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

CRISPR gene
editing andtranscriptional
activation inhuman iPS cells.
Authors
John A. Schiel
Travis HardcastleŽaklina Strezoska
Anja van Brabant Smith

Introduction
The CRISPR (clustered regularly interspaced short palindromic
repeats)-Cas9 (CRISPR-associated 9) gene editing system is a
powerful tool for creating mutations and insertions in a cell’s
genomic DNA (1,2,3). Co-delivery of a Cas9 nuclease and aDNA-targeting guide RNA causes a targeted double-strand break,prompting the cellular machinery for repair. Researchers havebeen able to expand the capability of the CRISPR-Cas9 system byincluding a repair template for use with homology-directed repair.
Additionally, an inactive, also known as a dead, Cas9 nucleasehas been demonstrated to inhibit or activate gene expression
when fused to an effector domain (4,5,6). The power behind
CRISPR-Cas9 is the ease of gene editing reagent construction andthe ability to use this technology in many different types of cells.
Induced pluripotent stem cells are a valuable research tool forability to be turned into virtually any cell type. With the adventof CRISPR-Cas9 researchers can modify the genome of the iPS
cells and subsequently differentiate them to see the affect thatthe genomic modification has upon the differentiated cell type.
Here we present the ability to modify the genome and modulate
expression of genes within a human induced pluripotent stem
(iPS) cell line with Dharmacon™ Edit-R CRISPR and CRISPRmod
CRISPRa reagents. We demonstrate the following:• Performing gene editing with Edit-R Cas9 nuclease andsynthetic RNA• Knockout of POU5F1(OCT4) to induce iPS cell differentiation
determined via live-cell fluorescent microscopy• CRISPRa mediated activation of gene transcription withCRISPRmod CRISPRa dCas9-VPR nuclease and synthetic RNA• HDR mediated knock-in of EGFP to create an EGFP
tagged protein

CRISPR gene editing and transcriptional activation in human iPS cells.

Results

iPS cells with Cas9 protein as part of an RNP complex.

Gene editing with Edit-R Cas9 nuclease andmarkers were used to determine differentiation caused

synthetic guide RNAby Cas9 targeting POU5F1 (Figure 2A). Sub-populations ofA human induced pluripotent stem (iPS) cell line was grownin a feeder cell-free culture environment and was validated
to be undifferentiated via live-cell fluorescent microscopy
before plating for transfection (Figure 1A). Undifferentiated
iPS cells were disassociated and transfected using theLonza Nucleofector with Edit-R Cas9 Nuclease protein,synthetic crRNA, and synthetic tracrRNA targeting thehuman PPIB gene. After nucleofection, cells were plated
into a 6-well dish and placed into a 37 °C incubator for

Seventy-two hours after transfection, live cell differentiation

iPS cells displaying increased differentiation staining werevisualized, while the untransfected and the non-targeting
crRNA controls showed very low amounts of differentiation(Figure 2A). After live cell imaging, each individual crRNA cell
population was lysed and a subsequent mismatch detection
assay (T7EI) was performed, which confirmed detectablelevels of editing (indels) at POU5F1(Figure 2B).

Aan additional forty-eight hours. A DNA mismatch detection
assay (T7 endonuclease I; T7EI) was performed and a 31%
indel (insertions or deletions) rate was observed for the PPIB
gene target (Figure 1B).

BAB

Figure 2: Inducing iPS cell differentiation with synthetic crRNAs
A. CD44 Alexa Fluor 488 and Tra-1-60 Alexa Fluor 594 stained
live iPS cells either untransfected or transfected with NTC1 crRNAof five different crRNAs targeting POU5F1. B. DNA mismatchdetection assay of NTC1 or POU5F1-targeting crRNA.

Activating gene expression with CRISPRmod
CRISPRa dCas9-VPR and synthetic guide RNACRISPR activation (CRISPRa) is a powerful gene modulation
Figure 1: Working with iPS cells – Checking for differentiation andgene editing. A. Determination of iPS cell differentiation using
live cell imaging of CD44 Alexa Fluor 488 (differentiated) andTra-1-60 Alexa Fluor 594 (undifferentiated) fluorescent labeledprimary antibodies to each cell surface marker. B. DNA mismatchdetection assay of PPIB-targeting crRNA.

tool that uses a nuclease-deactivated Cas9 (dCas9) fusedto the chimeric gene activation domain VPR (VP64, P65and Rta) to increase the transcription of an endogenous
gene target (8). CRISPRmod CRISPRa dCas9-VPR was usedin conjunction with CRISPRmod CRISPRa synthetic crRNAstargeting POU5F1, TTN, ASCL1, or EGFR. A plasmid driving

