Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual
(030619)
takarabio.com
Takara Bio USA, Inc.
Page 6 of 24
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)
o
50 µl
Guide-it Scaffold Template (1 ng/µl)
o
350 µl
Guide-it In Vitro Transcription Buffer
o
1 ml
RNase Free Water
o
150 µl
Guide-it T7 Polymerase Mix (33 U/µl)
o
625 µl
PrimeSTAR® Max Premix (2X)
o
100 µl
Recombinant DNase I (RNase-free) (5 U/µl)
•
Guide-it IVT RNA Clean-Up Kit (Cat. No. 632638)
o
13 ml
RNase Free Water
o
3 ml
IVT Binding Buffer
o
50
IVT RNA Clean-Up Spin Columns
o
2 x 6 ml
IVT Wash Buffer
o
50
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)
o
2 x 750 µl DEF-CS GF-1
o
500 µl
DEF-CS GF-2
o
500 µl
DEF-CS GF-3
