Droplet Digital™ PCR

ddPCR™ Genome Edit Detection Assays
ddPCR Genome Detection Assays offer a fast, precise, simple, and cost-effective
method for detection of genome editing events. Assays can be obtained for any target
using Bio-Rad’s easy-to-use, intuitive Digital Assay Site.
ddPCR Genome Edit Detection Assays:
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■■

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 nable rapid assessment of HDR and NHEJ edits generated by CRISPR-Cas9 orEother genome editing tools
Detect editing events present at frequencies of ≤0.5%
 rovide absolute quantification of genome editing events from as little as 5 ng
Pof total gDNA (cells from single well of a 96 well plate)■■Distinguish between homozygous and heterozygous edits in clonal populations■■

Results are easily analyzed using QuantaSoft™ Analysis Pro Software

Visit bio-rad.com/web/GenomeEditAssays for more information.

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Genome Editing
Genome editing with site-specific, programmable nucleases has
revolutionized the ability to edit the genome of any cell. Nucleases,
including zinc-finger nucleases (ZFNs), transcription activator-like
effector nucleases (TALENs), and clustered regularly interspaced
short palindromic repeat (CRISPR)–associated Cas nucleases,
produce site-specific DNA double-strand breaks (DSBs). DSBs canbe repaired by imprecise, error-prone non-homologous end joining(NHEJ) or by homology directed repair (HDR) pathways, leading totargeted mutagenesis (Figure 1).

ACas9gRNA(s)HDR donor (–n–)
NHEJ

HDR

Unedited cells

x

Cells with one allele editedNuclease-induced
double-strand break (DSB)
NHEJ

HDRDeletions

xx

Donor
templateInsertions

Cells with both alleles edited

B
Variable length
indelsPrecise insertion
or modification

Fig. 1. Targeted genome editing. DSBs introduced by nucleases can be repaired
by non-homologous end joining (NHEJ) or homology-directed repair (HDR). NHEJ
produces insertion and/or deletions (indels) of variable length at the site of the DSB.
HDR can introduce precise point mutations or insertions using a DNA donor template.

Creation of Recombinant Cell Lines using CRISPR-Cas9
Introducing CRISPR-Cas9 and guide RNA (gRNA) into cells (withor without a donor template) results in DSBs, which the cellularmachinery repairs by NHEJ or HDR. This results in a mixed
population of cells with heterogeneous indel errors and varying
allelic editing frequencies (Figure 2A). Transfected cells are
harvested and analyzed 24–72 hours post-transfection to measuregenome editing frequency at the desired locus. Clonal cell lines
can then be generated using serial dilutions to isolate single cells
followed by an expansion period. Each clonal cell line is thenassayed to verify the genome editing event (Figure 2B).

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Use Droplet Digital PCR to:■■Q uickly assess genome
edit efficiency■■
Evaluate guide RNAs,transfection reagents,and other experimental
conditions■■
Select pools for cell linecreation■■■■

 etect genome edits present
Dat low frequencies from cells
from a single wellAccelerate cell line
development by avoiding
expansion of cell populations
required to obtain large
amounts of DNAxxx■■

Distinguish between
homozygous andheterozygous clones

Fig. 2. Creation of clonal cell lines containing desired genome edits. A, CRISPRmediated genome editing can result in unedited cells, cells with one allele edited,and cells with both alleles edited. B, To isolate cells with both alleles altered by HDR,a mixed pool of cells is serially diluted. Subpopulations are screened using Droplet
Digital PCR (ddPCR), wells with cells of interest are selected and further subdivided
until a clonal population containing the desired edit is obtained.

© 2017 Bio-Rad Laboratories, Inc.

