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

Generating
functional protein
knockout in iPSCs
using Dharmacon
Edit-R reagents.

Introduction
Whether a researcher is studying normal human cell biology
or a disease state, primary human cell models are not always
available and can be challenging to work within the lab.
Induced pluripotent stem cells (iPSCs) are a useful research tool
because they are a renewable model for karyotypically normalcells. Additionally, they can be manipulated in the pluripotent
state and differentiated into various isogenic cell types.
Here we demonstrate the ability to modify the genome andcreate functional knockout models within a human iPSC linewith Dharmacon™ Edit-R™ CRISPR reagents.

Authors
Amanda Haupt
John Schiel
Revvity, Inc.

Generating functional protein knockout in iPSCs using Dharmacon Edit-R reagents.

Results
Electroporation of Edit-R Synthetic sgRNAs and Edit-R Cas9Edit-R Cas9 Nuclease mRNA and Dharmacon Edit-R

Nuclease mRNA in human iPSCs

Synthetic sgRNA targeting SOX2 induce loss-of-pluripotency

To demonstrate the use of Dharmacon Edit-R Cas9 Nuclease

phenotype in iPSCs

mRNA with GFP fluorescence in iPSCs, Cas9 Nuclease mRNA

Indel formation in a gene of interest may not always lead toco-expressing a GFP reporter was electroporated witha functional protein knockout. To demonstrate a functional

synthetic sgRNAs targeting four genes using previously

knockout phenotype, we targeted SOX2, a known controller

optimized conditions (see Methods). Six hoursof iPSC pluripotency (Rizzino 2013). Here, we use Dharmacon

post-transfection, GFP fluorescence was observed in a highEdit-R Cas9 Nuclease mRNA and three pre-designed

percentage of iPSCs, suggesting a highly efficient transfection

Dharmacon Edit-R Synthetic sgRNAs targeting the SOX2 locus(Figure 1A–B). Cells were harvested 5 days post-transfection

to identify the best target for functional protein knockout.

when they exhibited expected iPSC morphology (smooth

Individual synthetic sgRNAs were electroporated into healthy

rounded colonies with tightly packed cells) and a confluence

iPSCs with Cas9 Nuclease mRNA, and five days later,of approximately 70% (Figure 1C–D). Cells were lysed, andreplicate wells were lysed for PCR and Sanger sequencing

PCR primers were used to amplify approximately 300 bp on

required for TIDE analysis. Cells were also fixed and stained

each side of the sgRNA binding site. Sanger sequencing waswith antibodies against four iPSC pluripotency markers,

used to perform a TIDE analysis (see Methods), indicating

including the target gene of interest, SOX2.

that the percentage of indel formation at each target locus
was greater than 75% (Figure 1E). Cells were also fixedand stained with antibodies against four iPSC pluripotency
markers. Visual analysis confirmed no loss of pluripotency
marker expression in any of the treated samples comparedto the untreated control cells, which is expected since noneof the four proteins targeted had been reported to affect thepluripotency of iPSCs (Figure 1F).

Each sgRNA used caused greater than 80% indels at thetarget locus, and visible changes in iPSC morphology wereseen on the day of harvest (large, flat cells not growing
in tightly packed colonies, Figure 2A–B). Pluripotency
marker staining showed that the non-targeting control
sample expressed all four pluripotency markers (SSEA4,OCT4, SOX2, and TRA-1-60) as expected for healthy iPSCs(Figure 2C, left series). Cells treated with sgRNAs against
SOX2 show varying levels of diminished SOX2 expression(Figure 2C, third row highlighted in green). In SOX2 sgRNA
treated samples, cells also show loss of OCT4 and TRA-1-60
expression (Figure 2C). This result suggests a functionalloss of pluripotency is also achieved with the knockout ofSOX2 and agrees with previously published work describing
the highly interdependent network of factors that control
pluripotency (Rizzino 2013).

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2Generating functional protein knockout in iPSCs using Dharmacon Edit-R reagents.

