A P P L I C AT I O N N O T E

Homology-directed
repair with
Dharmacon Edit-R
CRISPR‑Cas9
reagents andsingle‑stranded
DNA oligos.

Abstract
CRISPR (clustered regularly interspaced short palindromic
repeats)-Cas9 (CRISPR-associated protein 9) is a revolutionary
tool that utilizes an RNA-guided nuclease for efficientsite‑directed genome engineering in various eukaryotic systems.
The double-strand breaks (DSBs) created by CRISPR-Cas9
are repaired in the cell by two predominant mechanisms:
imprecise non-homologous end joining (NHEJ) and precise
homology-directed repair (HDR). The Dharmacon™ Edit-R™
CRISPR-Cas9 gene engineering platform consists of bothsynthetic and expressed guide RNAs. In conjunction with Cas9
expression plasmids and lentiviral particles, it supports multiple
experimental workflows. Here we demonstrate an innovative
workflow to guide the insertion of 10-12 nucleotides into a
gene of interest by HDR with the Edit-R CRISPR-Cas9 system,
DharmaFECT™ Duo Transfection Reagent, and a single-stranded
donor DNA oligo (ssDNA).

Authors
John A. Schiel
Eldon Chou
Maren Mayer
Emily M. Anderson
Anja van Brabant Smith
Revvity, Inc.

Keywords
Homology-directed repair, homologous recombination,
non‑homologous end joining, gene editing, genome engineering,
Edit-R, Cas9, CRISPR, donor oligo, donor DNA, lipid transfection,
homology arms, insertion, repair, DSB, knockinHomology-directed repair with Dharmacon Edit-R CRISPR‑Cas9 reagents and single‑stranded DNA oligos.

Introduction

such as a sister chromatid or a foreign donor DNA sequence

The bacterial adaptive immune system, the CRISPR-Cas9

donors, one can rapidly design and synthesize a donor DNA

system, can be repurposed for targeted gene editing ofsequence containing the desired changes to the genomic

mammalian genomes.

location of interest.

(plasmid or DNA oligo). By using single-stranded DNA (ssDNA)

1,2,3

Derived from Streptococcus

pyogenes, the system uses the Cas9 nuclease proteinthat complexes with a tracrRNA and a targeting crRNA(which contains a 20 nucleotide guide sequence
complementary to the genomic target of interest).3
The genomic target is amenable to Cas9 targeting if it isupstream of a PAM (protospacer adjacent motif), which forS. pyogenes is NGG. The Edit-R CRISPR-Cas9 system mimics
the endogenous bacterial system by using a synthetic
tracrRNA and custom synthetic crRNA to guide Cas9 to a
specific target site in order to create a DSB three nucleotides
upstream of the PAM sequence. Once the DSB occurs,
the mammalian cell utilizes endogenous mechanisms torepair the broken genomic DNA, the most common methods
being NHEJ or HDR. While NHEJ repair of the DSB is often
imprecise and error-prone due to the generation of random
insertions and deletions (indels), HDR faithfully copies thegenetic information from a related DNA sequence,

The Edit-R CRISPR-Cas9 synthetic guide RNA experimental
workflow (Figure 1) consists of (1) a plasmid expressing
a mammalian codon-optimized gene sequence encoding
Cas9, (2) a synthetic crRNA designed to target a genomicsite of interest, and (3) a synthetic tracrRNA. The use ofchemically synthesized RNA components allows facile
generation of indels through NHEJ which can result in genedisruption (knockout). However, this same workflow canalso be combined with (4) an ssDNA donor oligo for rapid
genome engineering using HDR. Here, we first demonstrate
the application of Edit-R crRNA:tracrRNA in an experimental
workflow (Figure 2) including ssDNA oligos to knock inshort DNA sequences into two different genomic regionsof interest. Second, we establish lipid-based transfection
as an effective technique to accomplish HDR experimentswith knockin efficiencies as high as 25%. Last, we optimize
ssDNA oligo homology arm length to provide DNA donor
oligo design recommendations for use with the Edit-R
genome engineering platform. The methods presented
within this application note can be applied to HDR-based
insertion of epitope tags such as a FLAG™ tag, SNPs,
precise stop codons, and amino acid changes in the active
site of enzymes.