Knockout of POU5F1 using Edit-R Cas9 nuclease protein

the expression of dCas9-VPR was co-delivered with eachcauses iPS cells to differentiate

gene specific synthetic crRNA:tracrRNA. Seventy-two hours

POU5F1 has previously been shown to be essential forthe maintenance of iPS cell pluripotency (7). That led us topropose the following question: If crRNAs are designed
to program Cas9 to knockout POU5F1, will iPS cell
pluripotency be affected, and would iPS cells differentiate?
To investigate this, five synthetic crRNAs were designed totarget the coding region of POU5F1 with the proprietary
Edit-R guide RNA design algorithm. Using previously
described nucleofection conditions we individually delivered
each of the five different crRNAs into undifferentiatedwww.revvity.com

after nucleofection, RT-qPCR was used to determine
the levels of gene activation (Figure 3A). Target genetranscription levels of three gene targets were detected
at increased levels when compared to untransfectedcontrols (Figure 3A). We did not see further activationof POU5F1 in this experiment because it is known that
POU5F1 is highly expressed and essential to maintainpluripotency in iPS cells (7). Of the three gene targets thatwere successfully activated, ASCL1 transcript levels wereincreased to over 100-fold compared to untransfected cells.
2CRISPR gene editing and transcriptional activation in human iPS cells.

ASCL1 is a member of the basic helix-loop-helix (BHLH)

medium was applied, and cells were visualized on afamily of transcription factors and this protein has been

fluorescent microscope for proper localization (Figure 4).

described as an oncogene and suggested to be essential

After imaging, cells were disassociated and the population

for the survival of lung cancers (9). We wanted to checkof GFP positive cells was determined by flow cytometry.

if activation of ASCL1 would lead to downstream affects

The HDR-mediated knock-in of EGFP was observed in 2.6%

and activation of previously described gene targets (9).

of the cell population as measured by FACS.

Interestingly, we probed the gene expression levels of two
previously characterized downstream ASCL1 interaction
proteins (9), DLL1 and DLL3, and saw that the DLL1 transcript
was increased three-fold and the DLL3 transcript levels
increased by approximately 30-fold over untransfected
controls (Figure 3B). This combination of CRISPRa and iPS
cells represents an interesting experimental system as one
could begin to test questions associated with increased gene
expression levels on the differentiation of specific cell types.

Figure 4: Creating an EGFP reporter in iPS cells using HDR
EGFP fluorescence in iPS cells of HDR tagged EGFP-SEC61B

Discussion
Here we have demonstrated the effectiveness of Edit-R
Figure 3: Performing CRISPRa in iPS cells
A. dCas9-VPR and a synthetic crRNA targeting either POU5F1,TTN, ASCL1, EGFR, or a non-targeting control (NTC) were delivered
to iPS cells with nucleofection. Gene activation of POU5F1, TTN,
ASCL1, EGFR, or NTC was assessed at seventy-two hours post
nucleofection by RT-qPCR and is shown as fold transcriptional
activation compared to the NTC. B. Relative expression of DLL1,DLL3, or NTC upon CRISPRa transcriptional activation of ASCL1
analyzed by RT-qPCR and normalized to NTC samples.

Knock-in of EGFPCRISPR-Cas9 and CRISPRmod CRISPRa reagents for gene
editing and gene modulation in a human iPS cell line.
The use of the synthetic two-part crRNA:tracrRNA system in
iPS cells provides rapid reagent generation and compatibility
with different CRISPR-Cas9 systems. CRISPR-Cas9 mediatedknockout of genes required for the maintenance ofpluripotency, such as POU5F1, can be targeted to drive
differentiation into different cell types, or in a screen toidentify other gene targets important in the maintenance ofpluripotency. Researchers can also use CRISPR-Cas9 andUsing CRISPR-Cas9 to create knockout cell lines is an

iPS cells to create matched, isogenic cell lines whereby

important tool for teasing apart the function of a specifican unmodified iPS cell population can be compared togene target. Conversely, there are circumstances when one

a CRISPR-Cas9 targeted iPS cell population to study thewould want to repair a genetic defect, create a reporter

impact of a specific genotype on a cellular phenotype.

cell line, or tag an endogenous protein. Here iPS cells werenucleofected with Cas9 protein, SEC61B crRNA, tracrRNA,
and an EGFP repair plasmid. The repair plasmid was
designed to insert a single EGFP tag into the N-terminus ofSEC61B using 1,000 bp homology arms. Transfected cells
were grown for an additional 72 hours before imaging

