Ultra-Sensitive Quantification of Genome Editing
Events Using Droplet Digital™ PCR
Jennifer R. Berman,1 Samantha Cooper,1 Bin Zhang,1 George Karlin-Neumann,1 Claudia Litterst,1 Yann Jouvenot,1
Eli Hefner,1 Yuichiro Miyaoka, 2 Bruce R. Conklin 2
1
Digital Biology Center, Bio-Rad Laboratories, Pleasanton, CA
2
Gladstone Institute of Cardiovascular Disease, UCSF, San Francisco, CA

Droplet Digital PCR

Bulletin 6712

Abstract

Genome editing has become a widely used tool and allows specific modification of almost
any cell’s genome. However, the often low (<5%) frequency of gene edits, in particular forsome primary or induced pluripotent stem cell types, hinders facile and reliable detection
by gel- or sequencing-based methods, or requires laborious clonal isolation of edited cells.
Here we introduce an ultra-sensitive, cost-efficient Droplet Digital PCR–based method forthe detection of HDR and NHEJ alleles generated using the CRISPR/Cas9 and TALEN
genome editing systems.
Introduction
Genome editing tools including TALENs and the CRISPR/
Cas9 system have revolutionized our ability to edit the
genome of any cell, including human induced pluripotent
stem cells (iPSCs). Sequence-specific nucleases induce
double-strand breaks or nicks at target sites, activating the
DNA repair pathways of non-homologous end joining (NHEJ)
and homology-directed repair (HDR). NHEJ produces small
insertions or deletions (indels) and is useful for disrupting
gene function. HDR can induce precise gene repair of one
to thousands of base pairs in the presence of a homologous
donor molecule, allowing for correction of point mutations andintroduction of exogenous sequences. Genome editing is anincreasingly common part of the molecular biologist’s toolkit
and is being actively developed for therapeutic indications.
While powerful, the efficiency of gene editing is cell type–
dependent and often low (<5%), particularly in primary cells
or iPSCs. Sanger sequencing and gel-based methods lackthe sensitivity required to detect such events in non-clonalcell populations. Detection by next-generation sequencing(NGS) presents hurdles in terms of cost and workflow,
and NGS sample prep can introduce bias for or againstedited alleles.
Droplet Digital PCR (ddPCR™) enables ultra-sensitive absolute
quantification of genome editing events. Here we introduce
ddPCR assay strategies for the detection of HDR- and NHEJedited alleles. Using these assays we can detect alleles inedited samples present at frequencies of less than 0.5%.
These methods are useful for ultra-sensitive detection ofedited alleles and offer a rapid, low-cost readout for technicaloptimization of genome editing protocols.

Methods
Genome Editing

The disease-relevant loci RBM20 and GRN were targeted
for editing in HEK293T cells using TALEN, wild type (WT)Cas9, dCas9-FokI, or Cas9-nickase strategies. TALENs
were constructed using the Voytas laboratory’s Golden
Gate assembly system (Cermak et al. 2011) and the MR015
backbone vector (gift from M. Porteus and M. Rahdar,
Stanford University). Guide RNAs (gRNAs) were cloned
into the Zhang laboratory’s pX335 (nickase) and pX330(nuclease) vectors (Cong et al. 2013 and Ran et al. 2013).
Droplet Digital PCR

The QX200™ Droplet Digital PCR System (Bio-Rad
Laboratories) was used as instructed by the manufacturer.
ddPCR reactions were assembled using standard protocols:
ddPCR Supermix for Probes (no dUTP) (Bio-Rad Laboratories)
was combined with 5–125 ng of sample, 1 µl of 20x assay
mix (1x primers at 900 nM each, 1x probes at 250 nM each),
4 units of restriction enzyme (HaeIII for RBM20 or MseI for
GRN), and water for a final reaction volume of 20 µl. Reactions
were converted into approximately 20,000 one-nanoliter
droplets using the QX200 Droplet Generator and transferred
to a 96-well plate for thermal cycling per manufacturer
recommendation for this supermix. For GRN assays, a discrete
2 minute extension step at 72°C was included, generatinga three-step thermal cycling protocol that accommodates alonger amplicon (252 bp). After thermal cycling, droplets wereread on the QX200 Droplet Reader and assigned as positiveor negative based on fluorescence amplitude.

