Direct Quantification of Genome Editing Efficiency fromWhole Cells Using SingleShot Cell Lysis Buffer andddPCR Genome Edit Detection Assays
Meiye Wu and Madhuri Ganta
Bio-Rad Laboratories, Digital Biology Center, 5731 W Las Positas Blvd, Pleasanton, CA 94588, USA

Droplet Digital PCR

Bulletin 7155

Abstract

Genomic DNA extraction from transfected cell populations and single-cell clones for theassessment of genome editing efficiency is time consuming and a common pain point in gene
editing workflows. Here, we report a rapid, automation-compatible method that eliminates
the need for DNA extraction prior to determining gene-editing efficiency. Intact, whole cells
are lysed using diluted SingleShot Cell Lysis Buffer with proteinase K to release genomic DNA
from transfected cells. The cell lysate can be used directly to determine gene editing fractional
abundance using Droplet Digital PCR (ddPCR).
Introduction
With the advancement of CRISPR and other genome editing
technologies, modification of genomes to induce targetedchanges is opening up vast possibilities in gene therapy,
agriculture, and bioenergy. Though various methods of gene
editing reagent delivery and methods can be automated using
robotics, there remains a bottleneck where the induced genetic
changes are rapidly characterized. Currently, genomic edit
characterizations are performed on column-purified genomic
DNA (gDNA) from up to thousands of single-cell clones, greatly
reducing the efficiency and scalability of gene editing workflows.
Therefore, the need for a rapid, quantitative workflow to assess
genome edits at the population level to quickly provide metrics
for optimization of transfection and in vivo efficiency is critical togene editing scale-up and manufacturing.
Materials and Methods
Cell Culture

eHAP cells (catalog #C669; Horizon Discovery, Cambridge,UK) were maintained in IMDM (#1244053; Thermo Fisher
Scientific, Waltham, MA) plus 10% FBS (#26140-079; GIBCO,Gaithersburg, MD) supplemented with penicillin-streptomycin(#15070063; Thermo Fisher Scientific). Cells were trypsinizedand passaged at ~70% confluence. All transfections wereperformed on cells at ~40% confluence.

crRNA and ddPCR NHEJ Assay Design

CRISPR RNA (crRNA) targeting the earliest exon common
among all splice variants of STAT5A was designed using theBroad Institute’s Genetic Perturbation Platform. ddPCR NHEJ
assays targeting the crRNA putative cut sites were designed
according to manufacturer’s instructions using the automatedassay design portal found at bio-rad.com/digital-assays.
RNP Transfection

crRNA, transactivating RNA (tracrRNA) (#1073189; IDT, Skokie,IL), and Cas9 nuclease (#1074181, IDT) were transfected using
Lipofectamine RNAiMax Reagent (#13778075, Invitrogen,Carlsbad, CA). eHAP cells were thawed by diluting at 1:10 into
warmed growth media 24 hr prior to transfection. Transfectionconditions were 40,000 cells/well in 96-well cell culture plates.
Briefly, crRNA and tracrRNA were combined 1:1 and diluted
to 1 µM in NFDB buffer (#1072570, IDT). The 1 µM crRNA/tracrRNA solution was heated to 95°C for 5 min, then cooled
to room temperature (RT). Cas9 enzyme was diluted to
1 µM in OptiMEM (#31985070, Thermo Fisher Scientific) andcombined 1:1 with the crRNA/tracrRNA complex to generate
the ribonucleoprotein (RNP). The RNP was transfected at
40 nM with Lipofectamine concentrations at 1.5 µl/well. Thetransfected cells were incubated in a 5% CO2 incubator for
48 hr prior to harvesting for determination of NHEJ fractional
abundance by ddPCR.

Direct Quantification of Genome Editing Efficiency from Whole Cells Using SingleShot Cell Lysis Buffer and ddPCR Genome Edit Detection Assays

Assays (Figure 2). A FAM-labeled probe is designed to binda reference sequence distant from the nuclease target site
but still within the amplicon. A second HEX NHEJ/drop-off
probe binds the WT sequence at the nuclease target site,
such that NHEJ-induced indels block the probe from binding.
In a 2-dimensional view of the ddPCR analysis, droplets withsignal from both the FAM and HEX probes contain wild-typeamplicons while droplets that are FAM-positive but HEXnegative contain amplicons with mutations at the target site
(Figure 3). The ratio of FAM positive to FAM/HEX double
positive droplets provides the fractional abundance of NHEJ
mutations.

