delivery:
7
CRISPR/Cas9 mediatedgenomic deletions
Cas9/gRNA1 complex
5’ cut position
gRNA 13’ cut position
gRNA 2
Cas9/gRNA2 complex
Deletion junction
Single 5’ cut
NHEJ repair
Simultaneous 5’ and 3’ cut
NHEJ repair
Single 3’ cut
NHEJ repair
Target region (3- to 14-kb long)
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CRISPR/Cas9 mediatedgenomic deletions
5’ cut position
gRNA 13’ cut position
gRNA 2
Deletion junction
Single 5’ cut
NHEJ repair
Simultaneous 5’ and 3’ cut
NHEJ repair
Single 3’ cut
NHEJ repair
Target region (3- to 14-kb long)
Inversion
Cas9/gRNA1 complex
Cas9/gRNA2 complex
9
Outline
CRISPR/Cas9 mediated genomic deletions
Molecular tools to detect and quantify genomic deletions- PCR- Cellular cloning- qPCR
ddPCR Experimental applications
Droplet Digital PCR (EvagreenTM)
10
Molecular tools to detect genomic deletions
5’ cut position
gRNA 13’ cut position
gRNA 2
Deletion junction
Detecting deletion events by regular PCR:
A B
D
A BDeletion A + B
Inversion A + D
Cas9/gRNA1 complex
Cas9/gRNA2 complex
Target region (3- to 14-kb long)
11
Molecular tools to detect genomic deletions
5’ cut position
gRNA 13’ cut position
gRNA 2
Deletion junction
Detecting deletion events by regular PCR:
A B
D
A BDeletion A + B
Inversion A + D
LadderCas9
+ gRNAsCas9
Qualitative detection
Cas9/gRNA1 complex
Cas9/gRNA2 complex
Target region (3- to 14-kb long)
Cas9 + gRNAsCas9
DEL INV
12
Molecular tools to detect genomic deletions
Quantification of deletions by cellular cloning:
Cellular cloning by dilution:0,2 cell/well
Genotyping by regular PCR
% DEL / % INV / % WT
Cell linesHSPCsiPSCs
CRISPR/Cas9 system delivery
Viral delivery:
DNA delivery:
Cas9 gRNA1gRNA2
gRNA1
gRNA2
RNA delivery:
RNP delivery:
13
Molecular tools to detect genomic deletions
5’ cut position
gRNA 13’ cut position
gRNA 2
Deletion junction
A
C
B
D
A B
Cas9/gRNA1 complex
Cas9/gRNA2 complex
Quantification of deletions by cellular cloning:
Target region (3- to 14-kb long)
14
Molecular tools to detect genomic deletions
5’ cut position
gRNA 13’ cut position
gRNA 2
Deletion junction
A B
A BDeletion A + B
Cas9/gRNA1 complex
Cas9/gRNA2 complex
Quantification of deletions by cellular cloning:
C DTarget region (3- to 14-kb long)
15
Molecular tools to detect genomic deletions
5’ cut position
gRNA 13’ cut position
gRNA 2
Deletion junctionDeletion A + B
Cas9/gRNA1 complex
Cas9/gRNA2 complex
C
Inversion A + D
B
A B
A
D
Quantification of deletions by cellular cloning:
Target region (3- to 14-kb long)
16
Molecular tools to detect genomic deletions
5’ cut position
gRNA 13’ cut position
gRNA 2
Deletion junction
B
D
A B
Cas9/gRNA1 complex
Cas9/gRNA2 complex
Deletion A + B
Inversion A + D
WT A + C
A
C
Quantification of deletions by cellular cloning:
Target region (3- to 14-kb long)
17
Molecular tools to detect genomic deletions
Quantification of deletions by cellular cloning:
Examples:
Clone WT/WT
Clone DEL/WT
Clone DEL/DEL
WT PCR INV PCRDEL PCR
Time-consuming
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Molecular tools to detect genomic deletions
Deletion junction
qPCR A qPCR B CTRL F CTRL R
ΔΔCt quantification using intrachromosomal reference CTRL primers
Relative values must be interpolated from a standard curve
Quantification of deletions by qPCR:
gRNA2
19
Molecular tools to detect genomic deletions
Quantification of deletions by qPCR:
gRNA1
WT cell line
Clone DEL/DEL
