Takara Bio Cellartis iPSC rCas9 User Manual

Electroporation and single-cell cloning system

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Takara Bio USA, Inc.

Cellartis® iPSC rCas9

Electroporation and

Single-Cell Cloning

System User Manual

Cat. No. 632643

(030619)

Takara Bio USA, Inc.

1290 Terra Bella Avenue, Mountain View, CA 94043, USA

U.S. Technical Support:

techUS@takarabio.com

United States/Canada

Asia Pacific

800.662.2566

+1.650.919.7300

Europe

Japan

+33.(0)1.3904.6880

+81.(0)77.565.6999

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Summary of Contents for Takara Bio Cellartis iPSC rCas9

Page 1 Takara Bio USA, Inc. Cellartis® iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual Cat. No. 632643 (030619) Takara Bio USA, Inc. 1290 Terra Bella Avenue, Mountain View, CA 94043, USA U.S. Technical Support: techUS@takarabio.com United States/Canada Asia Pacific Europe...
Page 2: Table Of Contents
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual Table of Contents Introduction ..................................4 List of Components ................................. 6 III. Additional Materials Not Supplied ..........................7 A. Required ..................................7 Recommended ................................7 General Considerations ............................... 8 A. Storage and Handling ..............................8 Transferring Human iPS Cells to the DEF-CS Culture System ..................
Page 3 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.
Page 4: Introduction
(Kim et al. 2014). The editing component of the Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System contains: •...
Page 5: Figure 1. Schematic Of Crispr/Cas9-Based Editing To Create Gene Knockouts And Knockins
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.
Page 6: List Of Components
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.
Page 7: Additional Materials Not Supplied
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.
Page 8: General Considerations
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.
Page 9: Complete Experimental Workflow
& Guide-it IVT RNA Clean-Up Kit 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.
Page 10: Generating The Dna Template
Transcription Components v2 Up Kit 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.
Page 11: Figure 5. Designing A Forward Pcr Primer To Generate A Dna Template For An Sgrna Containing Your Target Sequence
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual Designing a 56- to 58-nt Forward PCR Primer The forward (sense) primer must contain the following four sequence elements, in the order shown in Figure 5, Panel B. a. A T7 promoter sequence plus four extra bases (21 total nt) at the 5’ end of the primer.
Page 12: Performing The In Vitro Transcription (Ivt) Reaction
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual PCR-Amplifying the sgRNA Template For use with the Guide-it sgRNA In Vitro Transcription Components v2. 1. Combine the following components in a 200-µl PCR tube. Briefly vortex and spin down to collect the reagents at the bottom of the tube.
Page 13: Figure 7. Sgrna Yield Over Time
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual Reagent Amount (µl) sgRNA PCR template (from Step B) Guide-it In Vitro Transcription Buffer Guide-it T7 Polymerase Mix RNase Free Water Total NOTE: If you require a higher amount of sgRNA, you can scale up the total reaction size (e.g., to 50 µl) without affecting the quality of the sgRNA.
Page 14: Purifying The Transcribed Sgrna
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual Purifying the Transcribed sgRNA For use with the Guide-it IVT RNA Clean-Up Kit. NOTE: Before purifying your sgRNA, prepare the IVT Wash Buffer by adding 24 ml of 96–100% ethanol.
Page 15: Editing Hips Cells By Electroporation
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual VII. Editing hiPS Cells by Electroporation Once your sgRNA has been prepared, the electroporation protocol can begin. We recommend using the Neon Transfection System or the Amaxa 4D-Nucleofector System to electroporate cells. Our electroporation protocol has been optimized using the Neon Transfection System to electroporate cells from Cellartis Human iPS Cell Line 18 (Cat.
Page 16: Table Ii. Preparation Of Coating Solution For A 48-Well Plate
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual 1. Preparing 48-Well Plates These plates will be used for seeding the electroporated cells. One well of a 48-well plate is needed for each electroporated sample. 1. Dilute the required volume of COAT-1 (not SCC-COAT-1) in D-PBS +/+ prior to use. Make a 1:20 dilution.
Page 17: Preparing The Rcas9/Sgrna Rnp Complex
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual NOTE: For electroporation, each sample requires 1 x 10 cells. However, due to the potential variation of pipette and tip volumes, we recommend preparing 1.5X the necessary volume of cell suspension (i.e., 1.5 x 10 cells) for electroporation with a 10-µl Neon Tip to ensure that there is sufficient volume.
Page 18: Electroporating Cells Using The Neon Transfection System
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual 3. Mix the reaction well by gently pipetting up and down. Incubate using a thermal cycler preheated to 37°C with the following program: 37°C 5 min 4°C hold 4. OPTIONAL: Add donor DNA and keep on ice until use.
Page 19: Single-Cell Cloning Of Edited Cells
Table IV describes a schedule of all media changes (volume and composition) necessary to create clonal lines in 24-well plates that are ready for culture with the Cellartis DEF-CS 500 Culture System. Table IV. Workflow for single-cell cloning and expansion using the Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System.
Page 20: Single-Cell Seeding Into A 96-Well Plate
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual Single-Cell Seeding into a 96-Well Plate To optimize the survival rate and expansion potential during single-cell seeding, use cells that are in an early proliferative state. We recommend starting with a confluent but not dense culture, corresponding to a density of 0.8–1.5 x 10...
Page 21: Table Vi. Preparation Of Medium For Dissociation And Single-Cell Seeding
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual 2. Preparing DEF-CS SCC Medium for Single-Cell Seeding Prepare the appropriate volume of DEF-CS SCC medium by adding DEF-CS GF-1 (dilute 1:333), GF-2 (dilute 1:1,000), and GF-3 (dilute 1:1,000) to Cellartis DEF-CS 500 Basal Medium according to Table VI.
Page 22: Culturing Single-Cell Colonies
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual Culturing Single-Cell Colonies After single-cell cloning, cells will proliferate into emerging colonies (Figure 10) that will be passaged for further expansion during scale-up. Figure 10. A single cell seeded in one well generates an emerging colony.
Page 23: Passaging Cells From The 96-Well Plate To A 48-Well Plate
Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System User Manual Figure 11. Clonal colonies, ready for transfer to larger wells and scale-up. The cells have the typical undifferentiated stem cell morphology (i.e., high nucleus-to-cytoplasm ratio, defined borders, and prominent nucleoli).
Page 24: Scaling Up From The 48-Well Plate
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