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

Fluorescenttagging of an
endogenous geneby homologydirected repair
using Dharmacon
Edit-R CRISPRCas9 reagents.

Abstract
CRISPR-Cas9 has increased the accessibility of genome
engineering due to its ease of use and ability to cause
double-strand breaks (DSBs) at almost any locus of interest. The
DSB that is created by Cas9, when guided to a specific genomic
locus by crRNA and tracrRNA, can be repaired by various
pathways, including homology-directed repair (HDR). The choiceof repair pathway depends on numerous factors, including thepresence of repair proteins and a repair donor template. Here
we utilize the HDR pathway in conjunction with Dharmacon™
Edit-R™ CRISPR-Cas9 reagents and a DNA donor plasmid tocreate a fluorescent reporter-gene fusion with an endogenous
gene target.

Keywords
Homology-directed repair, homologous recombination,

Authors
John A. Schiel
Maren M. Gross
Eldon T. Chou
Melissa L. Kelley
Anja van Brabant Smith
Revvity, Inc.

non-homologous end joining, gene editing, genome engineering,
Dharmacon Edit-R, Cas9, CRISPR, donor plasmid, donor DNA,homology arms, knockin, NHEJ, HDR

Introduction
When a double-strand break (DSB) is created in the genome,
it can be repaired by several different repair pathways,
predominantly non-homologous end joining (NHEJ),
microhomology-mediated end joining (MMEJ; also known
as alt-NHEJ), and homology-directed repair (HDR). Detailed
descriptions of these pathways have previously been
described1-3. Although researchers have been using HDR
to make modifications within cells for quite some time,Fluorescent tagging of an endogenous gene by homology-directed repair using Dharmacon Edit-R CRISPR-Cas9 reagents.

the HDR pathway has been characteristically inefficient,conversion tracts are extremely short; efficiency of insertionand has required the use of a donor construct containingof foreign DNA into a DSB by HDR decreases by 17% whenseveral kilobases (kb) of homologous DNA to create precise

the insert is > 10 nt away from the cut site, and as muchgenomic modifications. Since the advent of site-specificas 87% when the insert is 100 nt away from the cut site8.

nucleases, we have seen HDR efficiencies increase through

Therefore, it is important to assess the proximity of crRNAs

creation of targeted DSBs to force the cell to initiate DSB

to the insertion site and balance this with the crRNA cutting

repair pathways.

efficiency in order to determine the optimal crRNA for

With the ability to efficiently target most genomic lociprecise genome engineering.

for genome engineering with CRISPR-Cas9 technologies,

Donor design considerations: When designing a donor

researchers are able to more efficiently introduce exogenous

plasmid, it is important to remember that Cas9 can target

DNA into the genome. These changes are often insertions of

the donor template itself if it contains an intact protospacer

single nucleotide polymorphisms (SNPs), corrections of single

sequence next to a protospacer adjacent motif (PAM).

base mutations, insertions of fluorescent tags or reporter

Therefore, in order to avoid Cas9 cutting the donor

genes, or addition of genes or other pieces of DNA into a

template, it is important to design the donor template withsafe harbor site4-6. Depending upon the scientific question

one or more of the following changes (Figure 1).

at hand, and the size of the DNA to be inserted within
the genome, multiple factors will impact the efficiency oftargeted HDR.
Required components: There are several components thatneed to be introduced into a cell for a CRISPR-Cas9 HDR
experiment: Cas9 nuclease (plasmid, mRNA, recombinant
protein, or a stable cell line), guide RNA (gRNA) (synthetic
crRNA:tracrRNA, synthetic sgRNA, or sgRNA expressed froma plasmid), and a donor template (single-stranded DNA oligo
or plasmid). Depending on the source of Cas9, gRNA, donor
template, and cell type, the best delivery method should bedetermined and optimized. This optimization is essential tomaximize the number of DSBs for sequence insertion with
HDR as discussed further below.
Additionally, it is important to decide on the correct type ofdonor DNA template (single-stranded DNA oligo or plasmid),and the design of the homology arms for HDR. For short
insertions such as small nucleotide (nt) changes (< 50 nt),
synthetic donor oligos with 30-40 nt homology arms willbe the most efficient method7. For large DNA insertions,such as creating a reporter fusion, donor plasmids withindividual homology arm lengths between 500 and 1000 nt