Target region (14-kb long)
Validation of bi-allelic (DEL/DEL) clone by targeted sequencing
20
Molecular tools to detect genomic deletions
Creation of a standard curve using gDNA from genotyped clones
CloneDEL/DEL
Standard mixes
Example :CTRL primers: 21.401 CtDEL primers : 23.306 Ct ΔΔCt = 0.27711
Quantification of deletions by qPCR:
0.0 0.2 0.4 0.60
20
40
60
80
100
DDCt
Fra
ctio
n o
f d
ele
ted
alle
les
(%
)CloneWT/WT
Standard curve
36.1%
DEL % ΔΔCt
0% 0.00344
3% 0.02131
5% 0.03901
10% 0.08387
20% 0.15121
30% 0.22548
40% 0.31385
50% 0.39748
80% 0.59894
21
Molecular tools to detect genomic deletions
• qPCR gives poorly reproducible results for low frequency deletion events
• The use of a standard curve method
- requires the availability of genotyped clones - can represent a source of error- is time-consuming
22
Outline
CRISPR/Cas9 mediated genomic deletions
Molecular tools to detect and quantify genomic deletions- PCR- Cellular cloning- qPCR
ddPCR Experimental applications
Droplet Digital PCR (EvagreenTM)
23
Digital PCR
First presented by Sykers et al. in 1992
- Target limit dilution
- Qualitative all-or-none end point reactions
- Application of Poisson statistics
24
Digital PCR
First presented by Sykers et al. in 1992
10 target molecules
- Target limit dilution
- Qualitative all-or-none end point reactions
- Application of Poisson statistics
25
Digital PCR
First presented by Sykers et al. in 1992
- Target limit dilution
- Qualitative all-or-none end point reactions
- Application of Poisson statistics
10 target molecules
26
Digital PCR
First presented by Sykers et al. in 1992
10 target molecules
- Target limit dilution
- Qualitative all-or-none end point reactions
- Application of Poisson statistics
27
Digital PCR
First presented by Sykers et al. in 1992
10 target molecules
- Target limit dilution
- Qualitative all-or-none end point reactions
- Application of Poisson statistics
28
Digital PCR
First presented by Sykers et al. in 1992
6 Negative 13 Total
10 target molecules
- Target limit dilution
- Qualitative all-or-none end point reactions
- Application of Poisson statistics
C° = - Ln (6 / 13) / V
29
Droplet Digital PCR (EvagreenTM)
Principle of digital droplet PCR:
- Droplet partitioning enables thousands of digital measurements in a single well
One measurement Many thousands of discrete measurements
Uniform droplet generation (≅0.8nL/droplet)
30
Droplet Digital PCR (EvagreenTM)
Principle of digital droplet PCR:
- All-or-none end-point PCR reaction in each droplet
- The number of target present in each droplet does not change the fluorescence at the end of the reaction
31
Droplet Digital PCR (EvagreenTM)
Principle of digital PCR:
- Target concentration will influence the number of positive droplet
- Through a Poisson law distribution…
No target Low concentration
Medium concentration
High concentration
32
Droplet Digital PCR (EvagreenTM)
Principle of digital PCR:
- Application of Poisson statistics allows the calculation of the initial target concentration in each sample
Siméon Denis Poisson (1781-1840)
Target concentration formula:
C° = - Ln (Nneg / Ntot) / Vdroplet
Vdroplet = 0.8nLNneg = number of negative dropletsNtot = total number of droplets
33
Droplet Digital PCR (EvagreenTM)
Principle of digital PCR:
Example:
+ droplets
- droplets
Flu
ore
scen
ce
Droplet acquired along the time
34
Outline
CRISPR/Cas9 mediated genomic deletions