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We were able to show that CRISPRa reagents showed anincrease in gene expression of a lineage specific oncogene(ASCL1) and subsequent downstream transcriptional
activation of ASCL1 gene target, DLL3. CRISPRa provides
the ability to begin to ask questions related to elevatedexpression levels of specific and downstream gene targets,
3

CRISPR gene editing and transcriptional activation in human iPS cells.

and the compatibility of CRISPRa with synthetic crRNAFor the 2b nucleofection system, cells were then mixed

enables both a pooled and arrayed approach to test thesewith 500 ng dCas9-VPR plasmid and 500 ng custom sgRNAquestions. One such question to take this experimentplasmid. For HDR experiments 500 ng repair template

further would be to compare gene expression profiles,

was used. Nucleofections were carried out, as indicated,

protein interaction partners, etc. in elevated ASCL1 iPS cellswith either the Lonza Nucleofector™ 96-well Shuttle with

where ASCL1 is not oncogenic, to iPS cells differentiated into

the P3 Primary Cell Solution (Lonza, Cat# PBP3-02250)

pulmonary neuroendocrine cells where ACLS1 is oncogenic.

or the Lonza Nucleofector™ 2b with the Human Stem CellFinally, we were able to create a reporter iPS cell line using
Cas9 RNP and an EGFP repair plasmid to tag the N-terminus

Nucleofector Solution 2 (Lonza, Cat# VPH-5002) according
to the manufacturer’s protocol.

of SEC61B. The methods presented in the creation of theDNA mismatch detection assay: Cells (1 × 105 per sample)

reporter cell line can be adopted to create other precisely

were lysed in Phusion™ HF buffer (Thermo Scientific,tagged, or repaired gene targets such as the creation ofCat #F-518) with 5 µL each of Proteinase K (Thermodisease causing SNPs, or the creation of differentiated

Scientific, Cat #EO0492) and RNase A (Thermo Scientific,cell reporters.

Cat #EN0531), and incubated for 1 hour at 56 °C then heat

This application note demonstrates the utility of synthetic
crRNA, tracrRNA, and Cas9 protein as a convenient sourceof CRISPR reagents for effective genome editing in human
iPS cells.

inactivated at 98 °C for 10 minutes. Fifty microliter PCR
reactions were carried out using 0.5 µL Phusion Hot Start II
DNA Polymerase (Thermo Scientific, Cat #F-549S), 5X Phusion
HF buffer (Thermo Scientific, Cat #F-518), 200 µM each dNTP
(Thermo Scientific, Cat #F-549S), 0.5 µM forward and reverse
primers (PPIB: Forward 5’-ACCGTGTATTTTGACCTACGAAT-3’,

Materials and methods
Tissue culture: iPS cells (Thermo, Cat#A18945) weremaintained according to the manufacturer’s recommendationsin Essential 8™ medium (Thermo, Cat #A1517001),
on vitronectin coated culture plates (Thermo, Cat# A14700)
in serum-free conditions. Cells were passaged using ReLeSR,
an enzyme-free dissociation reagent (Stem Cell technologies,Cat# 05872) according to manufacturer’s recommendations.
During passaging, iPS cells were supplemented with RevitaCell
Supplement (Thermo, Cat# A26445). Essential 8 medium wasreplaced every twenty-four hours during culture.

Reverse 5’-AAACATTCGTAGGTCAAAATACA-3’; POU5F1:
Forward 5’-AGAAGGGCAAGCGATCAAGC-3’, Reverse
5’-TGAGCATTGGATATTCCCATCC-3’;) and 5 µL direct cell
lysis template. Touchdown PCR and an annealing programwith the following thermal cycling steps were run for eachsample: Denature/enzyme activation at 98 °C for 3 minutes
followed by 10 cycles of 98 °C for 10 seconds, 72 °C for
15 seconds -1 °C/cycle, and 72 °C for 30 seconds then
25 cycles of 98 °C for 10 seconds, 62 °C for 15 seconds
and 72 °C for 30 seconds and final extension at 72 °C for
10 minutes. Samples were heated to 95 °C for 10 minutes
and slowly cooled to re-anneal. Then, 10 µL of annealed
PCR products were combined with 5 units of T7EI enzyme

Electroporation: At the time of electroporation,and NEBuffer 2 (New England Biolabs, Cat #M0302L)

2 × 105 (96-well Shuttle Nucleofection System) or 8 × 105

and incubated at 37 °C for 25 minutes. Three microliters

iPS (Lonza 2b Nucleofection System) cells were collectedof 6X Orange Loading Dye (Thermo Scientific, Cat #R0631)

per reaction and centrifuged at 500 × g for 2 minutes.

was added to the T7EI reactions and the entire volume was

Cell pellets were washed with DPBS (GE Healthcare

loaded and ran on a 2% agarose gel. For more details see theHyclone, Cat #SH30264.01) and re-centrifuged