Figure 1: Design homology arms that avoid additional roundsof cutting & NHEJ repair after insertion of desired sequence
by HDR. Three types of DNA donor oligo design considerations
are presented to avoid Cas9 cutting. 1. A DNA donor oligo withhomology arms specific to the gene target (underlined) flanking
the insert to be integrated via HDR (blue text).The insert separates
all, or a part of, the crRNA target sequence from the PAM (redtext). 2. A DNA donor oligo with the right homology arm containing
a nucleotide substitution (green text and *) in the PAM. 3. A DNA
donor oligo with the right homology arm containing two nucleotide
substitutions (green text and *) in the crRNA target sequence.

are required6,9.

the crRNA-guided Cas9 nuclease. For HDR experiments,

1. Split the protospacer sequence (crRNA target sequence),or a portion of the protospacer sequence, and the PAM
between the two homology arms of the donor plasmid sothat the intended DNA insert is between these
two elements;

selection of the crRNA target site is a balance between the

2. Introduce silent SNPs in the S. pyogenes NGG PAM; or

Selection of cut site by CRISPR-Cas9: HDR starts andends with the creation and repair of a DSB. Therefore, it isimportant to ensure highly efficient generation of DSBs using

efficiency of the DSB caused by the Cas9 nuclease andthe distance of the DSB from the desired HDR insertion/
alteration site. It has been reported that mammalian genewww.revvity.com

3. Introduce silent SNPs in the crRNA target region mostproximal to the PAM to prevent subsequent recognition by
crRNA. (Figure 1)
2

Fluorescent tagging of an endogenous gene by homology-directed repair using Dharmacon Edit-R CRISPR-Cas9 reagents.

Finally, it is not always possible to find an efficient andactive crRNA, crRNA1, was 40% (data not shown). The Cas9

specific crRNA targeting sequence that is within ~ 100 nt

cleavage site for crRNA1 was at the intended TurboGFP

of your intended insertion site. In these circumstances,insertion site and split the protospacer motif to avoid future

the addition of a positive selection element to your donor

rounds of genomic cutting by Cas9, thus making this an ideal

plasmid, such as antibiotic selection, has been shownsite for use with HDR (Figure 2A).

to aid in selection of the modified cells that contain thedesired insertion10.

Design of DNA donor plasmid: The endogenous HDR
machinery must recognize a template with homology to

Workflow overview: By using the recommendations outlined

the target to commence precise repair3. We designed

above, we have developed an experimental HDR workflow

a TurboGFP donor plasmid flanked on each side within which we use Cas9 mRNA, synthetic crRNA:tracrRNA, andapproximately 1000 nt homology arms for SEC61B

a donor template to make precise HDR changes to a gene.

(Figure 2B). Homology arms were generated by amplifying

Specifically, we demonstrate knockin of a GFP reporter togenomic DNA from the cell lysate of the experimental U2OS

a gene target, creation of clonal cell lines, and confirmation

cell line. Therefore, any SNP(s) present in the PCR ampliconof the precise insertion of GFP by Sanger sequencing andfrom this cell line will be included in the donor plasmid.

fluorescent microscopy.

Upon successful knock in, the TurboGFP insert will be

The HDR workflow outlined here uses a lipid transfection

placed in-frame at the N-terminus of SEC61B (Figure 2B).

method for delivery of the required components.
sequence include:A

3. Identifying the optimal amount of donor template that
does not significantly decrease the amount of DSBs
4. Determining the optimal time point for analysis in order toavoid potential background
5. Using the appropriate assays to confirm precise insertionin clonal cell lines

Exon 2Cutsite

1. Optimizing the creation of Cas9-mediated DSBs byidentifying the crRNA with highest editing efficiency
2. Designing an effective DNA donor plasmidExon 1

Protospacer

B

GFP insertion site
Start
ATG

DNA

the endogenous gene SEC61B (Gene ID: 10952) byinserting TurboGFP™ (Evrogen, Moscow, Russia)
in-frame at the N-terminus of the protein. Two crRNAsequences (crRNA1-CCCUCAUCUCCAAUAUGGUA andcrRNA2-CCAUACCAUAUUGGAGAUGA) nearest to the intended
TurboGFP insertion site were chemically synthesized andtransfected individually, each with Dharmacon Edit-R synthetic
tracrRNA, into a U2OS-CAG-Cas9-integrated cell line. We
characterized the editing efficiency of each crRNA to create
DSBs in the absence of a donor plasmid by a mismatchdetection assay using T7 endonuclease I (T7EI). The resulting
percentage of insertions and deletions (indels) for the mostwww.revvity.com

