Droplet Digital PCR: a tool to quantify CRISPR/Cas9...

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)

8

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

18

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

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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

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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

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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

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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)

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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

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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

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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

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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

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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

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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

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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

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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.

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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…)

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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.

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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