This document describes a workflow for generating clonal CRISPR-edited human induced pluripotent stem cell (hiPSC) lines. Key aspects of the workflow include: 1) Developing a hiPSC line that stably expresses Cas9 to facilitate efficient genome editing. 2) Optimizing delivery methods for CRISPR/Cas9 editing tools and improving single cell clone survival and isolation using laminin-521 and StemFlex medium. 3) Applying the workflow to generate hiPSC lines carrying disease-relevant mutations and testing the cell models in assays, finding increased sensitivity to stress in models of Parkinson's disease.

1. Chetana Revankar, Erik Willems, Rex Lacambacal, Tufan Gokirmak, Jordan Dizon, Jacquelyn Webb, Raquel Vega and David Piper; Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008 USA
RESULTS
Figure 5: A stringent gating strategy was used to identify single, viable and pluripotent hiPSCs which were
seeded as indicated. Effects of hiPSC growth medium were investigated. The novel workflow based on these
results is shown. Factors that may improve survival after sorting were then tested to understand if single cell
cloning in feeder-free conditions was at all possible. Through extending the RevitaCellTM exposure from 24h to
72h we were able to dramatically increase clone survival in Essential8TM when seeding limited amounts of cells
into a 96-well. Clone survival from seeding one cell was, however, limited and therefore the effect of matrix
protein and media system was further investigated. Sorting cells on rhLaminin-521TM further increased clone
recovery from single cells, up to 15% and this improvement was enhanced when sorting cells into StemFlexTM
medium, yielding up to 40% clone survival from single cell seeds.
Figure 6. Single cell-derived pluripotent clones were successfully derived from all hiPSC lines tested with
different clonal survival rates, but approaching at least 25%. Individual clones are shown and stained for TRA 1-
60 to demonstrate their pluripotent nature. Cloning efficiency measured by confluency or PrestoBlue® for the
different lines is also indicated.
ABSTRACT
Induced pluripotent stem cell (iPSC)-derived cells are widely used in disease models, safety testing and
phenotypic and functional assays. The CRISPR/Cas9 system has revolutionized genome editing thus
enabling rapid generation of disease models. Cutting edge technologies like genome editing paired with
iPSC-derived cell models have reshaped our approach to disease modeling in vitro. Although,
CRISPR/Cas9 has had huge impact on generation of complex cellular models, there are challenges in
replicating disease-in-a-dish. One of the challenges is the implementation of genome editing tools in
iPSC, particularly issues associated with editing tools delivery, cell recovery and clonal isolation of
genome-edited cells. Here, we describe a workflow that facilitates the generation and isolation of clonal
CRISPR-edited iPSCs through the use of a novel toolset. We observed high genome editing efficiency
using an iPSC line that stably expresses the Cas9 protein and obtained improved survival of the iPSCs
after delivery of the editing tools through culture in StemFlex™ medium, a novel PSC culture system.
Furthermore, we have demonstrated that single edited iPSCs can be isolated through the combination
of an optimized cell sorting method and expanded as single cell clones with reagents that support
single cell survival. Through this workflow we have generated several iPSC lines carrying disease
relevant SNP mutations. These tools and workflows not only contribute to improved success in the
derivation of homogenous genome edited human iPSC clones but also provide novel alternatives to
study disease-causing mutations in vitro. Additionally, we have also tested these cell models in
commonly used cell health assays. These cell models can be further used to generate organoids to
mimic conditions found in tissues. iPSC- derived cell models not only permit the study of disease
mechanism in the cell types specifically affected in the disease but also deepen our understanding of
the molecular mechanisms and in turn help development of novel therapeutics.
INTRODUCTION
Reprogramming permits the derivation of hiPSCs from diseased patients, and allows us to model
diseases in vitro. Furthermore, with the advent of CRISPR mediated genome editing, we can now
mimic disease mutations in control hiPSC lines to study the biological effect of just those mutations.
hiPSCs can then be differentiated into specified cell types such as neurons which can be used to
develop assays for drug safety screening or can be used to model disease phenotypes in a dish to
discover new drugs.
The implementation of CRISPR/Cas9 in hiPSC disease models has proven challenging due to
difficulties with efficient editing tool delivery, survival after delivery and clonal isolation. The more
efficient the delivery and clonal isolation is, the higher the success rate in the derivation of the edited
hiPSC line of choice. Therefore, we highlight several new tools that can be used to improve the
derivation of CRISPR/Cas9-based disease models through use of a hiPSC line stably expressing Cas9
or use of TrueCut™ Cas9 protein and an improved workflow for clonal isolation of edited hiPSC, which
hinges on the use of rhLaminin521TM matrix protein and StemFlexTM medium.
CONCLUSIONS
 With hiPSC-based disease modeling at the forefront of drug discovery, the ability of genome editing has taken
an important place in hiPSC-based disease model generation. We therefore sought to develop new tools and
methods that improve the efficiency of genome editing and clonal isolation to facilitate generation of isogenic
hiPSC lines or hiPSC lines carrying disease causing mutations to facilitate the generation of disease models
and validate their functionality.