CDS

1 kb 5' Homology arm
1 kb 3’ Homology arm
Primers for 5'GFP
arm insertion site
Primers for 3' arm

Start

CDS
DNA 3' HA PCR product

ATG

1 kb 5' Homology arm
Primers for 5' arm

1 kb 3’ Homology arm
Primers for 3' arm

5' HA PCR product

tGFP

3' HA

most efficient crRNA: We set out to add a tag to

PAM

Protospacer

tGFP

Optimizing creation of DSBs by selection of the

Cut
site

TGCCACCCTCATCTCCAATATGgtatggcgg

5' HA PCR productResults

PAM

TGCCACCCTCATCTCCAATATGgtatggcgg

5' HA

Important steps for successful insertion of the desired

3' HA PCR producttGFP

Figure 2. Target gene for insertion of TurboGFP through a donor
plasmid and the HDR pathway. A. Illustration of the first two exonsof SEC61B. Introns are indicated by lines and lowercase sequence.
Exons are indicated by blue boxes and uppercase sequence. The
ATG start codon is in green text. The protospacer sequence isrepresented by the underlined sequence. B. Primers were designed
to amplify 1 kb 5’ and 3’ homology arms for insertion of GFP on the
N-terminus of SEC61B and subsequently cloned into a vector alongwith TurboGFP (tGFP) to generate the donor plasmid. Primers used
for construction of the donor plasmid, on average, were 45 nts inlength and designed whereby half the primer sequence was specificto the area needed to amplify the homology arm (5’ arm gray arrows
and bars; 3’ arm purple arrows and bars) and the other half was
specific to the donor plasmid backbone (5’ arm red bars; 3’ armorange bars). HA = homology arm.
3

Fluorescent tagging of an endogenous gene by homology-directed repair using Dharmacon Edit-R CRISPR-Cas9 reagents.

Determination of assay time point: Optimization of the time

we grew 135 individual, flow cytometry-sorted,interval between transfection and detection of HDR events

TurboGFP-positive cells in 96-well plates and determined

will be dependent on the experiment-specific reagents.

that 98.5% (N=133) of those cell clones displayed the

As controls, donor plasmids containing TurboGFP with orcorrect localization of SEC61B. These results are inwithout homology arms were transfected without Cas9,

agreement with our previous observation that expression

crRNA, or tracrRNA into U2OS cells. While the TurboGFP

of TurboGFP from the donor plasmid is only present in aplasmid without homology arms did not result in any GFPvery small fraction of the total TurboGFP-expressing cells

expression, approximately 10% of the cells transfected

when analyzed at seven days post-transfection.

with the donor plasmid containing homology arms for
SEC61B displayed cellular GFP expression 24 hours after
transfection (Figure 3A). Expression of GFP from the donor
plasmid is due to the presence of endogenous promoter
region sequences for SEC61B in the ~ 1000 nt 5’ homology
arm11. By monitoring the transfections over the course of
seven days after the initial transfection, GFP expression fromthe donor plasmid diminishes each day post-transfection,with the nadir occurring seven days post-transfection(Figure 3B). Thus, in order to avoid this background GFPsignal from the donor plasmid, subsequent GFP detection
assays were performed at seven days post-transfection.
Detection of TurboGFP integration into the target locus:
Various methods can be used to assess integration ofexogenous DNA into a target locus. The methods ofdetection that we tested were high-content imaging,flow cytometry, PCR, and Sanger sequencing. Below we
describe the detection of TurboGFP insertion at the desired
locus following transfection of Cas9 mRNA, synthetic
crRNA:tracrRNA, and donor plasmid into U2OS cells.

Detection of donor plasmid integration by PCR
amplification and Sanger sequencing: Primers that were
optimized for the mismatch detection assay were used toamplify genomic DNA from lysate of untreated U2OS cells
and TurboGFP-SEC61B positive and negative (determinedby FACS analysis) clonal lines (Figure 6). PCR amplicons forthe untreated control and TurboGFP-negative cell clone
were detected at the expected length of 507 bp (clones
UN and 1), whereas positive integration of TurboGFP at
SEC61B was detected at the expected size of 1242 bp(clones 2-4; Figure 6A). In some cases, additional bands
were also observed indicating indel formation by the NHEJ
pathway in different alleles (clones 3 and 4; Figure 6A).
PCR amplicons from two individual GFP-positive
cells (clones 3 and 4; Figure 6A) were cloned into a
plasmid, transformed, and single bacterial colonies
were sequenced. While GFP-positive clone 3 did have
TurboGFP insertion in at least one allele, another allele
did not have TurboGFP integrated and contained a 107 bp
deletion (Figure 6A and C). Interestingly, GFP-positive clone