Results
Genome engineering using CRISPR-Cas9 requires
expression of the Cas9 nuclease with the crRNA andtracrRNA. This can be achieved by co-transfection of aplasmid expressing Cas9 and crRNA:tracrRNA (Figure 2A),
or by creation of a cell line in which the Cas9 cassette is
Figure 1: Edit-R CRISPR-Cas9 genome engineering. Illustrationof Cas9 nuclease (1) bound to the synthetic crRNA (2):tracrRNA(3) complex and targeted to genomic DNA by the guide sequencein the crRNA, adjacent to the PAM (red). Repair of DSBs canbe achieved by NHEJ in the absence of a donor template,or by HDR when a donor template such as a single-stranded
DNA oligo (4) is present.

delivered using lentiviral particles and stably integrated
and expressed prior to transfection with crRNA:tracrRNA.
For HDR and creation of knockins, it is important to optimize
experimental conditions to obtain the maximal levels of
DSBs. Therefore, experiments to optimize DSBs without adonor template should be performed (Figure 2A) to assess
and maximize levels of gene editing. These experiments
should be performed prior to experiments using thedonor template (Figure 2B) and be used as a preliminary
assessment for overall levels of DSB generation.

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Homology-directed repair with Dharmacon Edit-R CRISPR‑Cas9 reagents and single‑stranded DNA oligos.

Genome engineering at the VCP locus: A crRNA wasdesigned to target exon 2 of the human VCP gene andinsert a DNA sequence containing two restriction sites for
detection of the knockin (Figure 3A). First, a U2OS cell line
was co-transfected with a hCMV-PuroR-Cas9 nuclease
expression plasmid and the synthetic crRNA:tracrRNAcomplex using DharmaFECT Duo Transfection Reagent
according to transfection conditions optimized for maximal
indel formation (Figure 2A). Transfected cell populations
were either not selected or selected with puromycin
to enrich for Cas9 expression. Additionally, U2OS cells
stably expressing Cas9 were transfected with the
VCP crRNA:tracrRNA. All cell populations were assessed
72 hours post-transfection with a DNA mismatch detection
assay using T7 endonuclease I (T7EI) to estimate relativeA

indel formation (Figure 3B).
The above transfections were also performed in thepresence of a ssDNA donor oligo containing homology arms
and a 12 nucleotide sequence for insertion that contains
two adjacent restriction enzyme sequences (Figure 3A).
Transfected cells were assayed for knockin of the inserted
sequence 72 hours post-transfection by a Restriction
Fragment Length Polymorphism (RFLP) assay (Figure 3C).
The highest amounts of HDR knockin were observed in
conditions corresponding to the highest amount of gene
editing as measured by indels using the mismatch detection
assay (Figure 3B and C). This result highlights the importanceof optimizing experimental conditions to ensure maximal
creation of CRISPR-Cas9-mediated DSBs.

B

Figure 2: HDR-based experimental workflow for co-transfectionof synthetic RNAs, Cas9 expression plasmid and synthetic DNA
oligo donor. A Cas9 expression plasmid co-transfection workflow
with DharmaFECT Duo Transfection Reagent. Detection of
double-strand breaks (A) prior to performing the HDR transfection(B) ensures maximal double-strand breaks for HDR repair.
Optimization of maximal DSBs (A and dashed arrows) is necessary
to increase the amount of DSBs to be repaired by HDR.

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Homology-directed repair with Dharmacon Edit-R CRISPR‑Cas9 reagents and single‑stranded DNA oligos.