Droplet Digital PCR (ddPCR) for Ultra-Sensitive
Quantification of Genome Editing Events
The efficiency and mutation spectrum generated by genome
editing varies substantially depending on the type of cells used,transfection method, target sequence, and many other factors.
Therefore a simple, quick method is required to accurately
characterize and quantify genome-editing events.
The T7 Endonuclease 1 or Surveyor assays are commonly used
for detecting genome editing events. However, these methods
are semi-quantitative, offer limited sensitivity, are prone to falsepositives, and suffer from high background signal when sequencepolymorphisms are present. These assays require a large amountof starting material necessitating significant cellular expansion.
Further, these methods have limited utility for screening of singlecell derived clones. For a typical diploid target locus, a clone
with both alleles successfully altered via genome editing, will beindistinguishable from a clone with one mutated allele and one
wild-type allele.
Next generation sequencing is a comprehensive method forassessing genome editing but the turn around time is several
weeks, analysis is complex, and costs are high. This makes thismethod unsuitable for routine screening.
Droplet Digital PCR offers a superior ultrasensitive, rapid, and lowcost method for absolute quantification of genome editing events.
ddPCR allows absolute quantification from very small amounts ofstarting material. Clones harboring homozygous and heterozygous
edits can be distinguished and ddPCR Genome Edit Detection
Assays can be easily designed for any target of interest. This
enables a much more rapid and efficient workflow and provides theability to quickly identify and focus on desired clones.
ddPCR Genome Edit Detection Assays
Genome edit detection assays, optimized specifically for ddPCR, canbe ordered instantly for any target of interest on Bio-Rad’s easy-to-use
Digital Assay Site (Figure 3). Bio-Rad’s assay design algorithms
take into consideration everything from primer/probe placement
and melting temperature to GC content and even genomespecificity to design ddPCR Genome Edit Detection Assays withsuperior sensitivity, specificity, and accuracy, that accelerate yourtime to results.

Detection of HDR Edits using ddPCR HDR Genome Edit
Detection Assays
ddPCR Homology Directed Repair Genome Edit Detection (HDRGED) Assays use a modified mutation detection assay strategy.
The ddPCR HDR GED Assay contains primers, a FAM probe thatis designed to bind the HDR edit, and a non-fluorescent probethat prevents binding of the FAM probe to unedited alleles. ddPCR
HDR GED Assays must be run duplexed with a reference assay
containing a HEX probe. Thus, two amplicons are generated(Figure 4). FAM positive droplets represent the population of alleles
with the desired HDR edit and the HEX positive droplets represent
the total number of alleles. Ratio of FAM positive droplets to HEX
positive droplets provides the fractional abundance of HDR editedalleles (Figure 5).
ddPCR HDR Genome Edit Detection Assays:
■■

■■

■■

 an be designed to detect single base pair substitutions,C
insertions, and deletions up to 25 bp
Can be designed for donor homology arms ≤450 bp
 re available with amplicon length-matched reference assays
Afor human, mouse, and rat■■

Quantify precise edits with sensitivity ≤0.5%■■

Are quantifiable using QuantaSoft Analysis Pro Software

Target Assay

HDR probe

Non-fluorescent probe

FAMACA/C
Edit siteDonor

Reference Assay

Reference probe
HEX

Reference GeneFig. 4. HDR assay design.
6,000

FAM amptitude

5,000
4,000
3,000
2,0001,0000Fig. 3. Order ddPCR Genome Edit Detection Assays for any target on theDigital Assay Site. Easy, step by step workflow allows design of NHEJ and HDR
Genome Edit Detection assays in minutes.

01,000

2,000

3,000

4,000 5,000
HEX amptitude

6,000

7,000

8,000Fig. 5. HDR assay readout. HDR mutation positive control (WT + 20% gblocks genefragment containing a single base pair substitution) with HDR GED + HDR REF Assay.
The HDR edits are FAM+HEX-, while the HEX+ and FAM+HEX+ positive clusters
represent the total number of copies, including WT, NHEJ, and HDR edits.

Bulletin 6872

Detection of NHEJ Edits Using ddPCR NHEJ GenomeEdit Detection Assays
NHEJ assays are duplexed primer probe-based ddPCR assays(Figure 6). A FAM-labeled probe is designed to bind a reference
sequence distant from the nuclease target site but still within theamplicon. A second HEX NHEJ/drop-off probe binds WT sequenceat the nuclease target site, such that NHEJ-induced indels block theprobe from binding. In a 2-dimensional view of the ddPCR analysis,
droplets with signal from both the FAM and HEX probe contain wildtype amplicons while droplets that are FAM-positive but HEX-negative
contain amplicons with mutations at the target site (Figure 7). The
ratio of FAM positive to FAM/HEX double positive droplets provides
the fractional abundance of NHEJ mutations.