AB

ECas9-GFP mRNAC

PPIB Control sgRNAF

SEA4

DNon-Targeting sgRNA #2

NTC #2

OCT4

Nuclei

SOX2

NucleiTFRC

SEA4OCT4Nuclei

TRA-1-60

SOX2

NucleiUntreated

SEA4

OCT4

Nuclei

TRA-1-60

SOX2

TRA-1-60

NucleiFigure 1: Gene Editing in iPSCs with Cas9-mRNA Nuclease and synthetic sgRNAs delivered by nucleofection is an effective editing methodthat is non-perturbing to iPSC morphology and pluripotency. A–B) PPIB Control sgRNA sample imaged 6 hours post-transfection whereB) shows GFP signal from the Cas9-GFP mRNA and indicates successful delivery. C–D) Phase images post-transfection after 5 days
(passaged 1 time). Cells show expected iPSC morphology. A–D) Scale bars 100 μM. E) Percent indel formation by TIDE analysis. F) Staining
for iPSC pluripotency markers SSEA4, OCT4, SOX2, and TRA-1-60 5 days after nucleofection of Non-Targeting Control #2 Synthetic sgRNA(NTC#2, left series), TFRC Synthetic sgRNA (middle series), and Untreated cells (right series).

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Generating functional protein knockout in iPSCs using Dharmacon Edit-R reagents.

AC

SEA4

B

SOX2

NucleiSEA4

OCT4Nuclei

Nuclei

TRA-1-60

Cas9 mRNA only

Untreated

SOX2 sgRNA #1

SOX2 sgRNA #2

SOX2 sgRNA #3

SOX2 sgRNA #1

NTC #2

OCT4

NTC #2SOX2Nuclei

SOX2 sgRNA #2

SEA4

OCT4Nuclei

TRA-1-60

SOX2Nuclei

SOX2 sgRNA #3

SEA4

OCT4Nuclei

TRA-1-60

SOX2

TRA-1-60

NucleiFigure 2: Gene Editing in iPSCs with Cas9-mRNA Nuclease and synthetic sgRNAs delivered by nucleofection to target pluripotencytranscription factor SOX2 generates a functional knockout with high efficiency. A) Percent indel formation by TIDE. B) Knockout of SOX2
induces changes in iPSC morphology. No changes to morphology are seen with Non-targeting control, or Cas9 mRNA only compared tountreated iPSCs. Images were taken 5 days after nucleofection, including one passage. Scale bars are 100 μM. C) Pluripotency markerstaining of fixed cells 5 days after electroporation. Scale bars are 100 μM.

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4Generating functional protein knockout in iPSCs using Dharmacon Edit-R reagents.

AB

ECas9-GFP mRNAC

PPIB Control sgRNA

DNon-Targeting sgRNA #2

Figure 3: Gene Editing in iPSCs with Cas9-mRNA Nuclease and synthetic sgRNAs delivered by DharmaFECT Duo Transfection Reagent is
an alternative delivery method that is non-perturbing to iPSC morphology. A–B) TFRC sgRNA sample imaged 20 hours post-transfectionwhere A) is the phase image and B) shows GFP signal from the Cas9-GFP mRNA and indicates successful delivery. C–D) Phase images
post-transfection after 72 hours. Cells show expected iPSC morphology. A–D) Scale bars 100 µM. E) Percent indel formation by TIDE analysis.

Lipid transfection of Edit-R Synthetic sgRNAs andDharmacon Edit-R Cas9 Nuclease mRNA in iPSCs
While electroporation is the optimal method for deliveryof Dharmacon Edit-R Cas9 Nuclease mRNA and synthetic
sgRNAs for iPSCs, the optimal methods for each cellline may require specific equipment that a lab may nothave access to such as a nucleofection instrument. As analternative, lipid transfection can also be used to deliver
Edit-R gene editing reagents to iPSCs.
Here, optimized transfection conditions using DharmaFECT™
Duo Transfection Reagent (see Methods) were used to deliver
Dharmacon Edit-R Cas9 Nuclease mRNA and synthetic
sgRNAs targeting three genes that are either expected tonot affect iPSC viability or morphology (TFRC, CLYBL) orare not expressed in iPSCs (TTN). After transfection, GFP
signal from the Cas9-GFP mRNA is observed, indicatingsuccessful delivery (Figure 3A–B). On the day of harvest, cells
exhibit expected iPSC morphology (Figure 3C–D) and highviability compared to untreated controls (Figure 3E). Cells
were lysed, and PCR products were Sanger sequenced andsubjected to TIDE analysis, which resulted in indel formation
between 12–18% for samples treated with sgRNAs targeted
to each of the three genes analyzed (Figure 3E).

