Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
takarabio.com
Takara Bio USA, Inc.
Page 9 of 24
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