Ultra-sensitive quantification of genome editing events by Droplet Digital™ PCR

Allele Quantification

For HDR assays, cluster thresholding and quantification
was performed using QuantaSoft™ Software. For NHEJ
drop-off assay analysis, cluster thresholds were assigned
using QuantaSoft and a modified data analysis strategy
was used for quantification.
Drop-off assays result in a two-dimensional plotNwith threeC ), single positiveV
clusters: droplets with no template (NemptyNdroplets containing NHEJ alleles (N NHEJ), and droplets that
contain WT alleles (N WT+), which are positive for both FAM
and HEX signal and appear on the 45° axis. (Figure 1). At low
sample loading, for example ~2,000 copies/well (~5 ng humangenomic DNA), most NHEJ mutant copies will occur in single
positive (FAM-only) droplets. However, as sample loadingor NHEJ fractional abundance increases, double positive
WT+NHEJ droplets appear as a “tail” extending leftward
from the WT-only cluster. In most cases, separation between
WT-only and double positive droplet clusters is inadequate
for robust thresholding because a distinct boundary isabsent. For this reason, and because the mathematicalanalysis described below accounts for it, the WT+NHEJ double
positive tail is grouped with the WT-only (green) cluster aspart of the WT+ droplet group. It is not advisable to eyeball athreshold for double positives; this is subjective and will likely
be incorrect. Future iterations of QuantaSoft Software willautomate this analysis.

FAM amplitude

10,000NNHEJ
NWT+

8,000

6,000

Tail
4,000

NEMPTY

2,000
0
0

1,000

2,000

3,000

4,000

HEX amplitudeFig. 1. Droplet group definitions for drop-off assay analysis.

The standard formula for quantification of Droplet Digital PCR
data is:VNempty = the number of droplets in the double negative
clusterlabeled “empty” (black)
N neg
■■ N
N total NHEJ = the number of droplets in the cluster labeled
C “NHEJ” (blue)■
V■dropletNWT+ = the total number of droplets in the cluster
labeled “WT+” (green)■■+

For NHEJ quantification, note that only NHEJ single positive
droplets are used for analysis. New analysis tools are indevelopment that will allow NHEJ quantification directlyfrom properly thresholded QLP files.
Results
HDR Edit Detection Using Droplet Digital PCR

12,000C

Because WT and WT+NHEJ droplet populations cannot beeasily separated, it becomes difficult to define Nneg and Ntotal.
To quantify NHEJ and WT copies from a drop-off assay, anappropriate subset of droplets is used to calculate N neg
and N total where:

N
N

V

N neg
N total

N neg
N totalC
VdropletC
Vdroplet© 2015 Bio-Rad Laboratories, Inc.

We developed assay strategies for the sensitive detectionof small (point mutations and <20 bp) and larger (>20 bp)
precise edits introduced via HDR (Figure 2). Using an allelicdiscrimination TaqMan Assay and Droplet Digital PCR,
Miyaoka et al. (2014) previously demonstrated ultra-sensitive
detection of edit-induced point mutations in iPSC cells,with detection sensitivity as low as 1 in 5,000 (0.02%). Thisapproach uses one primer pair that amplifies the intended edit
region and two competitive probes labeled with FAM and HEX
that are designed to bind edited (mutant) or WT sequences,respectively. Note that one primer is positioned outside thedonor molecule sequence to ensure quantification of integratededited sequences (Figure 2A).
Larger exogenous sequences, for example GFP or FLAGtags, can also be introduced by HDR-mediated editing. To
detect these rare integrated alleles, one reverse primer andprobe (FAM) is designed near the 5' end of the intended fusionsite inside the exogenous sequence (Figure 2B). The forward
primer is gene-specific and sits upstream of the intended
fusion site but outside of the homology arm sequence toensure quantification of integrated alleles only. To count the
total number of genomes interrogated, a standard reference
assay, such as the PrimePCR™ ddPCR Copy Number Assay