Discussion
Here we have demonstrated the effectiveness of Dharmacon
Edit-R Cas9 Nuclease mRNA and Synthetic sgRNAs in editing
human iPSCs. Optimized electroporation protocols are
ideal for the delivery of these reagents into this cell type,though exact conditions may vary by cell line. Alternatively,reagents can also be delivered by lipid transfection. To study
the impact of a specific protein of interest on cellular
phenotype or function populations of edited iPSCs exhibitinga high percentage of indel formation and functional protein
knockout can be used directly in comparison with unedited
isogenic controls, or alternatively, clonal cell lines can begenerated.
Using Revvity’s DNA-free reagents prevents unwanted
random genomic integration, preserving the integrityof the iPSC models’ genetic composition. Additionally,synthetic sgRNA and Cas9 Nuclease mRNA are activein the cell for less time than plasmid-based reagents,
which reduces the risk for off-target editing activity.
Using Cas9 Nuclease mRNA also precludes the needfor using a reagent with a cell-line specific optimized
promoter. This application note demonstrates the utility ofsynthetic sgRNA and Cas9 Nuclease mRNA as a convenientsource of CRISPR reagents for effective genome editing inhuman iPSCs.

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5Generating functional protein knockout in iPSCs using Dharmacon Edit-R reagents.

Materials and methods

After transfection was performed, 80 µL mTeSR™1 Without

Cell culture

supplemented with 10 µM ROCK inhibitor molecule Y-27632

A Human Episomal human iPSC Line (Cat # A18945,Thermo Fisher Scientific) found to be karyotypicallynormal was cultured on non-tissue culture treated plastics(recommended for use with Vitronectin coating) coatedwith truncated Vitronectin (VTN-N) Recombinant HumanProtein (Cat # A14700, Thermo Fisher Scientific) accordingto manufacturer’s recommendations. Cells were cultured inantibiotic-free mTeSR™ Plus media (Cat # 05825 StemCell
Technologies). Cultures were routinely maintained byclump passaging two times a week with Gibco™ Versene
Solution (Cat #15040066, Thermo Fisher Scientific)
per manufacturer’s instructions. Cells were incubated at
37 °C and 5% CO2.
Electroporation
Prior to electroporation, cells were treatedwith 10 µM ROCK inhibitor molecule Y-27632 (Cat #72302,StemCell Technologies) for 1–4 hours to promote viability
post-electroporation. Cells were passaged into a singlecell suspension using StemPro Accutase Cell Dissociation
Reagent (Cat # A1110501 Thermo Fisher Scientific)
and resuspended in mTeSR™ Plus media supplemented with
10 µM ROCK inhibitor molecule Y-27632. Cell line-specific(see Cell Culture section for specific lineage) electroporation
conditions, previously optimized with pMax-GFP expression
plasmid, were as follows: Lonza™ P3 Primary Cell
4D-Nucleofector™Kit (Cat # V4SP-3096) was used totransfect 3.4 × 105 cells per reaction in an Amaxa™ 96-wellShuttle™ Device (Cat # AAM-1001S) using setting CM-113.
Each reaction contained 1 µg Dharmacon Edit-R EGFP Cas9
Nuclease mRNA (Cat # CAS11860) and 2 µM Edit-R syntheticsgRNA in 20 µL reactions.

Phenol Red media (Cat # 05876 StemCell Technologies)
was added to each reaction well, and 25 µL of eachreaction was plated into a prepared Vitronectin-coated96-well plate containing 0.2 mL pre-equilibrated mTeSR™1Without Phenol Red media supplemented with 10 µM
ROCK inhibitor molecule Y-27632. Cells were incubatedat 37 °C and 5% CO2. After 6–20 hours, cells were imagedfor GFP fluorescence from the Edit-R EGFP Cas9 mRNA
as an indication of transfection efficiency. Twenty-fourhours post-transfection, media was changed to mTeSR™
Plus, and cells were fed daily until the time of harvest or
passaged one time before harvest.
Lipid transfection
One day before transfection, healthy iPSC cultures with
65-75% confluence were passaged into a single cellsuspension using StemPro Accutase Cell Dissociation
Reagent and resuspended in mTeSR™ Plus media
supplemented with 10 µM ROCK inhibitor molecule Y-27632.
Cells were replated into non-tissue culture treated 96-wellplates coated with Vitronectin at a density of 6 × 103 cells/wellin 0.1 mL mTeSR™ Plus media supplemented with 10 µM
ROCK inhibitor molecule Y-27632 and incubated at 37 °C
and 5% CO2. This seeding density was optimized for this
iPSC line to give 60-75% confluence on the day of harvest.
The day after plating, the transfection mix was preparedso that each reaction contained 200 ng Edit-R EGFP Cas9Nuclease mRNA, 50 nM Edit-R synthetic sgRNA, and 0.3 µg/mL
DharmaFECT Duo Transfection Reagent (Cat #T-2010-01)(the amount of transfection reagent needed should be
empirically determined through optimization experiments
for each iPSC line). Reagents were pre-complexed for
20 minutes at room temperature in HyClone MEM-RS(Cat #SH30564, Fisher Scientific) before mTeSR™1 without