Molecular tools to detect and quantify genomic deletions- PCR- Cellular cloning- qPCR
ddPCR experimental applications
Droplet Digital PCR (EvagreenTM)
35
ddPCR Experimental applications
Application of ddPCR to quantify CRISPR/Cas9 mediated deletions:
Deletion junction
Chr11
qPCR A qPCR B CTRL F CTRL R
Absolute quantification of both deletion and an intrachromosomal control
36
ddPCR Experimental applications
Example of titration in clones
Validation in mono-allelic (DEL/WT) and bi-allelic (DEL/DEL) clones
gRNA1 gRNA2
WT cell line
Clone DEL/WT
Clone DEL/DEL
Target region (14-kb long)
37
ddPCR Experimental applications
Example of titration in clones
Validation in mono-allelic (DEL/WT) and bi-allelic (DEL/DEL) clones
DELprimers
CTRLprimers
DELprimers
CTRLprimers
2751
12286
5220
9579
Deletion frequency: 46.5%
5296
10988
4009
8212
Deletion frequency: 98.9%
238copies/μL
512copies/μL
463copies/μL
468copies/μL
Clone DEL/WT Clone DEL/DEL
Poisson modelisation =>
Ratio DEL/CTRL =>
Useful tool to screen bi-allelic clones
38
ddPCR Experimental applications
Quantification of low frequency deletion events
Standard curve: mix of gDNA from DEL/DEL clone and WT cell line:
StandardsDeletion
frequency (%)
100% 101 ± 0.001
50% 46.3± 1.10
40% 35.9 ± 0.14
25% 24.9 ± 1.07
20% 17.5 ± 0.02
10% 9.73 ± 0.4
5.0% 4.64± 0.03
2.5% 2.19 ± 0.02
1.0% 096 ± 0.01
0.5% 0.55± 0.02
0.0% 0.10± 0.01
0 20 40 60 80 1000
25
50
75
100
Theoretical deletion frequency (%)
De
letio
n fre
qu
en
cy
by
dd
PC
R (%
)
R2 = 0.9962
39
ddPCR Experimental applications
Quantification of low frequency deletion events
Standard curve: mix of gDNA from DEL/DEL clone and WT/WT cell line:
StandardsDeletion
frequency (%)
40% 35.9 ± 0.14
25% 24.9 ± 1.07
20% 17.5 ± 0.02
10% 9.73 ± 0.4
5.0% 4.64± 0.03
2.5% 2.19 ± 0.02
1.0% 0.96 ± 0.01
0.5% 0.55± 0.02
0.0% 0.10± 0.010 10 20 30 40
0
10
20
30
40
Theoretical deletion frequency (%)
De
letio
n fre
qu
en
cy
by
dd
PC
R (%
)
R2 = 0.9938
Zoom
40
ddPCR Experimental applications
Quantification of deletion events in HSPCs
DEL CTRL
Cas9
0.1 % 21.4 %4.5 %
Protocol 2Protocol 1
DEL CTRL DEL CTRL
41
ddPCR Experimental applications
Evaluation of deletion frequency by multiplex ddPCRPerformed on precious and/or low concentrated samples (modified HSPCs, iPSCs…)
CTRL primers70nM181bp
DEL primers40nM105bp
Ch
ann
el 1
Channel 2
NEGATIVE
DEL +
CTRL +
DEL + / CTRL +
Differential primer concentration changes droplet fluorescence after end-point PCR reaction.
42
ddPCR Experimental applications
DEL / CTRL simplex titration DEL / CTRL multiplex titration
DEL Chr11
(copies/μL) (copies/μL) % DEL
54 553 9.76
DEL Chr11
(copies/μL) (copies/μL) % DEL
30,3 284 10.7
DEL +
CTRL +
DEL + / CTRL +
NEGATIVE
CTRLprimers
DELprimers
Evaluation of deletion frequency by multiplex ddPCRPerformed on precious and/or low concentrated samples (modified HSPCs, iPSCs…)
43
Conclusions
EvagreenTM ddPCR technology:
• Precise absolute quantification of genomic deletions and inversions.
• No requirement of standard curves.
• Rapid readout.
• Easy to optimize.
• Cost-effective.
• Multiplexable.
44
Acknowledgments
Lab members:
Annarita MICCIOChiara ANTONIANIVasco MENEGHINIOriana ROMANO
BIO-RAD:
Martial SAUMIERSylviane PACHECOKristen SLAWINSKIMargaret FANAGAN
Genomic & bio-informatic core facilities:
Olivier ALIBEUChristine BÔLE-FEYSOTMélanie PARISOTAurore POULIET Cécile FOURRAGEMohammed ZARHRATE