A

A

B

C

Figure 3: Gene editing at the VCP locus. Expressed Cas9 andsynthetic crRNA:tracrRNA were used to target the VCP locus for
HDR genome engineering in U2OS cells. A. Schematic of the VCP
locus. Exons are indicated by the blue boxes. The guide sequencein the synthetic crRNA is indicated by the gray highlighted region,
the PAM is indicated in red, and the cut site is indicated by theblue arrow. The inserted sequence is indicated in blue. B. Analysisof the efficiency of indel mutations using mismatch detection
analysis of untransfected cells and transfected cells without(no selection) and with (puro selection) puromycin selection or
using a Cas9-expressing cell line (Cas9 integrated). The percent
editing is indicated below the gels. C. Analysis of the efficiency
of HDR-based insertion of a HindIII and NheI restriction enzyme
sites introduced using a 30 nucleotide homology arm DNA oligo.
Percent HDR editing was calculated using an RFLP assay with NheI
digestion of VCP PCR amplicons. co-Tfx = co-transfection.

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B

C

Figure 4: Gene editing at the EMX1 locus. Expressed Cas9 andsynthetic crRNA:tracrRNA were used to target the EMX1 locus
for HDR genome engineering in U2OS cells. A. Schematic of
the EMX1 locus. Exons are indicated by the blue boxes anduppercase lettering; introns are indicated by the lowercase
lettering. The guide sequence in the crRNA is indicated by thegray highlighted region, the PAM is indicated in red, and thecut site is indicated by the blue arrow. The inserted sequenceis indicated in blue, and the endogenous stop codon is green.
B. Analysis of the efficiency of indel mutations using mismatchdetection analysis of untransfected cells and transfected cells
without (no selection) and with (puro selection) puromycin
selection or using a Cas9-expressing cell line (Cas9 integrated).
The percent editing is indicated below the gels. C. Analysis of theefficiency of HDR insertion of a HindIII and NheI restriction enzyme
sites introduced using a 30 nucleotide homology arm DNA oligo.
Percent HDR editing was calculated using an RFLP assay with NheI
digestion of EMX1 PCR amplicons. co-Tfx = co-transfection.

4

Homology-directed repair with Dharmacon Edit-R CRISPR‑Cas9 reagents and single‑stranded DNA oligos.

Genome engineering at the EMX1 locus: DharmaFECTDuo Transfection Reagent was used to co-transfect
U2OS cells with a hCMV-PuroR-Cas9 nuclease expression
plasmid and synthetic crRNA:tracrRNA complex targetingexon 3 of the human EMX1 gene, and to transfectsynthetic crRNA:tracrRNA into a Cas9-integrated U2OS
cell line (Figure 4A). Transfections were performed
as described above, including a ssDNA donor oligo.
Total gene editing (indel %) and HDR knockin in thenonselected, puromycin‑selected, and Cas9-integrated U2OS
cell populations were analyzed 72 hours post‑transfection(Figure 4B and C). Consistent with the results fromtargeting VCP, the highest amounts of HDR knockin at the
EMX1 locus were observed in conditions corresponding to
the highest amount of gene editing as measured by indel
formation using the mismatch detection assay.
Concentration of synthetic single-stranded donor DNA
oligo: In order to determine the optimal concentration of
the donor template for HDR, we performed a dose curveof the ssDNA oligo targeting the EMX1 locus. Non-selected
and puromycin-selected U2OS cells were transfected with
fixed amounts of the hCMV-PuroR-Cas9 nuclease expression
plasmid (200 ng/ well), synthetic crRNA:tracrRNA complex(25 nM final/well), and increasing amounts of ssDNA donor
oligo (0.5, 1.0, 2.5, 5.0, 10.0, 20.0, 40.0, 60.0, and 100.0
nM final/well) in a 96-well plate. Cas9-integrated U2OS
cells were transfected with fixed amounts of the synthetic
crRNA:tracrRNA complex (25 nM final/well), and increasing
amounts of ssDNA donor oligo (0.5, 1.0, 2.5, 5.0, 10.0,
20.0, 40.0, 60.0, and 100.0 nM final/well) in a 96-wellplate. Cells were harvested 72 hours post-transfectionand HDR knockin was detected using the RFLP assay.
Both non‑selected and puromycin-selected U2OS cell
populations display the highest levels of HDR knockin using
a final concentration range of 2.5 nM to 10.0 nM ssDNA
donor oligo (Figure 5). Similarly, the Cas9-integrated U2OS
cell line exhibited the highest levels of HDR knockin using a