Bulletin 6712+

Ultra-sensitive quantification of genome editing events by Droplet Digital™ PCR

A. HDR Edit Detection Assay (Point and Small (<20 bp) Mutations)
HDR probe

W T probe

FAM

HEX

B. Standard Two-Step Protocol
400 bp 600 bp
A02

10,000

B02

1 kb
C02

2.5 kb
E02

3.7 kbD02

60,00080,000100,000 120,000

1.5 kb

F02

4.4 kb
G02

5.7 kb
H02

8,000
CH1 Amplitude

for RPP30 in HEX (assay ID dHsaCP1000485), may be usedin duplex if amplicon lengths are similar or in a second well
if lengths differ. Using a reference assay of amplicon length
similar to the designed edit detection target assay avoids
biased amplification in double positive droplets and servesto control for sample integrity.

6,000
4,000
2,000
0
0

20,00040,000140,000

Event number

Fig. 3. A three-step thermal cycling protocol with long extension times
improves amplification of long amplicons. A plasmid was quantified
keeping one primer constant but varying the amplicon length by changing
the location of the forward primer. Longer amplicons amplified more efficiently
with a three-step protocol with 2 minute extension (A) compared to a standard
two-step protocol (B), as evidenced by a significant improvement in separation
between negative (blue) and positive (grey) droplet populations.

B. HDR Edit Detection Assay (Large (>20 bp) Insertions)
HDR probe
FAM

Target Assay

Table 1. Recommended three-step extension times for long amplicons.
Length windows are approximate, will vary with sequence, and should bevalidated empirically. Use of ddPCR Supermix for Probes (no dUTP) andthe deep-well C1000™ Thermal Cyler is advised.

Reference Assay

Amplicon Length, kb
150 –1.5
1.5–2.5
2.5–4

Reference probe
HEX

Extension Temp,°C
72
72
72

Extension Time, min
2
4
6

NHEJ Edit Detection Using Droplet Digital PCR

Fig. 2. Primer and probe design for HDR edit detection assays.

Some donor molecules have long homology arms, which
influences edit detection assay amplicon length. We foundthat amplification efficiency is reduced when amplicon lengths
exceed 200 bp. This manifests as reduced amplitude of
positive droplets. A three-step thermal cycling protocol withincreased extension time (Table 1) improved performance
significantly in those cases (Figure 3).
A. Three-Step Protocol with 2 Minute Extension
400 bpA02

10,000600 bp
B02

1 kb
C02

1.5 kbD02

2.5 kb
E02

3.7 kb
F02

4.4 kb
G02

5.7 kbH02

CH1 Amplitude

8,000
6,000
4,000
2,000
0
0

20,00040,00060,00080,000

Event number© 2015 Bio-Rad Laboratories, Inc.

100,000 120,000

140,000

The introduction of indels can be a desired outcome of NHEJ
when gene inactivation (knockout) is intended. NHEJ-induced
indel alleles can also occur when specific edits mediated by
HDR using a donor template are sought. In either case, NHEJ
indel mutations can provide a readout of genome editing
nuclease activity during genome editing.
The challenge with detecting NHEJ edits is that a numberof different alleles can be generated from a single treatmentof cells with genome editing nucleases (Ran et al. 2013). One
edited sample can thus contain NHEJ alleles with deletions
and/or insertions of varying sizes. To quantify the indel alleles
generated, we used a drop-off assay strategy. This is an assaythat identifies mutated alleles based on the absence of a
WT signal.
In this assay, one primer pair amplifies the sequence that
includes the predicted cut site (Figure 4A). The cut site
could be, for instance, a site 3 bp upstream of a CRISPR
protospacer-adjacent motif (5'-NGG PAM) or the sequence
between two TALEN sites. A FAM-labeled reference probe
distant from the site of potential NHEJ is designed to countall copies of the amplicon. A HEX-labeled drop-off probe isdesigned to bind WT DNA and targets the predicted cut site.
This probe will not bind, or will “drop off,” when the allele has
an insertion or deletion at this site. It is the loss of this HEX