Detection of TurboGFP integration and expression by

4 did have TurboGFP insertion in at least one allele, while

high-content imaging: U2OS cells were imaged at sevenanother allele contained an incomplete copy of TurboGFP

days post-transfection in order to determine expression(Figure 6A and D).

of TurboGFP and correct localization of the target gene.
and can also be observed in the post-ER compartment.
Cells containing TurboGFP-tagged SEC61B were detected
and shown to co-localize with antibody stained SEC61B(Figure 4), supporting in-frame insertion of the TurboGFP.
Detection of TurboGFP expression by flow cytometry:
U2OS cells were analyzed by flow cytometry sevendays post-transfection to determine the percentage ofthe population expressing TurboGFP. We determinedthat the donor plasmid-only control (no Cas9 mRNA,
crRNA, or tracrRNA) contained 0.13% TurboGFP positive
cells (Figure 5A), whereas the cells transfected with all
required components for HDR insertion (Cas9 mRNA,
crRNA:tracrRNA, and donor plasmid) contained 5.06%
TurboGFP-positive cells (Figure 5B). Additionally,
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AB
GFP cells/intensity (%)

SEC61B is part of the endoplasmic reticulum (ER) membrane

12
10
8
6
4
2
0

1

2
3
4
5
6
Days post-transfection

7

Figure 3. Characterizing TurboGFP expression from a donor
plasmid. The donor plasmid for insertion of TurboGFP at the
N-terminus of SEC61B (without Cas9, crRNA, or tracrRNA) wastransfected into U2OS cells and TurboGFP expression wasdetermined by fluorescent microscopy and flow cytometry.
A. Fluorescent imaging at three days post-transfection shows
expression of GFP. B. Flow cytometry-based detection of GFP
positive cells at one to seven days post-transfection.
4

Fluorescent tagging of an endogenous gene by homology-directed repair using Dharmacon Edit-R CRISPR-Cas9 reagents.

Figure 4. Co-localization of endogenous SEC61B with TurboGFP-tagged SEC61B in GFP-positive cell clones. A TurboGFP-positive SEC61B
U2OS cell line was fixed and co-stained with a primary antibody to confirm correct TurboGFP localization.

SEC61β donor alone

Increasing tGFP

B

SEC61β HDR

Increasing

IncreasingA

Increasing tGFP

Figure 5: FACS detection of GFP-positive cells from plasmid donor transfected cells. U2OS cells were analyzed by FACS so that viable, single
GFP-positive cells were gated, quantified, and sorted. GFP intensity is measured along the x-axis and auto fluorescence detection at 575 nm is
measured along the y-axis. A. SEC61B donor only transfected cells (~ 25,000 cells measured). B. SEC61B HDR transfected cells (~ 18,000 cells
measured). A-+ and A++ represent unquantified quadrants containing zero FACS detected cells.

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5Fluorescent tagging of an endogenous gene by homology-directed repair using Dharmacon Edit-R CRISPR-Cas9 reagents.

GFP -

A

GFP +

1242 bp1057 bp
507 bp400 bpClone UN

B

1

2

3

4

Start codon

C
PAM

D

Figure 6: PCR-based detection of TurboGFP integration. A. PCR was performed across the intended insertion site for single cell clones ofTurboGFP-SEC61B to detect a shift in the size of the PCR amplicon due to the TurboGFP insertion. UN = untreated. B. Sanger sequencing
results of each TurboGFP integration junction in TurboGFP-SEC61B clone 2. C. Sanger sequencing results showing the indel from the 400 bp
amplicon from TurboGFP-SEC61B clone 3. D. Sanger sequencing results showing the deletion of 185 bp (resulting in the 1057 bp band) from
TurboGFP-SEC61B clone 4.