 We developed a hiPSC line that stably expresses Cas9, which not only allows efficient genome editing in the
hiPSCs themselves, but also allows to study CRISPR/Cas9 mediated perturbations after differentiation.
 We also validated other novel tools that facilitate editing in any hiPSC line. The combination of TrueCut Cas9,
TrueGuide gRNA and the clonal isolation method provide a series of novel tools that dramatically increase the
success of CRISPR/Cas9 edited hiPSC lines, as we demonstrated for three individual targets.
 To facilitate the clonal isolation of genome edited hiPSCs, we developed a new method that allows isolation of
single cell derived clones via FACS. Key components of post survival are extracellular factors such as the
rhLaminin-521TM matrix protein and StemFlexTM medium.
 Clonal CRIPSR-edited iPSCs differentiated into different cell types provide a platform to model a disease
phenotype in vitro and should contribute to the adoption of disease models in research and drug discovery.
Generation of Clonal CRISPR/Cas9-edited Human iPSC Derived Cellular Models and its Applications in
Physiologically Relevant Assays
Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • www.thermofisher.com
Day 4
Day 4
HPRT gRNAB2M gRNA
LRRK2 gRNAHPRT gRNA
Cas9
hiPSC
Floorplate
progenitors
FACS on PI-
/Tra 1-60+
hiPSCs
Seed single cells
onto rhLam521TM
into StemFlexTM
medium +
RevitaCellTM
Media change
with
StemFlexTM at
day 3/6/9/12
post sort
Clone expansion
for downstream
work from day 12
post sort
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
1 3 5
%CloneFormation
Cells seeded per 96-well
Essential 8
mTesr1
StemFlex
Scatter gate
(exclude debris)
SSC gate
(exclude doublets)
FSC gate
(exclude doublets)
PI-/Tra 1-60+ gate
(select viable
pluripotent cells)
Most available CRISPR/Cas9 editing methods relying on DNA and mRNA based tools do not allow
efficient genome editing in hiPSC, resulting in screening of hundreds of clones to find a correctly edited
hiPSC line. Common approaches to facilitate isolation of genome edited hiPSC clones is to introduce a
positive selection marker with the desired edit into the genome to enrich for correctly edited hiPSCs, after
which hiPSCs are seeded at limited dilutions to isolate potentially single cell derived clones. This approach
however typically requires removal of the selection cassette. We thus developed a hiPSC line stably
expressing Cas9, as a backbone to easily generate hiPSC-based disease models (Figure 2). Through
lentiviral delivery we introduced Cas9 into hiPSCs (Cas9 hiPSCs) and after selection a stable cell line
was obtained (Figure 2). The cell line generation process did not affect the biology of the hiPSCs as the
cells maintained their normal morphology, karyotype and expression of pluripotent markers. Additionally,
we also evaluated the differentiation potential of the Cas9 hiPSCs both through spontaneous
differentiation as embryoid bodies and directed differentiation to dopaminergic neurons (Figure 2). http://www.thermofisher.com/stemflex
http://www.thermofisher.com/askdiscovery
Figure 3. To test editing capabilities of the Cas9 hiPSCs, in vitro transcribed gRNA were delivered through
electroporation, yielding a high degree of indel induction. When single stranded oligos as donor are co-delivered
with the gRNA into the Cas9 hiPSCs, homology driven repair was efficient in most cases, although target
dependent. Here we show that floor plate progenitors can be edited through delivery of IVT gRNA via RNAiMaxTM .
Up to 75% indel induction is observed with as little as 50ng gRNA both in Cas9 hiPSCs (top left panel) and Cas9
hiPSC-derived floor plate progenitors (bottom left panel). Indel induction up to 75% and homology driven repair
(HDR) up to ~40% was observed in Cas9 hiPSCs. The ability to edit in Cas9 hiPSC-derived differentiated cell types
allows for functional screening in a variety of cell types and opens up avenues to use gRNA collections such as the
Invitrogen LentiArrayTM libraries to identify important biological targets for the model of choice.
TRA1-60BF
DF1
TRA1-60BF
BS3
TRA1-60BF
GIBCO
0%
10%
20%
30%
40%
50%
GIBCO BS3 DF1
%CloneFormation
Cell Line
PrestoBlue
Confluency
Target
SNCA_1
SNCA_2
SNCA_3
SNCA_4
LRRK2_1
LRRK2_2
B2M
HPRT
%Indels
38 30 63 23 73 78 62 49
%HDR 37 N/D 34 N/D 26 14 N/D N/D
Figure 7. Cas9 hiPSCs were used to introduce SNPs known to be associated with Parkinson’s disease (LRRK2
G2019S, LRRK2 I1371V and SNCA A30P). We obtained 11-41% homology driven repair or SNP introduction in the
edited cell pool. Single cell clones were then isolated from each of the pools and yielded 17%-37% surviving clones.
These clones were then screened by Sanger sequencing for the presence of homozygous and heterozygous SNPs
as well as indels (pie charts). Selected single cell clones were then further analyzed by next generation sequencing
to understand if the isolated clones were really originating from a single cell. Ratios of 100% WT or SNP are expected
for unedited or homozygotes, whereas for heterozygotes, both the WT and SNP allele should be represented by
equal amounts. Across all targets and all the derived clonal lines, this is indeed the case (bottom table),
demonstrating that a single round of clonal isolation via FACS is sufficient to obtain single cell derived clonal lines.