Figure 5: Donor DNA oligo concentration optimization.
DharmaFECT Duo Transfection Reagent was used toco-transfect U2OS cells with a Cas9 expression plasmid,
EMX1 crRNA:tracrRNA, and increasing concentrations ofa donor DNA oligo with 30 nucleotide homology arms.
Alternatively, a Cas9-integrated U2OS cell line was transfectedwith crRNA:tracrRNA to target EMX1, and increasing
concentrations of a donor DNA oligo with 30 nucleotide
homology arms. The RFLP assay was used to determine theamount of HDR knockin for each concentration of donor
DNA oligo in each transfection. Data presented is from three
independent transfections.

Effect of homology arm length on HDR efficiency:
When targeting VCP or EMX1 (Figures 3 and 4), the ssDNA
oligo donor sequences contained 30 nucleotides ofhomology on each arm. In order to determine how homology
arm length can affect HDR efficiency, Cas9-integrated U2OS
cells were transfected with complexed crRNA:tracrRNAand ssDNA donor oligos with increasing homology arm
lengths. Donor DNA oligo homology arms were designed
to have 10 nucleotides of homology per arm, increasingin 10 nucleotide increments up to 70 nucleotides total
per homology arm. Cell populations were assayed at
72 hours post-transfection and the percent HDR knockin
was determined by RFLP (Figure 6). HDR was detected withhomology arms as short as 20 nucleotides at a knockinpercentage comparable to the longer homology arms thatwere tested (30-70 nucleotides).

final concentration range of 2.5 nM to 10.0 nM ssDNA donor
oligo (Figure 5).

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5Homology-directed repair with Dharmacon Edit-R CRISPR‑Cas9 reagents and single‑stranded DNA oligos.

Discussion

To this end, we were able to identify the optimal amounts

The Edit-R CRISPR-Cas9 genome engineering platform

for maximal levels of HDR in U2OS, for both co-transfection

employing chemically synthesized RNAs is a quick and easyof Cas9 plasmid plus synthetic crRNA:tracrRNA complex,

method to test multiple guide sequences for optimizing %and transfection of synthetic crRNA:tracrRNA complex into

indel formation through NHEJ, and achieving functionala Cas9-integrated cell line (Figure 5). Our data show thatgene knockouts. Here, we have demonstrated the ability to

HDR is possible with donor homology arms as short asutilize the same experimental workflow to rapidly create

20 nucleotides for each arm. However, the variability of theknockins through the HDR pathway simply by including

knockin is potentially greater at this arm length; therefore,a single-stranded DNA oligo donor template. We have

we recommend each individual homology arm length to befurther demonstrated the use of lipid-based transfection

at least 30 nucleotides flanking the desired insert.

using DharmaFECT Duo Transfection Reagent to achieveprecise sequence insertion through the HDR pathway,
whereas, in the literature, this is commonly achieved throughexpensive electroporation methods and use of costly Cas9
nuclease reagents. Recommended experimental conditions
for performing similar successful sequence insertion
experiments using these Edit-R CRISPR-Cas9 reagents andworkflows are shown in Table 1.

of ssDNA donor required (ranging from 2.5 nM to 10.0 nM)

When designing donor oligos for HDR, one must consider
where the Cas9 DSB occurs with respect to the homology
arms of the oligo donor. If the CRISPR protospacer andPAM sequence occurs in one of the homology arms of theoligo donor, silent mutations must be made at one of thetwo guanine bases of the NGG PAM and/or SNPs in the
protospacer sequence proximal to the PAM to prevent
Cas9 from cutting the donor oligo-repaired HDR site.