Discussion
Here we have demonstrated that fluorescent tagging of

Additional biological factors may also contribute to variable

an endogenous protein can be accomplished with the

HDR insertion, such as genomic accessibility, DSB and insert

Dharmacon Edit-R CRISPR-Cas9 genome engineeringsite distance, gRNA activity, and essential gene function.

platform and a donor plasmid template. We inserted
TurboGFP in-frame with the endogenous SEC61B gene andselected individual clonal cell lines, which were verified
by Sanger sequencing. Transfection optimization is one of
the most important experimental factors when performing
HDR experiments since the HDR machinery will only repair
a fraction of the DSBs created by Cas9 nuclease. It is also
necessary to perform a donor-only transfection to evaluate
false-positive effects from the donor; in the example
provided, GFP-positive background was detected from thedonor plasmid alone and not due to true genomic insertionof GFP. Importantly, GFP expression from a plasmid donoralone will diminish over time as the plasmid is degraded anddiluted during cell culture maintenance.
When choosing a DNA donor for CRISPR-Cas9 HDR
experiments, one must consider the size of the exogenous
DNA to be inserted. In our experience, short inserts (< 50 nt)
with DNA oligos can be up to three times more efficientthen large inserts (e.g., GFP reporter) with donor plasmids.
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The Dharmacon CRISPR design tool and the Dharmacon
Edit-R HDR donor designer can assist in identifying anefficient guide RNA target site and proper design of thedonor template.
Interestingly, when characterizing the HDR clonal celllines, we detected expression of TurboGFP by fluorescentimaging; however, when amplifying the integration site, we
detected smaller bands that differed from both the expected
unintegrated PCR amplicon size and positive integration
PCR amplicon size. Smaller bands from the unintegrated
PCR amplicons likely occurred through the NHEJ pathway,
while the integrated truncated version of TurboGFP might
have originated from other repair pathways such as the
microhomology-mediated end joining pathway. These results
underscore the necessity of generating many clonal celllines and fully characterizing each clonal cell line before
deciding to progress to future experiments with your desired
knockin cells.
6Fluorescent tagging of an endogenous gene by homology-directed repair using Dharmacon Edit-R CRISPR-Cas9 reagents.

In conclusion, we have discussed and shown best practices

Endogenous SEC61B was stained with a polyclonal rabbit

for HDR workflows, including crRNA site selection, DNA

antibody (ProteinTech Group, Cat #15087-1-AP) at 1:50

donor design, transfection optimization, assay time pointin permeabilization buffer for 1 hour, washed twice inassessment, and detection of successful integration

PBS, followed by a secondary antibody (Alexa 568 goatusing several methods resulting in precise tagging of ananti-rabbit; Invitrogen, Cat #A11010) incubation at 1:250

endogenous protein, SEC61B, with TurboGFP. These

dilution in permeabilization buffer for 30 minutes at room

methods and recommendations are applicable to othertemperature. All cells were stained with Hoechst 33342

HDR-mediated insertions of large DNA into different(Molecular Probes, Cat #H-3570) in PBS at a 1:2500 dilution

genomic locations.

for 10 minutes followed by two final washes in PBS.
Flow cytometry: U2OS cells were trypsinized, resuspended

Materials and methods

in cell sorting medium at 10 million cells/mL, and stored on

Tissue culture: All U2OS cells were maintained in standard

sorting instrument by the Flow Cytometry Core at

growth medium per manufacturer’s recommendations

The University of Colorado Cancer Center [Cancer Center(ATCC, Cat #HTB-96).

Support Grant (P30CA046934)]. Sorted cells were placed

Transfection: U2OS and U2OS-CAG-Cas9-integrated cells
were seeded in a 96-well plate at 10,000 cells per wellice until sorting. Cells were sorted on a Moflo XDP 100 cell

into tubes and 96-well plates, using FBS enriched medium(FBS:U2OS culture medium at 1:1 ratio).

one day prior to transfection. Dharmacon Edit-R synthetic

Isolation of individual cell clones: A Moflo XDP100 flow

crRNAs (Dharmacon, custom synthesis) and tracrRNA

cytometer was used to sort single TurboGFP-positive(Cat #U-002000-20) were individually resuspended in 10 mM

cells into individual wells of five 96-well plates for

Tris-HCl (pH 7.5) to a concentration of 100 μM. crRNA andcolony expansion. Penicillin-Streptomycin (2%; HyClone,

tracrRNA were combined at equimolar ratio and diluted to

Cat #SV30010) was added to normal U2OS growth medium

2.5 μM using 10 mM Tris-HCl (pH 7.5). A final concentration

as a precaution from exposure to the open air duringof 25 nM crRNA:tracrRNA complex (25 nM of each crRNAsorting. Cells were maintained in culture and visuallyand tracrRNA) was used for transfection. Cells wereobserved for GFP fluorescence for three weeks.

transfected with 200 ng of Dharmacon Edit-R Cas9 Nuclease
plasmid (Cat #U-005100-120) or 200 ng Dharmacon Edit-R
Cas9 Nuclease mRNA (Cat #CAS11195) using 0.3 μL/wellof DharmaFECT™ Duo transfection reagent (Dharmacon
Cat #T-2010-03) for co-transfections with crRNA:tracrRNAcomplex, with or without 200 ng donor plasmids.