Bulletin 6712

Ultra-sensitive quantification of genome editing events by Droplet Digital™ PCR

signal (while maintaining FAM signal) that indicates presenceof an NHEJ allele (Figure 4B). Since this assay produces
three-cluster 2-D fluorescence intensity plots, the resulting
data cannot be directly quantified by QuantaSoft Software,
up to and including versions 1.7 and 1.8. Instructions forquantification are presented in the Methods section. An
automated analysis mode for this type of experiment will
be included in the next version of the software.
A
Reference probe

HEX

FAM

A
105

Drop-off assay schematic

HEX

FAM

Concentration, copies/μl

Drop-offprobe

edit detection assay. RBM20 R636S was detected down tofrequencies of less than 0.05% in 100 ng WT DNA (Promega
Corporation) spiked with a 2x serial dilution of gBlocks
Fragment synthetic template containing the C>A substitution(Figure 5A). The same RBM20 R636S edit detection assay
was used on HEK293T cells that had been treated with either
CRISPR/Cas9- or TALEN-based editing reagents to introduce
the C>A mutation for cell-based studies. Cell pools with <1%edited RBM20 alleles were easily identified (Figure 5B).

WT template – both probes bind

104
103
102
101
1
10

–1

10 –2
NTC

NHEJ template – only referenceprobe binds

B
WT-only (100 ng)
12,000
FAM amplitude

10,000
8,000
6,000

5

2.5 1.25 0.625 0.31
Percent mutant

0.16

0.08

0.04

TALEN-Edited
SampleCRISPR-Edited Samples

3
2
1
0
R3

R5

R11

R8

R9

Sample

2,000
0

4

R1

4,000

WT

B
Fractional abundance, %

FAM

WT

0

1,000

2,000

3,000

4,000

H E X amplitude
WT (100 ng) + 1% NHEJ Mutation
12,000Fig. 5. Quantification of HDR alleles in synthetic mutant templates andedited HEK293T cells. A, single-well measurements of gBlocks Fragment
synthetic mutant template 2x-diluted into a constant background of 100 ng
WT DNA demonstrates detection of mutant template present at frequenciesof less than 0.05%. The assay sensitivity is approximated by the red dotted line;
B, CRISPR- and TALEN-induced point mutations were detected in edited samples
at frequencies less than 1%.

FAM amplitude

10,000High-Sensitivity Quantification of NHEJ Alleles

8,000
6,000
4,000
2,000
0

0

1,000

2,000

3,000

4,000

HEX amplitudeFig. 4. NHEJ drop-off assay. A, primer and probe design for the NHEJ
drop-off assay; B, 2-D droplet fluorescence intesity plots of an NHEJ drop-off
assay using WT-only DNA and a Gene Fragment NHEJ mutation-positive control(WT + 1% gBlocks Gene Fragment containing a one base pair deletion at
the predicted cut site). The WT cluster (green) is positive for both FAM and HEX,
while the NHEJ mutant single-positive droplets (blue) are positive for FAM only.

High-Sensitivity Quantification of HDR Alleles

Dilated cardiomyopathy and sudden early death is associatedwith heterozygous germline mutations in RBM20, which
encodes an RNA-binding motif protein (OMIM: 613172).
Sensitive detection of the RBM20 R636S C>A pathogenic
point mutation was performed using an allelic discrimination© 2015 Bio-Rad Laboratories, Inc.