Cell Lysis and Proteinase K Digestion of Transfected Cells Usinga Modified SingleShot Protocol

SingleShot Cell Lysis Buffer without DNase (#1725080,Bio-Rad Laboratories, Hercules, CA) was diluted 1:6using sterile DI water. Proteinase K was added to a final
concentration of 40 µg/ml. Each well of transfected cells
was washed with 200 µl of PBS and incubated with 30 µlof diluted SingleShot Cell Lysis Buffer with proteinase K for
10 min at RT. After incubation, the cell lysates were transferred
to a clean 96-well PCR plate and incubated for 5 min at 37°C.
The proteinase K was then inactivated at 75°C for 5 min.
The resultant cell lysates were used immediately for ddPCR
analysis at 2 µl/reaction. The cell lysates can also be stored at–20°C to be used at a later time. See Figure 1 for a detailed
workflow using diluted SingleShot Buffer.

In a mixed population of cells containing both WT and edited
cells, the WT cells exhibit two fluorescent signals and theNHEJ edited population exhibits the reference probe signal
only (Figure 2). The 2-D plot of the ddPCR NHEJ Drop-Off

Population NHEJ Editing Assessment by ddPCR(Column Purification vs. SingleShot Lysate)

From the same STAT5A RNP transfection experiment, halfof the transfected wells were subjected to column-based
gDNA purification using the Quick-DNA Plus Kit (#D4074,Zymo Research, Irvine, CA) and eluted into 30 µl DI water. The
other half were lysed using the modified SingleShot protocol
detailed previously. Two microliters of lysate and 2 µl of
purified gDNA were used for each NHEJ genome edit probe
assay. SbfI HF (1:4,000 final concentration) was added to eachreaction to increase access to gDNA. The NHEJ assays wereperformed using the ddPCR Supermix for Probes (no dUTP)(#1863024; Bio-Rad) in the T100 Thermal Cycler (Bio-Rad)
using ddPCR 96-Well Plates (#12001925; Bio-Rad). Cycling
parameters were as recommended by the manufacturer.
After thermal cycling, the droplets were analyzed on theQX200 Droplet Digital PCR System (#1864100, Bio-Rad). The
fractional abundance of NHEJ DNA was determined using
QuantaSoft Analysis Pro Software (visit bio-rad.com for free
software download). The percent fractional abundance ofNHEJ events was calculated as: a/(a+b)*100, where a is thenumber of FAM positive droplets (NHEJ) and b is the numberof FAM/HEX double positive droplets (WT).

WT cells

Transfection ofgene editing reagents

xMixedpopulationxxxxxx

A

B

Cell lysis using
SingleShot Lysis Buffer
diluted with DI waterResults
We designed a modified protocol using the SingleShot
Cell Lysis Buffer, originally designed for performing bulk
RT-qPCR in whole cells as input (Figure 1), to enable ddPCR
assessment of population gene editing efficiency fromwhole cells immediately after transfection. To validate ourmethod, the STAT5A gene was subjected to CRISPR/Cas9RNP–mediated NHEJ editing. At 48 hr post-transfection, thetransfected cells were lysed using the diluted SingleShot Cell
Lysis Buffer and the fractional abundance of the edited cells
was quantified using a ddPCR Drop-Off Assay designed using
the assay design portal (bio-rad.com/digital-assays). Briefly,
NHEJ assays are duplexed primer probe-based ddPCR© 2019 Bio-Rad Laboratories, Inc.

xx

Columnpurificationof gDNA

ddPCR analysis

Fig. 1. Experimental workflow comparing the quantification of genome
editing efficiency between direct cell lysis and column-purified DNA.
A, gene edited cells were lysed directly into diluted SingleShot Lysis Buffer withproteinase K, followed by incubation at 37°C for 5 min and heat inactivation
at 75°C for 5 min. The lysate was used at 2 µl per 20 µl reaction of ddPCR
genome editing assay. B, gDNA from the gene edited cells was first extracted
by column purification and then analyzed by Droplet Digital PCR using addPCR NHEJ Drop-Off Assay.

