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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

    -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 gRNA1 gRNA2

    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 gRNA1 gRNA2

    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 gRNA1 gRNA2

    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

    Ct 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 Ct = 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 % Ct

    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

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