Frontotemporal dementia is a neurodegenerative diseasecaused by mutations in the progranulin gene GRN (OMIM:
607485). To detect NHEJ indels generated by CRISPR/Cas9
we designed a series of drop-off assays. A one base pairdeletion in synthetic templates was detected down to a fractional
abundance of approximately 0.1% (Figure 6A). In this experiment,
100 ng WT DNA per well was spiked with a 2x serial dilutionof a synthetic gBlocks Fragment DNA template containing a
one base pair deletion at the predicted Cas9 cut site. Similarsensitivity was observed on a synthetic gBlocks Fragment
DNA template with a one base pair insertion (data not shown).
The drop-off assay was then used on HEK293T cell samples
treated with editing reagents, showing detection of NHEJ
events present at frequencies as low as 0.3% (Figure 6B).

Bulletin 6712

Ultra-sensitive quantification of genome editing events by Droplet Digital™ PCR

Linearity of measurements of NHEJ events in HEK293T cells
over a 2x dilution series is shown for one sample (Figure 6C),
demonstrating the ability of this assay to consistently quantify
genome editing events across a range of sample inputs. A recent
publication confirmed the accuracy of this approach by using
drop-off assays in Droplet Digital PCR to quantify NHEJ alleles
in TALEN-edited T cells, demonstrating strong concordance to
NGS and flow cytometry data (Mock et al. 2015).
A. GRN R493X: NHEJ Quantification by Drop-Off Assay
10,000Concentration, copies/μl

1,000
100
10
1
.10

NHEJ
WT

.01

WT

5

2.5

1.25
0.63
Percent mutant

0.31

0.16

0.08

B. GRN R493X: NHEJ Fractional Abundance in Edited Samples
5.0
Fractional abundance, %CRISPR-Edited Samples

4.0
3.0

2.0
1.0
0

0.0%

0

1

2

3
4
5
GRN R493X edited sample

6

7

Quantification of Cut Site Usage and Biasin Nickase-Edited Samples

Advanced computational tools have been developed that
identify candidate gRNAs for efficient targeting of CRISPRbased nucleases. However, there has been limited empiricalvalidation of these computational tools and of predicted gRNA
efficiency in general. To examine potential bias in gRNA usage
by Cas9 nucleases, we compared NHEJ allele generation
at two different gRNA sites used for Cas9 nickase–based
editing. In this system, paired Cas9 nickases use two proximal
gRNA sites to nick a DNA locus targeted for editing (Ran etal. 2013). A nickase-edited sample was assayed with three
types of drop-off assay (Figure 7): one with a probe over eachnick site (nickase assay) and two using just a single drop-off
probe positioned over one of the two nick sites (CRISPR_A
and CRISPR_B assays). Note that HEX amplitude varies asa function of the type and number of NHEJ probes present.
The nickase assay has 2 NHEJ probes, CRISPR_A andCRISPR_B, and maximal HEX amplitude is roughly the sumof the individual probe amplitudes (Figure 7A). For this reason,an NHEJ event at one site will lose only partial HEX amplitudewith the nickase assay (Figure 7A, blue droplet cluster). As
seen in the 2-D plot (Figure 7A) and quantitatively (Figure
7B), in this sample gRNA CRISPR_B produces the majority
of NHEJ events, suggesting better nicking efficiency at thissite compared to the gRNA CRISPR_A site. Similar trends
were observed in a second edited sample in which GRN wastargeted. These data demonstrate the ability of Droplet Digital
PCR to quantify, empirically validate, and compare gRNA
efficacy in cells targeted for editing. This approach therefore
offers a quantitative readout useful for empirical validation ofgRNA site efficacy predictions made by computational tools.

C. GRN R493X: Dilution Series
1,000

NHEJ or WT, copies/µl

NHEJ
WT

100

10

1

.1
0

1

2

3
4
Sample dilution

5

6

7

Fig. 6. Quantification of NHEJ alleles in synthetic mutant templates andedited HEK293T cells. A, two merged well analyses of 100 ng of WT gDNA
per well with 2x serial dilution of synthetic 1 bp NHEJ deletion gBlocks Fragment
template at the GRN locus demonstrates detection down to 0.1%; B, CRISPRinduced point mutations in HEK293T cells can be detected when present at
frequencies as low as 0.3%; C, linearity of NHEJ detection across a 2x dilution
series in an edited HEK293T sample.