Edit-R Synthetic crRNA Positive Controls Protocol. The level

at 500 × g for an additional 2 minutes. Cells wereof editing was calculated using densitometry (% gene editing)resuspended in 100 µL Buffer P3 (Lonza, Cat # PBP3-in Image J and gene editing was estimated with the equation

00675). For the 96-well shuttle system, cells were then(1-√(1-(a+b)/(a+b+c)))*100.

mixed with 30 pmol Edit-R Cas9 Nuclease protein NLS(Dharmacon, Cat #CAS11729; 40 µM), and 60 pmol
tracrRNA (Dharmacon, Cat #U-002000-120) with 60 pmol
predesign Edit-R PPIB crRNA (Dharmacon, Cat #CM004606-02-0020) or 60 pmol Edit-R crRNA Nontargeting
Control #1 (Dharmacon, Cat # U-007501-01-20).
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Gene expression analysis (RT-qPCR): Total RNA was
isolated using an SV 96 Total RNA Isolation System(Promega, Cat #Z3500) per manufacturer’s instructions.
cDNA was synthesized using a Maxima First Strand cDNA
Synthesis Kit (Thermo Scientific, Cat#K1672).
4

CRISPR gene editing and transcriptional activation in human iPS cells.

Briefly, 1 µL of 10X dsDNase Buffer, 1 µL of dsDNase, and8 µL of total isolated RNA were combined and incubated atReferences

37 °C for 2 minutes. Next, 4 µL of 5X Reaction Mix, 2 µL of

1. M. Jinek, et al., A Programmable Dual-RNA-Guided DNA

Maxima Enzyme Mix, and 4 µL of nuclease-free water wereEndonuclease in Adaptive Bacterial Immunity. Science.

added to each reaction. Each reaction was incubated at337(6096), 816-821 (2012).

25 °C for 10 minutes followed by 30 minutes at 50 °C andterminated by incubating at 85 °C for 5 minutes. Relativegene expression was determined using TaqMan GeneExpression Assays. For each assay, 7.5 µL of TaqMan MasterMix was combined with 2 µL of undiluted cDNA, 0.75 µLof respective TaqMan Probe, and 4.75 µL of nuclease-freewater. Samples were mixed and 10 µL of each reaction was

2. P. Mali, et al. RNA-guided human genome engineering via
Cas9. Science. 339(6121), 823-826 (2013).
3. L. Cong, et al., Multiplex Genome Engineering Using
CRISPR/Cas Systems. Science. 339(6121), (2013).
4. L.S. Qi, et al., Repurposing CRISPR as an RNA-guided

added to individual wells on a 384-well plate and loaded

platform for sequence-specific control of gene expression.

onto a Roche LightCycler 480 II. The following thermo cycling

Cell. 152(5): p. 1173-83 (2013)

steps were performed on each sample: Enzyme activation
at 95 °C for 10 minutes followed by 40 cycles of denaturing
at 95 °C for 15 seconds and annealing at 60 °C for
1 minute. Each sample was performed in technical triplicate.

5. L.A.Gilbert, et al., CRISPR-mediated modular RNA-guided
regulation of transcription in eukaryotes. Cell.
154(2): p. 442-51 (2013).

The relative expression of each gene was calculated with the

6. A.W. Cheng, et al., Multiplexed activation of endogenous

ΔΔCq method using GAPDH as the housekeeping gene andgenes by CRISPR-on, an RNA-guided transcriptional

normalized to a non-targeting control.

activator system. Cell Res. 23(10): p. 1163-71 (2013).

Fluorescence microscopy: CD44 Alexa Fluor

7. Y. Yeom, et al., Germline regulatory element of Oct-4

488 (Thermo, Cat #A25528) and Tra-1-60 Alexa

specific for the totipotent cycle of embryonal cells.

Fluor 594 (Thermo, Cat #A24882) were used accordingDevelopment. 122(3), 881–894 (1996).

to manufacturer’s recommendations. CD44 Alexa
Fluor 488 and Tra-1-60 Alexa Fluor 594 were added tothe iPS cell culture media, after a 30-minute incubation at37 °C, culture media was washed twice with FluoroBriteDMEM (Thermo, Cat #A1896701) and immediately imaged.

8. A. Chavez et al., Highly efficient Cas9-mediatedtranscriptional programming. Nat. Methods.
12, 326–328 (2015).
9. A. Augustyn, M. Borromeo. et al., ASCL1 is a lineage
oncogene providing therapeutic targets for high-gradeneuroendocrine lung cancers. PNAS. 111(41),14788-93 (2014).

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