Optimizing gene editing experimental conditions using a

Lastly, it has been found that SNP correction rates are as

mismatch detection assay is essential before performing

much as four‑fold lower when the DSB occurs 100 bp away

HDR applications because of the generally lower baselinefrom the SNP;4 thus, try to design your crRNA to create a

rate of HDR when compared to NHEJ for repair ofCas9‑mediated DSB as close to the insertion site as possible

double-strand DNA breaks. One method for increasing

for maximal HDR efficiency.5

the percent editing in a population of transfected cells is
to subject the cells to the selective pressure of antibiotic
resistance, e.g., puromycin selection. We have observed thattransfected cell populations selected for Cas9 expression
by application of puromycin correlated with higher levelsof knockin through HDR. Furthermore, we have observed
higher levels of knockins when using a cell line thatstably expresses Cas9 nuclease. We observed that whencomparing the genomic engineering of the two targets
presented in this study, the mismatch detection assay andthe RFLP assay results did not correlate, which is consistent

Conclusion
The Edit-R CRISPR-Cas9 genome engineering platform
simplifies the experimental workflow by removing tediouscloning steps prior to testing gene knockout, gene deletionand HDR. With careful optimization of editing efficiency ineach desired cell line, precise insertion of short sequencesusing synthetic DNA oligo donors can be achieved utilizing
the HDR cellular process.

with the overall lower efficiencies of HDR compared to the
NHEJ. Yet, for each gene target tested, the highest amountof gene editing, as estimated by mismatch detection,is associated with the highest amount of HDR knockin.
It is important to note that careful transfection optimization
is a critical process to maximize HDR and decrease theoverall effort of screening individual clonal lines to find thecorrect HDR-induced modification. For example, one would
need to screen ten times more individual clonal lines when
HDR knockin levels are observed at 1% compared to 10%.
Additionally, expression of HDR machinery in cell lines andcell types can vary, which can affect HDR efficiency.

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Materials and methods
Lipid transfection: U2OS and Ubi-GFP U2OS-Cas9 cells(Thermo Scientific™ BioImage™ Proteasome Redistribution
Assay Cat #R0402102) were seeded in a 96-well plate
at 10,000 cells per well. Edit-R synthetic crRNAs andtracrRNA were individually resuspended in 10 mM
Tris-HCl (pH7.5), to 100 μM. crRNA and tracrRNA werecombined and the RNA was further diluted to 2.5 μM
using 10 mM Tris-HCl (pH7.5). A final concentration of6Homology-directed repair with Dharmacon Edit-R CRISPR‑Cas9 reagents and single‑stranded DNA oligos.

25 nM crRNA:tracrRNA complex (25 nM of each crRNA andtracrRNA) was used for transfection. Cells were transfectedwith 0.4 μL/well DharmaFECT Duo Transfection Reagent(Dharmacon Cat #T-2010-03) using 200 ng of Cas9 plasmid(for co-transfections) and 25 nM crRNA:tracrRNA complex.
A final concentration range of 0.5 to 100 nM ssDNA donor
oligo was used for transfections.

Note: Conditions were optimized in U2OS cell lines. Conditions mayneed optimization in your cells of interest.