© 2015 Bio-Rad Laboratories, Inc.

Bulletin 6712

Ultra-sensitive quantification of genome editing events by Droplet Digital™ PCR

A. Assays Run on Nickase-Edited Samples
RBM20 nickase – edited sample

Nickase assay
Reference probe

Note the nickase assay has CRISPR_A + CRISPR_B NHEJ probes

CRISPR_A assay
Reference probe

Conclusions
We have shown that Droplet Digital PCR is a powerful
technology for ultra-sensitive quantification of NHEJ- andHDR-edited alleles in heterogeneous cell pools. The assay
strategies for detection of HDR and NHEJ events describedhere can detect CRISPR- and TALEN-mediated edits present
at frequencies of less than 0.5%. This rapid, cost-effective
method is amenable to high throughput and provides aconvenient readout for the detection of edited alleles andoptimization of editing protocols.
References

CRISPR_B assay

Cermak T et al. (2011). Efficient design and assembly of custom TALEN
and other TAL effector-based constructs for DNA targeting. Nucleic Acids
Res 39, e82.

Reference probe

Cong L et al. (2013). Multiplex genome engineering using CRISPR/Cas
systems. Science 339, 819 – 823.
Miyaoka et al. (2014). Isolation of single-base genome-edited human iPS
cells without antibiotic selection. Nat Methods 11, 291– 293.
B. NHEJ Production Is gRNA-Dependent
16

RBM20 Sample

NHEJ, copies/µl

14Mock et al. (2015). mRNA transfection of a novel TAL effector nuclease(TALEN) facilitates efficient knockout of HIV co-receptor CCR5. Nucleic
Acids Res 43, 5660–5571.

GRN Sample

Ran FA et al. (2013). Double nicking by RNA-guided CRISPR Cas9
for enhanced genome editing specificity. Cell 154, 1380–1389.

12
10
8

Visit bio-rad.com/ddPCRGenomeEditing
for more information.

6
4
2

FAM is a trademark of Applera Corporation. gBlocks is a trademarkof Intergrated DNA Technologies Inc. TaqMan is a trademark of
Roche Molecular Systems, Inc.

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C1000 Thermal Cyclers are covered by one or more of the following
U.S. patents or their foreign counterparts owned by Eppendorf AG:
U.S. Patent Numbers: 6,767,512 and 7,074,367.

WT, copies/µl

C. Wild-Type Copies/µl per Reaction
2,000
1,800
1,600
1,400
1,200
1,000
800
600
400
200
0

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The QX200 Droplet Digital PCR System and/or its use is covered by claimsof U.S. patents, and/or pending U.S. and non-U.S. patent applications
owned by or under license to Bio-Rad Laboratories, Inc. Purchase of the
product includes a limited, non-transferable right under such intellectual
property for use of the product for internal research purposes in the fieldof digital PCR only. No rights are granted for diagnostic uses. No rights
are granted for use of the product for commercial applications of any kind,including but not limited to manufacturing, quality control, or commercial
services, such as contract services or fee for services. Information
concerning a license for such uses can be obtained from Bio-Rad
Laboratories. It is the responsibility of the purchaser/end user to acquire
any additional intellectual property rights that may be required.

Fig. 7. Quantitative comparison of gRNA usage in nickase-edited
samples. A, primer and probe design strategy and representative 2-D
plots; B, Using the nickase, CRISPR_A and CRISPR_B assays on RBM20
and GRN edited samples reveals a clear difference in efficacy of different
gRNAs, as measured by NHEJ allele generation; C, for each locus,
nickase-edited sample were prepared using one sample preparation thatwas split across three wells for NHEJ quantification. The measurementof WT copies/µl demonstrates, by locus, that equivalent amounts ofsample were assayed in each well.

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