2Bulletin 7155

Direct Quantification of Genome Editing Efficiency from Whole Cells Using SingleShot Cell Lysis Buffer and ddPCR Genome Edit Detection Assays

Assays shows WT cells in the top-right corner, with positivesignals in both FAM (Y-axis) and HEX (X-axis) channels, andthe NHEJ-edited cell population shifts to the left, with only theFAM reference signal remaining (Figure 3). The column-purified
gDNA from the same transfection experiment was used as a
Drop-off
probe

Reference
probe

HEX

FAM

benchmark for comparing the two workflows (Figures 3 and 4).
The substitution of diluted SingleShot Lysis Buffer for column
gDNA purification did not significantly alter the NHEJ fractional
abundance measurement (Figure 4A) nor did it impact thedroplet number (Figure 4B).

Drop-off assay schematicHEX

FAM
WT template — both probes bindFAM
NHEJ template — only reference probe bindsIndelFig. 2. Assay schematic for ddPCR NHEJ Drop-Off Assay. The ddPCR NHEJ assay comprises a pair of primers flanking the putative edit site and two
fluorescent probes, one reference probe that binds to wild-type (unedited) DNA, and a drop-off probe that will not bind if an editing event occurs at the Cas9
cut site. The NHEJ drop-off assay is designed using the web assay design portal bio-rad.com/digital-assays.
AB9,000

NHEJ

WT

8,000

NHEJ

WT

9,000

7,000

8,000

6,000

Channel 1 amplitude

Channel 1 amplitude

11,00010,000

5,000
4,000
3,000
2,000

7,000
6,000
5,000
4,000
3,000
2,000

1,000

1,000

00SingleShot Buffer–1,00000

B

1614

NHEJ fractional abundance, %1,000 2,000 3,000 4,000 5,000 6,000 7,000
Channel 2 amplitude12

14

1,000 2,000 3,000 4,000 5,000 6,000 7,000
Channel 2 amplitude

20,00018,000

16,25017,43016,0001214,000
10Droplet countA

Column Purification–1,000Fig. 3. 2-D plots of SingleShot lysate
versus column-purified gDNA. The
WT population is shown in orange and ispositive in both FAM and HEX channels,
whereas the edited NHEJ population isshifted to the left of the plot (blue). Empty
droplets are shown in grey. A, 2-D plot
from SingleShot lysate ddPCR reaction;
B, data from column-purified gDNA
obtained from the same transfection.

8612,000
10,0008,000
6,000

44,00022,00000SingleShot Buffer© 2019 Bio-Rad Laboratories, Inc.

Column Purification

SingleShot Buffer

3Column Purification

Fig. 4. Absolute quantification ofNHEJ events. A, NHEJ fractional
abundance was calculated using
QuantaSoft Pro Analysis Software withthe formula a/(a+b)*100 where a = NHEJ
population, b = WT population. There
was no significant difference in thefractional abundance observed between
the SingleShot and column-purification
workflows. B, the number of droplets
was also not impacted when using
SingleShot lysate.

Bulletin 7155

Direct Quantification of Genome Editing Efficiency from Whole Cells Using SingleShot Cell Lysis Buffer and ddPCR Genome Edit Detection Assays

Conclusion
Here, we present a workflow that combines the SingleShot
Cell Lysis Buffer with ddPCR Genome Edit Detection Assays
for a streamlined workflow that can directly assess populationlevel gene editing efficiency from whole cells. The workflow
bypasses the need for limiting dilution of single cells andcolumn purification for gDNA extraction. The entire process
can be performed immediately upon conclusion of transfection
and provide quantitative measurement of genome edit eventsin the population in less than four hours, with less than an hourof hands-on time.
Acknowledgements:

The authors would like to thank Steven Okino and Yan Wang
of Bio-Rad’s Life Sciences Group for insightful discussions
regarding CRISPR workflow process.
Visit bio-rad.com/GenomeEditing for more information.
Bio-Rad, Droplet Digital PCR, and ddPCR are trademarks ofBio-Rad Laboratories, Inc. in certain jurisdictions.
The QX200 Droplet Digital PCR System, and the consumables and reagents
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