Puromycin selection: Twenty-four hours post-transfection,
cells were exposed to 1.5 μg/mL of puromycin(Invivogen Cat #ant-pr-5). Cells surviving selection were
harvested, analyzed, and are referred to as Puro selection.
ssDNA donor oligos: DNA oligos were synthesized
in-house using standard solid-phase DNA synthesis anddesalting procedures. Each DNA oligo used contained 2
phosphorothioate linkages on the first two and last two DNA
bases. Oligos were ethanol precipitated and resuspended(100 μM) in 10 mM Tris-HCl (pH7.5). Oligos were further
diluted to 1 μM prior to transfection.
Genomic DNA isolation and DNA mismatchdetection assay: Genomic DNA was isolated 72 hours
post‑transfection by direct lysis of the cells in Phusion™
HF buffer (Thermo Scientific Cat #F-518L) proteinase

Figure 6: Optimizing homology arm length for maximal HDRknockin. DharmaFECT Duo Transfection Reagent was used totransfect a Cas9-integrated U2OS cell line with crRNA:tracrRNAtargeting EMX1 or VCP and a donor DNA oligo. Different donor
DNA oligos were used, each with increasing homology arm length.
A RFLP assay was performed on three independent transfections
to determine HDR-based knockin for each donor DNA oligo withdifferent homology arm lengths.
Table 1: Recommendations for HDR-based gene editing using
Edit-R expressed Cas9 and synthetic crRNA:tracrRNA in 96-well
cell culture dishes.

K (Thermo Scientific Cat #EO0491) and RNase A(Thermo Scientific Cat #EN0531) for 1 hour at 56 °C
followed by heat inactivation at 96 °C for 5 minutes.
PCR was performed with primers flanking the cleavage sites.
PCR products (500 ng) were treated with T7 endonuclease
I (T7EI; NEB Cat #M0302L) for 25 minutes at 37 °C
and the samples were separated on a 2% agarose gel.
Percent editing in each sample was calculated using ImageJsoftware (NIH, imagej. nih.gov/ij, 1997-2014).
Restriction Fragment Length Polymorphism assay:

Cells seeded at a density that gives 70-90% confluency on

Genomic isolation was performed as described abovetransfection day

72 hours post‑transfection. PCR was performed with

• Stable cell lines can be created by transduction using

primers flanking the restriction enzyme knockin site andEdit-R Cas9 Lentiviral Particles

outside of the homology arms used for each donor DNA

DharmaFECT Duo Transfection Reagent

oligo. PCR products were further purified and eluted in(optimized concentration for each cell line)

water. PCR products (500 ng) were digested in FastDigest

25 nM/well Edit-R synthetic crRNA:tracrRNAOligo concentration:• U2OS cells (co-transfection): 2.5-10 nM/well donor
DNA oligo• U2OS-Cas9 cells (integrated): 2.5-10 nM/
well donor DNA oligo

Buffer and 1 U of FastDigest NheI enzyme (Thermo Scientific
Cat FD0974) for 30 minutes at 37 °C and the samples
were separated on a 2% agarose gel. HDR knockin foreach sample was calculated using ImageJ software(NIH, imagej nih.gov/ij, 1997-2014).

30 nucleotide homology arms for small insertions(< 50 nucleotides)

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Homology-directed repair with Dharmacon Edit-R CRISPR‑Cas9 reagents and single‑stranded DNA oligos.

References
1. L. Cong, F.A. Ran, et al. Multiplex Genome Engineering
Using CRISPR/Cas Systems. Science. 339, 819-823 (2013).
2. P. Mali, L. Yang, et al. RNA-guided human genome
engineering via Cas9. Science. 339 (6121), 823-826 (2013).
3. M Jinek, K. Chylinski, et al. A Programmable
Dual‑RNA‑Guided DNA Endonuclease in Adaptive Bacterial
Immunity. Science. 337, 816-821 (2012).
4. B. Elliott, C. Richardson,e t al. Gene Conversion Tracts
from Double-Strand Break Repair in Mammalian Cells. Mol.
Cell Biol. 18, 93-101 (1998).
5. F.A. Ran, P.D. Hsu, et al. Double Nicking by RNA-Guided
CRISPR Cas9 for Enhanced Genome Editing Specificity.
Cell. 155, 1380-1389 (2013).

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