Ordering Information

ddPCR Genome Edit Assays for NHEJ:

Unique Assay ID

Gene NamedHsaRFS19383242
dHsaRFS83653015
dHsaRFS57922788
dHsaRFS32192561
dHsaRFS96462334
dHsaRFS70732107

Rpp30, human
Rpp30, humanRpp30, human
Rpp30, human
Rpp30, human
Rpp30, humandHsaRFS45001880
dHsaRFS19271653
dHsaRFS83541426
dHsaRFS57811199
dHsaRFS32080972
dHsaRFS96350745

EIF2C1, human
EIF2C1, human
EIF2C1, human
EIF2C1, human
EIF2C1, human
EIF2C1, humandRnoRFS70620518
dRnoRFS44890291
dRnoRFS19160064
dRnoRFS83429837
dRnoRFS57699610
dRnoRFS31969383

Rpp30, rat
Rpp30, rat
Rpp30, rat
Rpp30, rat
Rpp30, rat
Rpp30, rat

dRnoRFS96239156
dRnoRFS70508929
dRnoRFS44778702
dRnoRFS19048475
dRnoRFS83318248
dRnoRFS57588021

ApoB, rat
ApoB, rat
ApoB, rat
ApoB, rat
ApoB, rat
ApoB, ratdMmuRFS31857794
dMmuRFS96127567
dMmuRFS70397340
dMmuRFS44667113
dMmuRFS18936886
dMmuRFS83206659

Rpp30, mouse
Rpp30, mouse
Rpp30, mouse
Rpp30, mouse
Rpp30, mouse
Rpp30, mousedMmuRFS57476432
dMmuRFS31746205
dMmuRFS96015978
dMmuRFS70285751
dMmuRFS44555524
dMmuRFS18825297

ApoB, mouse
ApoB, mouse
ApoB, mouse
ApoB, mouseApoB, mouse
ApoB, mouse

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 etect indels up to 40 bp in length symmetrically on either side
Dof the cut-site, or 80 bp asymmetrically
Quantify precise edits with sensitivity ≤0.5%
 re quantifiable using the “drop-off assay” analysis option
Ain QuantaSoft Analysis Pro
Drop-off
probe

Reference
probe

HEX

FAM
Drop-off assay schematic

Cut site

HEX

FAM
WT template – both probes bindFAM
NHEJ template – only referenceprobe binds

indelFig. 6. NHEJ assay design.
9,000
8,000

FAM amplitude

7,000
6,000
5,000
4,000
3,000
2,000
1,00000

1,000 2,000

3,000 4,000 5,000 6,000 7,000 8,000 9,000
HEX amplitude

Catalog #Description

12002312	ddPCR HDR Gene Edit Assay, 100 rxns
12002313
ddPCR HDR Gene Edit Assay, 500 rxns
12003796	ddPCR HDR Gene Edit Pkg*, 1,000 rxns
12003805
ddPCR HDR Ref Assay, predesigned, 100 rxns
12003806
ddPCR HDR Ref Assay, predesigned, 500 rxns
12003793
ddPCR HDR Ref Pkg, predesigned, 1,000 rxns
12002314
ddPCR NHEJ Gene Edit Assay, 100 rxns
12002315ddPCR NHEJ Gene Edit Assay, 500 rxns
12003794
ddPCR NHEJ Gene Edit Pkg, 1,000 rxns
* Sequences of primers and probes of genome edit detection assays are provided
Available HDR Reference Assays
Use Unique Assay ID to find desired reference assay at
bio-rad.com/web/GenomeEditAssays

Fig. 7. NHEJ assay readout. NHEJ mutation-positive control (WT + 1% gblocks
gene fragment containing a one base pair deletion at the predicted cut site). The
WT cluster is positive for both FAM and HEX, while the NHEJ mutant single-positivedroplets are positive for FAM only.

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Life ScienceGroup

Bulletin 6872 Ver A

US/EG16-10300317

Sig 1216