The specific guide RNA sequences used were:

Phenol Red media was added to the transfection mix for• Dharmacon Edit-R Human TFRC synthetic sgRNA,

was replaced with 0.1 mL transfection mix, and cells wereCat# SG-003941-02-0003• Dharmacon Edit-R Human SOX2 synthetic sgRNA,
Cat # SG-011778-01-0003, SG-011778-02-0003,
and SG-011778-03-0003

a final volume of 0.1 mL/reaction. Expired growth media
returned to the incubator. After 6–20 hours, cells wereimaged for GFP fluorescence from the Edit-R EGFP Cas9mRNA as an indication of transfection efficiency. Cells werefed every 24 hours with mTeSR™ Plus media until the day of
harvest (typically 72 hours post-transfection).

• Dharmacon Edit-R Human CLYBL synthetic sgRNA,
Cat # SG-018275-01-0003• Dharmacon Edit-R Human TTN synthetic sgRNA,
Cat # SG-005395-01-0003
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Generating functional protein knockout in iPSCs using Dharmacon Edit-R reagents.

Viability assay(2018) to estimate the likely indel frequencies present

Replicate wells were harvested for viability analysis by
CellTiter-Glo® Luminescent Cell Viability Assay (Cat #G7572,Promega) per the manufacturer’s instructions. Luminescence
was read on an Envision 2 Plate Reader.

within a Sanger sequence trace. Samples are first assessed
and filtered on the basis of sequence trace quality,
and the analysis parameters determined for each individual
input. Treated samples are then aligned to and comparedagainst untreated controls to calculate the sample editing

TIDE analysis

efficiency, the frequency of indels, the rate of indel

On the day of harvest, replicate wells were washed 1X inindel within the specified window of detection was

DPBS (Cat #SH30028, Fisher Scientific) and lysed inindividually tested to determine the likelihood of such an

0.1 mL/well Thermo Scientific Phusion HF Buffer (Cat #F-518L,

indel being explained by the observed Sanger trace data,Thermo Fisher Scientific) with 5 µL each of Proteinase K

and a confidence estimate was then assigned to this indel.

occurrence, and corresponding p-values. Each possible(Cat# E00492, Thermo Fisher Scientific) and RNase A(Cat #EN0531, Thermo Fisher Scientific), and incubatedfor 1 h at 56 °C then heat- inactivated at 98 °C for 10 min.
Fifty microliter PCR reactions were carried out in 1× Phusion
HF buffer (Cat #F-518, Thermo Fisher Scientific), using 0.5 µLPhusion Hot Start II DNA Polymerase (Cat #F-549S,Thermo Fisher Scientific), 200 µM each dNTP (Cat #F-549S,

Immunocytochemistry
Replicate wells were washed once with DPBS and thenfixed and stained with the Invitrogen™ Pluripotent Stem
Cell 4-Marker Immunocytochemistry Kit (Cat# A24881,
Thermo Fisher Scientific) per the manufacturer’s instructions.

Thermo Fisher Scientific), 0.5 µM forward and reverse
primers, and 5 µL direct cell lysis template. Touchdown PCR
cycling was run for each sample: denature/enzyme

References

activation at 98 °C for 3 min followed by 10 cycles of

1. A. Rizzino. The Sox2-Oct4 Connection: Critical players in a98 °C for 10 s, 72 °C for 15 s –1 °C/cycle, and 72 °C

much larger interdependent network integrated at multiple

for 30 s, then 25 cycles of 98 °C for 3 min followed bylevels. Stem Cells. 31(6): 1033–1039 (2013).

10 cycles of 98 °C for 10 s, 62 °C for 15 s, and 72 °C for
30 s, and a final extension at 72 °C for 10 min. Each PCR
product was verified to be single amplicons by running 5 µLof the reaction on 1.2% agarose gels before proceeding

2. E. Brinkman et al. Easy Quantification of template-directed
CRISPR/Cas9 editing. Nucleic Acids Research.
46(10): e58 (2018).

with Sanger sequencing. The resulting AB1 files wereanalyzed by an in-house program that utilizes an adapted
version of the TIDER algorithm described in Brinkman et al.

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