Takara Bio Cellartis iPSC rCas9 User manual

Takara Bio USA, Inc.
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Takara Bio USA, Inc.
Cellartis® iPSC rCas9
Electroporation and
Single-Cell Cloning
System User Manual
Cat. No. 632643
(030619)

Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
takarabio.com
Takara Bio USA, Inc.
Page 2 of 24
Table of Contents
I. Introduction.....................................................................................................................................................................4
II. List of Components.........................................................................................................................................................6
III. Additional Materials Not Supplied.................................................................................................................................7
A. Required......................................................................................................................................................................7
B. Recommended.............................................................................................................................................................7
IV. General Considerations...............................................................................................................................................8
A. Storage and Handling..................................................................................................................................................8
B. Transferring Human iPS Cells to the DEF-CS Culture System..................................................................................8
V. Complete Experimental Workflow.................................................................................................................................9
VI. In Vitro Transcription of an sgRNA Containing the Desired Target Sequence..........................................................9
A. Protocol Overview ......................................................................................................................................................9
B. Generating the DNA Template.................................................................................................................................10
C. Performing the In Vitro Transcription (IVT) Reaction.............................................................................................12
D. Purifying the Transcribed sgRNA.............................................................................................................................14
VII. Editing hiPS Cells by Electroporation ......................................................................................................................15
A. Protocol Overview ....................................................................................................................................................15
B. Preparing Media and Cells........................................................................................................................................15
C. Preparing the rCas9/sgRNA RNP complex ..............................................................................................................17
D. Electroporating Cells Using the Neon Transfection System.....................................................................................18
VIII. Single-Cell Cloning of Edited Cells..........................................................................................................................19
A. Protocol Overview ....................................................................................................................................................19
B. Single-Cell Seeding into a 96-Well Plate..................................................................................................................20
C. Culturing Single-Cell Colonies.................................................................................................................................22
IX. Passaging Cells from the 96-Well Plate to a 48-Well Plate......................................................................................23
A. Coating a 48-Well Plate............................................................................................................................................23
B. Preparing DEF-CS SCC Medium for Passaging.......................................................................................................23
C. Passaging...................................................................................................................................................................23
X. Scaling up from the 48-Well Plate................................................................................................................................24
XI. References.................................................................................................................................................................24

Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
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Table of Figures
Figure 1. Schematic of CRISPR/Cas9-based editing to create gene knockouts and knockins...............................................5
Figure 2. Using DEF-CS technology to generate edited clonal cell lines...............................................................................6
Figure 3. Workflow for gene editing of hiPS cells using components of the Cellartis iPSC rCas9 Electroporation and
Single-Cell Cloning System....................................................................................................................................................9
Figure 4. Workflow for generating sgRNA using components of the Cellartis iPSC rCas9 Electroporation and Single-Cell
Cloning System.....................................................................................................................................................................10
Figure 5. Designing a forward PCR primer to generate a DNA template for an sgRNA containing your target sequence .11
Figure 6. Gel electrophoresis of the PCR product ................................................................................................................12
Figure 7. sgRNA yield over time..........................................................................................................................................13
Figure 8. Workflow for electroporation-based delivery of rCas9/sgRNA complexes..........................................................15
Figure 9. Recommended density of starting culture used for single-cell cloning.................................................................20
Figure 10. A single cell seeded in one well generates an emerging colony..........................................................................22
Figure 11. Clonal colonies, ready for transfer to larger wells and scale-up..........................................................................23
Table of Tables
Table I. Examples of sgRNA concentration and yield using different elution volumes.......................................................14
Table II. Preparation of coating solution for a 48-well plate................................................................................................16
Table III. Preparation of DEF-CS medium for dissociation and recovery............................................................................16
Table IV. Workflow for single-cell cloning and expansion using the Cellartis iPSC rCas9 Electroporation and Single-Cell
Cloning System.....................................................................................................................................................................19
Table V. Preparation of coating solution for a 96-well plate................................................................................................20
Table VI. Preparation of medium for dissociation and single-cell seeding. .........................................................................21
Table VII. Preparation of coating solution for a 48-well plate. ............................................................................................23

Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
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Takara Bio USA, Inc.
Page 4 of 24
I. Introduction
The Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System allows efficient, footprint-free gene
editing of human induced pluripotent stem cells (hiPSCs) using the CRISPR/Cas9 system, followed by successful
clonal expansion of single, edited human iPSCs. Importantly, this system maintains karyotype and pluripotency
during the whole editing and culturing process.
The CRISPR/Cas9 system has emerged as a powerful tool for gene editing because of its high targeting
specificity, editing efficiency, and ease of use. The power of this technology derives from its simplicity, since all
it requires is a Cas9 nuclease enzyme combined with a single guide RNA (sgRNA) that determines its target
specificity (Jinek et al. 2012). This RNA-programmable method exploits the error-prone nature of the non-
homologous end joining DNA repair pathway (NHEJ) to generate gene knockouts (via insertion/deletion). The
method can also be used to generate knockins via the homology-directed repair (HDR) pathway (Figure 1).
CRISPR/Cas9 system components have been delivered successfully into target cells through a variety of
approaches, including vector-based expression systems, transfection of RNA, and introduction of Cas9-sgRNA
ribonucleoprotein (RNP) complexes (Sander and Joung 2014). Delivery of Cas9-sgRNA RNPs via
electroporation, the method described in this manual, provides a fast turnaround for gene-editing experiments
while minimizing the likelihood of off-target effects compared to vector-based approaches (Kim et al. 2014).
The editing component of the Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System contains:
•Recombinant wild-type Cas9 nuclease [Guide-it™Recombinant Cas9 (Electroporation-Ready)]
oThis recombinant wild-type Streptococcus pyogenes Cas9 nuclease was expressed with a C-
terminal nuclear-localization signal (NLS) and purified from E. coli for use in CRISPR/Cas9-
mediated gene editing experiments.
oThe rCas9 protein solution has been verified to be sterile and well-tolerated by mammalian cells
when electroporated as an RNP with a sgRNA for knockout experiments, or as an RNP with a
donor repair template for knockin experiments.
•The Guide-it sgRNA In Vitro Transcription Components v2 is used to produce high yields of sgRNAs
from in vitro transcription (IVT) reactions followed by the Guide-it IVT RNA Clean-Up Kit, used to
quickly purify sgRNA in a phenol-free manner.

Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
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Figure 1. Schematic of CRISPR/Cas9-based editing to create gene knockouts and knockins. The CRISPR/Cas9 system is a simple,
RNA-programmable method to mediate gene editing in mammalian cells. The CRISPR/Cas9 system relies on a single guide RNA
(sgRNA) directing the rCas9 endonuclease to induce a double-strand break at a specific target sequence three base-pairs upstream of a
PAM sequence in genomic DNA. This DNA cleavage can be repaired in one of two ways: 1) non-homologous end joining (NHEJ), which
can result in gene knockout due to error prone repair (orange), or 2) homology directed repair (HDR), which can result in gene knockin if a
homologous repair template (purple) is co-delivered.
Once the rCas9/sgRNA RNP complexes have been delivered by electroporation and cells have recovered, single
cells must be isolated and expanded into clonal cell lines in order to isolate and screen for the genotype of interest
(Figure 2). Traditionally, the establishment of a clonal population from hiPS cells grown and passaged as colonies
is inefficient, challenging, and time-consuming; often, it results in cell death or premature differentiation.
However, the cell culture component of the Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System
contains a defined culture system (the Cellartis DEF-CS™culture system, composed of basal medium, coating,
and additives) for efficient single-cell cloning and expansion of edited hiPSC clones. The DEF-CS culture system,
a monolayer-based culture system, bypasses the challenges of colony-based culture by allowing single-cell
passaging, promoting survival and further expansion of plated single cells, and preserving the pluripotency of
these cells.

Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
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Figure 2. Using DEF-CS technology to generate edited clonal cell lines. Human induced pluripotent stem (hiPS) cells can be cultured,
edited, and clonally expanded using the Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System. Initially, hiPS cells are
adapted to the DEF-CS culture system, which maintains cells as a karyotypically stable and pluripotent monolayer. Next, electroporation-
based delivery of rCas9 and an sgRNA (together as a ribonucleoprotein complex) are used to edit the cells. Using FACS or limiting
dilution, edited cells can be individually seeded into wells of a 96-well plate (Panel A) and expanded into clonal lines. If desired, edited
cells can be first sorted as a population by flow cytometry, prior to being seeded into wells of a 96-well plate, and finally expanded into
clonal lines (Panel B).
All procedures described in this manual are optimized for Cellartis human iPS cell lines. If you wish to use the
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System for other human iPS cell lines or for
Cellartis iPS cells grown in another system, please be aware that these cell lines will need to be adapted to the
DEF-CS culture system before editing (see Section IV.B).
II. List of Components
The Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System (Cat. No. 632643) contains:
•100 µg Guide-it Recombinant Cas9 (Electroporation-Ready) (Cat. No. 632641) (3 µg/µl)
•Guide-it sgRNA In Vitro Transcription Components v2 (Cat. No. 632637) (Not sold separately)
o50 µl Guide-it Scaffold Template (1 ng/µl)
o350 µl Guide-it In Vitro Transcription Buffer
o1 ml RNase Free Water
o150 µl Guide-it T7 Polymerase Mix (33 U/µl)
o625 µl PrimeSTAR® Max Premix (2X)
o100 µl Recombinant DNase I (RNase-free) (5 U/µl)
•Guide-it IVT RNA Clean-Up Kit (Cat. No. 632638)
o13 ml RNase Free Water
o3 ml IVT Binding Buffer
o50 IVT RNA Clean-Up Spin Columns
o2 x 6 ml IVT Wash Buffer
o50 Collection Tubes (2 ml)
•2 x 800 µl Cellartis iPSC Single-Cell Cloning DEF-CS COAT-1 (Cat. No. Y30018) (Not sold separately)
•500 ml Cellartis DEF-CS 500 Basal Medium (Cat. No. Y30011) (Not sold separately)
•Cellartis iPSC Single-Cell Cloning DEF-CS Additives (Cat. No. Y30019) (Not sold separately)
o2 x 750 µl DEF-CS GF-1
o500 µl DEF-CS GF-2
o500 µl DEF-CS GF-3

Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
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III. Additional Materials Not Supplied
A. Required
Mammalian Cell Culture Supplies
Use the Cellartis DEF-CS 500 Culture System (Takara Bio, Cat. No. Y30010) for maintaining hiPS cell
lines 1) prior to using the Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System and 2) for
scaling up clonal lines created using this system.
The following tissue culture materials are required but not supplied:
•96-well plates, flat bottom, cell-culture treated (Corning, Cat. No. 3595 or equivalent)
•48-well plates, flat bottom, cell-culture treated (Corning, Cat. No. 3548 or equivalent)
•TrypLE Select Enzyme (1X), no phenol red (Thermo Fisher Scientific, Cat. No. 12563011)
•PBS Dulbecco's with Ca2+ & Mg2+ (D-PBS +/+) (Sigma, Cat. No. D8662 or equivalent)
•PBS Dulbecco's w/o Ca2+ & Mg2+ (D-PBS –/–) (Sigma, Cat. No. D8537 or equivalent)
General Supplies
•96–100% ethanol (Sigma, Cat. No. E7023 or equivalent)
•Isopropanol (Sigma, Cat. No. I9516 or equivalent)
•NanoDrop 2000 spectrophotometer (Thermo Fisher Scientific, Cat. No. ND-2000 or equivalent)
Electroporation Supplies
Use of this product requires an electroporator, electroporation chamber (typically cuvettes or tips), and
an electroporation buffer that is suitable for hiPSCs. In this manual, we provide a protocol for
electroporation using the Neon Transfection System (Thermo Fisher Scientific, Cat. No. MPK5000).
If using the 4D-Nucleofector System (Lonza, Cat. No. AAF-1002B), please refer to the
manufacturer’s website for operating instructions.
B. Recommended
sgRNA Efficacy Testing Supplies
If you to wish to test the efficacy of different sgRNAs in vitro prior to using them in editing experiments,
try the Guide-it sgRNA Screening Kit (Cat. No. 632639).
Genotype Confirmation Supplies
These items are recommended for determining the efficiency of gene editing after electroporation (Cat.
Nos. 631443 & 631448), the genotype (Cat. No. 632611), and the sequence of the edits (Cat. No. 631444)
in the clonal cell lines:
Cat. No.
Product
Size
631443
Guide-it Mutation Detection Kit
100 rxns
631448
Guide-it Mutation Detection Kit
25 rxns
632611
Guide-it Genotype Confirmation Kit
100 rxns
631444
Guide-it Indel Identification Kit
10 rxns

Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
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IV. General Considerations
A. Storage and Handling
•Guide-it sgRNA In Vitro Transcription Components v2
oStore at –20°C.
oAvoid repeated freeze/thaw cycles.
•Guide-it IVT RNA Clean-Up Kit
oStore at room temperature.
•Guide-it Recombinant Cas9 (Electroporation-Ready)
oStore at –70°C.
oAt first use, thaw and aliquot.
oAvoid repeated freeze/thaw cycles.
•Cellartis iPSC Single-Cell Cloning DEF-CS COAT-1
oStore at 2–8°C.
oShelf life is specified on the product label.
•Cellartis DEF-CS 500 Basal Medium
oStore at 2–8°C.
oContains penicillin and streptomycin.
•Cellartis iPSC Single-Cell Cloning DEF-CS Additives
oStore at –20°C.
oShelf life is specified on the product label.
oAt first use, thaw provided vials, mix each vial gently, and aliquot each component separately
into appropriate volumes. Store aliquots at –20°C until the expiration date on the original
vial. Thawed vials may be stored at 2–8°C for up to one week. Do not re-freeze aliquots after
thawing.
B. Transferring Human iPS Cells to the DEF-CS Culture System
It is strongly recommended to transfer cells from other culturing systems to the Cellartis DEF-CS 500
Culture System (Cat. No. Y30010) before editing and single-cell cloning with the Cellartis iPSC rCas9
Electroporation and Single-Cell Cloning System. Human iPS cells maintained in other culture systems
can be readily transferred: fresh cultures can be transferred at passage and cryopreserved cultures can be
thawed directly into the Cellartis DEF-CS 500 Culture System. Cells should be passaged at least five
times in the DEF-CS system prior to using the Cellartis iPSC rCas9 Electroporation and Single-Cell
Cloning System.
Expected Morphology of Human iPS Cells in the DEF-CS System
DEF-CS technology uses enzyme-based passaging in conjunction with a coating to promote single-cell
survival, rapid expansion, and easier passaging. When transferring iPS cells to this system, you will
notice that some cell characteristics differ from those of iPS cells cultured in your previous system. In
contrast to commonly used colony-based culture systems, the DEF-CS culture system yields a monolayer
of evenly spaced cells. Newly passaged cells grown in the DEF-CS culture system tend to spread out;
however, as cells proliferate, the culture gets more confluent, and cells display typical undifferentiated
stem cell morphology (i.e., high nucleus to cytoplasm ratio, defined borders, and prominent nucleoli).

Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
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V. Complete Experimental Workflow
The Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System provides all materials for generating
and isolating edited clonal hiPS cell lines using CRISPR/Cas9. The workflow for this system (Figure 3) consists
of four main steps:
1. Generation and purification of the sgRNA via in vitro transcription
2. Formation of the rCas9/sgRNA RNP followed by its electroporation into hiPS cells
3. Isolation of edited single cells by FACS or limiting dilution
4. Expansion of the clonal cell lines
Figure 3. Workflow for gene editing of hiPS cells using components of the Cellartis iPSC rCas9 Electroporation and Single-Cell
Cloning System. The purple boxes indicate the sections containing the relevant protocols.
VI. In Vitro Transcription of an sgRNA Containing the Desired Target Sequence
CRISPR/Cas9 gene editing requires a custom sgRNA with a user-designed targeting sequence that is homologous
to the target gene or genomic region of interest. Selecting an appropriate DNA sequence at the target region is
critical for maximizing the potential for efficient cleavage at the target site and for minimizing the likelihood of
non-specific cleavage events. For many applications, it is advisable to design and test several different sgRNAs
against the same genomic target region. Candidate sgRNAs must be produced in sufficient quantity for the
generation of functional rCas9/sgRNA ribonucleoproteins.
A. Protocol Overview
The Guide-it sgRNA In Vitro Transcription Components v2 and Guide-it IVT RNA Clean-Up Kit can be
used to synthesize sgRNAs as follows (Figure 4):
1. Generate a DNA template that contains your sgRNA-encoding sequence under the control of a T7
promoter by performing a PCR reaction with the included Guide-it Scaffold Template and a
primer you design.
2. In vitro transcribe this template with the included Guide-it T7 Polymerase Mix to create an
sgRNA containing your target sequence.
sgRNA
production Cas9/sgRNA
delivery into cells
(electroporation) Single-cell
cloning Expansion of clonal cell lines
Step 1
Step 2
Step 3
Step 4
Guide-it sgRNA
In Vitro
Transcription
Components v2
&
Guide-it IVT RNA
Clean-Up Kit
Cellartis iPSC single-cell cloning DEF-CS
coating, basal medium, and additives
Guide-it
Recombinant Cas9
(Electroporation
Ready)
VI
VII
VIII
IX & X

Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
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3. Purify your sgRNA after digestion with Recombinant DNase I (RNase-Free) using the Guide-it
IVT RNA Clean-Up Kit; measure its concentration using a NanoDrop 2000 spectrophotometer or
equivalent.
4. If you wish to determine cleavage efficiency before proceeding with editing (Figure 4, gray
boxes), we recommend using the Guide-it sgRNA Screening Kit (sold separately).
Figure 4. Workflow for generating sgRNA using components of the Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning
System. Steps 1–3 describe the workflow for using Guide-it sgRNA In Vitro Transcription Components v2 and the Guide-it IVT RNA
Clean-Up Kit to synthesize and purify sgRNAs. The purple boxes indicate the sections containing the relevant protocols. The gray boxes
indicate optional steps of in vitro sgRNA screening.
B. Generating the DNA Template
Guidelines for Designing PCR Primers
Use the following guidelines to design a forward primer to be used in a PCR reaction with the included
Guide-it Scaffold Template to create a DNA template for in vitro transcription of your sgRNA. This
primer should contain the T7 promoter sequence, followed by your sgRNA target sequence, and the
Guide-it Scaffold Template-specific sequence (Figure 5).
Choosing the Correct DNA Target Sequence
Choose the DNA target sequence that will correspond to your actual sgRNA target sequence as
shown in Figure 5, Panel A, according to the following guidelines:
a. The DNA target sequence you choose must end with the proto-spacer adjacent motif
(PAM) sequence, NGG, on its 3’ end. Only DNA sequences that are 20 nucleotides
upstream of a PAM sequence can be used for CRISPR/Cas9.
b. Any target sequence can be used as long as the sequence is followed by the PAM
sequence, NGG. However, to minimize off-target cleavage events, the entire target
sequence (including the PAM) should have at least three base mismatches with any other
non-targeted genomic sequence. Off-target events should be especially low if the
mismatches are in, or adjacent to, the PAM. Most online tools for sgRNA design will
predict off-target sequences for a given sgRNA target sequence. To learn more, visit
http://www.takarabio.com/sgRNA-design-tools.
Use PCR to
generate an
sgRNA-
encoding
template
In vitro
transcribe
sgRNA
from the
template
Purify and
quantify the
sgRNA via
spin column
Use PCR to
create a
DNA
cleavage
template
Perform
cleavage
reactions
with Cas9-
sgRNA
Analyze
cleavage
efficiency
on an
agarose gel
VI.B
VI.C
VI.D
Guide-it sgRNA In Vitro
Transcription Components v2
Guide-it sgRNA Screening Kit (recommended)
Guide-it IVT
RNA Clean-
Up Kit
Step 1
Step 2
Step 3