-8 -7 -6 -5
0.0
0.1
0.2
0.3
0.4
0.5
A 3 0 P S NP /S NP
A 3 0 P S NP /W T
A 3 0 P W T /W T
A 3 0 P W T /in d e l
G lu co se starv ed
L o g [S tau ro sp o rin e] M
CellHealth
(PrestoBlue/NucBlue)
-9 -8 -7 -6 -5 -4
0.0
0.5
1.0
1.5
2.0
A30P SNP/SNP
A30P SNP/WT
A30P WT/WT
A30P WT/indel
+ Glucose
Log [Staurosporine] M
CellHealth
(PrestoBlue/NucBlue)
-9 -8 -7 -6 -5 -4
0
1
2
3
4
A 3 0 P S N P /S N P
A 3 0 P S N P /W T
A 3 0 P W T /W T
A 3 0 P W T /in d e l
+ G lu c o s e
L o g [S ta u r o s p o r in e ] M
CellHealth
(PrestoBlue/NucBlue)
- 8 - 7 - 6 - 5
0 . 0
0 . 5
1 . 0
1 . 5
2 . 0
2 . 5
A 3 0 P S N P / S N P
A 3 0 P S N P / W T
A 3 0 P W T / W T
A 3 0 P W T / in d e l
G lu c o s e s t a r v e d
L o g [ S t a u r o s p o r in e ] M
CellHealth
(PrestoBlue/NucBlue)
Neural Stem Cells Floor Plate Progenitors
Figure 8. Clonal CRISPR-edited iPSCs were differentiated into neural stem cells and floor plate progenitors.
Sensitivity to the cell death inducing agent staurosporine was assessed under normal glucose and glucose
starved conditions. Cell viability was measured through the plate-reader based PrestoBlue® assay. Under normal
glucose conditions, the selected disease lines behaved similarly to the control NSC and FPP after staurosporine
treatment. However, under glucose starved condition, diseased lines showed a significant higher sensitivity to
staurosporine, demonstrating that a Parkinson’s disease phenotype can be replicated in differentiated cells
derived from clonal-edited iPSCs.
Figure 4. Delivery of TrueCut™ Cas9 and TrueGuide™ gRNAs via Neon electroporation yield similar indel
frequencies to stable Cas9 hiPSC (two top panels). Lipid based delivery using Lipofectamine™ Stem can also be
used, but yield lower editing efficiencies (bottom panel). In addition to a stable Cas9 hiPSC line, we now have tools
available for efficient editing in any hiPSC line, including TrueCut Cas9 protein, Lipofectamine Stem for lipid based
delivery of editing tools and TrueGuide gRNAs for reduced gRNA toxicity
Figure 2. Cas9 hiPSCs were generated by transducing Human iPSCs with a Cas9 expressing lenti-vector after
which the hiPSCs were selected for presence of the lenti-vector. ICC for EB (B3T and SMA) and directed
differentiation to DA neurons (B3T and TH) demonstrates that the hiPSC Cas9 Line can be differentiated further.
Human Episomal
hiPSC
Cas9 Line
Lentivirus
containing Cas9
Human Episomal
hiPSC Line
NucBlue
TRA 1-60
NucBlue
B3T
NucBlue
SMA
NucBlue
B3T
TH
0
20
40
60
no gRNA CR:TR sg IVT
%Indels
CDK4
TrueCut Cas9 RNP Stable Cas9
0
20
40
60
no gRNA CR:TR sg IVT
%Indels
HPRT
TrueCut Cas9 RNP Stable Cas9
0
20
40
LRRK2 G2019S LRRK2 I2020T SNC5A E1053K TNNT2 R141W KCNH2 A422T
Gibco iPSC BS3-iPSC
%HDR
SNP
Neon Lipofectamine Stem
Human Episomal hiPSC Cas9 line (Cas9 hiPSCs
Genome editing is highly efficient in Cas9 hiPSCs and
Cas9 hiPSC-derived differentiated cells
Other tools for genome editing in hiPSCs
Improvements to increase the recovery of single cell clones
Single cell clone generation from different hiPSC lines
Generation of disease models in Cas9 hiPSCs
LB-043
Cell health assays of disease models in differentiated cell types
REFERENCES
1. Mercola et al. Induced Pluripotent Stem Cells in Cardiovascular Drug Discovery. Circulation Research 2013.
ACKNOWLEDGEMENTS
We would like to thank Uma Lakshmipathy and Xiquan Liang for donation of hiPSC cell lines. We would also like to
thank Rhonda Newman and David Kuninger for their contribution to this poster.
TRADEMARKS/LICENSING
© 2017 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are the property of Thermo Fisher Scientific and
its subsidiaries unless otherwise specified. Essential 8™ is a trademark of Cellular Dynamics International, Inc.
mTeSRTM1 is a trademark of Stem Cell Technologies Inc. For Research Use Only. Not for use in diagnostic procedures.