Genomic DNA isolation, PCR, and 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 K(Thermo Scientific, Cat #EO0491) and RNase A (Thermo
Scientific, Cat #EN0531) for 20 minutes at 56 °C followed

Cell imaging: Live cell colonies were imaged with an

by heat inactivation at 95 °C for 5 minutes. PCR was

IN Cell Analyzer 2200 Imaging System (GE Healthcare)

performed with Phusion High-Fidelity DNA Polymerase

after replacing the culture medium with imaging medium(Thermo Scientific, Cat #F530S) and primers flanking(HBSS, 20 mM HEPES, 16.8 mM D-glucose, pH 7.2).

the cleavage/insertion sites. PCR products (500 ng) were

A 10x objective was used to identify TurboGFP-positive

treated with T7EI (NEB, Cat #M0302L) for 25 minutes atcells and then reimaged with a 20x objective for localization

37 °C and the samples were separated on a 2% agarose gel.

analysis. For immunofluorescent microscopy, cells were fixed

Percent editing in each sample was estimated using ImageJwith 4% paraformaldehyde (Electron Microscopy Services,

software (NIH, imagej.nih.gov/ij, 1997-2014).

Cat #15710) for 15 minutes at room temperature, washedtwice with PBS, then incubated in permeabilization buffer(PBS containing 200 mg saponin, 2% FBS, and 100 µg BSA;

Chromatograms were analyzed with Geneious Version 8.1.3,
created by Biomatters (geneious.com)

Sigma, Cat #A3059) for 30 minutes at room temperature.

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7

Fluorescent tagging of an endogenous gene by homology-directed repair using Dharmacon Edit-R CRISPR-Cas9 reagents.

References
1.

2.

3.

J. Chapman, M. Taylor, et al., Playing the end game:Mol. Cell. 47(4), 497-510 (2012).

sequencing. Cell Rep. 7(1), 293-305 (2014).

K. Chiruvella, Z. Liang, et al., Repair of double-strand

10. S. Mansour, K. Thomas, et al., Disruption of the

breaks by end joining. Cold Spring Harb. Perspect.

proto-oncogene int-2 in mouse embryo-derived stem

Biol. 5(9592), 1-21 (2013).

cells: a general strategy for targeting mutations to

R. Ceccaldi, B. Rondinelli, et al., Repair pathway choices
Trends Cell Biol. 26(1), 52-64 (2016).
P. Mali, L. Yang, et al., RNA-guided human genome
engineering via Cas9. Science. 339(6121), 823-826 (2013).

5.

A. Hendel, E. Kildebeck, et al., Quantifying
genome-editing outcomes at endogenous loci with SMRT

and consequences at the double-strand break.

4.

9.

DNA double-strand break repair pathway choice.

non-selectable genes. Nature. 336(6197), 348-352 (1988).
11. R. Dreos, G. Ambrosini, et al., The Eukaryotic Promoter
Database: expansion of EPDnew and new promoter
analysis tools. Nucleic Acids Res. 43 (Database issue),
D92-D96 (2014).

H. Yang, H. Wang, et al., One-step generation of micecarrying reporter and conditional alleles by CRISPR/Casmediated genome engineering.
Cell. 154, 1370-1379 (2013).

6.

M. Ratz, I. Testa, et al., CRISPR/Cas9-mediatedendogenous protein tagging for RESOLFT superresolution microscopy of living human cells.
Sci. Rep. 5, 1-6 (2014).

7.

B. Elliott, C. Richardson, et al., Gene conversion tracts
from double-strand break repair in mammalian cells.
Mol. Cell Biol. 18(1), 93-101 (1998).

8.

J. Schiel, E. Chou, et al., Homology-directed repair with
Dharmacon Edit-R CRISPR-Cas9 reagents andsingle-stranded DNA oligos. GE Healthcare, Dharmacon
2015; dharmacon.horizondiscovery.com/uploadedFiles/
Re-sources/hdr-dna-oligo-text-appnote.pdf.

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