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Products >  Stem_Cell_Research >  Gene_Editing >  Human_iPS_Cell_Editing_Systems

Start-To-Finish hiPSC Editing and Single-Cell-Cloning Systems

Human induced pluripotent stem cells (hiPSCs) are a powerful tool for studying and modeling disease due to their ability to be expanded, renewed, and differentiated into multiple cell types. Moreover, human iPS cells can recapitulate disease phenotypes and genotypes depending on the individual from which they are derived. Combining CRISPR/Cas9-based gene editing with hiPSCs allows the study of specific genetic alterations that can influence function in isogenic cell lines, as edited hiPSCs are a renewable source of diseased and healthy cells with no genetic variability.

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.

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). Direct delivery of Cas9/sgRNA RNPs 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).

Once the Cas9/sgRNA RNP complexes have been delivered, single cells must be isolated and expanded into clonal cell lines in order to isolate and screen for the genotype of interest. Traditionally, the establishment of a clonal population from edited 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 Cellartis gene editing and single-cell cloning systems contains a defined 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 (Asplund et al. 2016), a monolayer-based culture system, bypasses the challenges of colony-based culture by allowing single-cell passaging and promoting survival and further expansion of plated single cells.

The Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit (Cat. # Y30021) is a complete system for efficient expansion and scale-up manufacturing of hiPSCs in a feeder-free and defined environment. The kit contains all the necessary reagents to go from seeding single hiPSCs into 96-well plates to expansion of clones into 48-well plates. Following clonal expansion, hiPSCs can continue to be cultured using the Cellartis DEF-CS 500 Culture System (Cat. # Y30010).

The Cellartis iPSC rCas9 Electroporation and Single-Cell Cloning System (Cat. # 632643) is a complete system that allows efficient gene editing of hiPSCs via electroporation followed by single-cell cloning and expansion into 48-well plates. This system provides recombinant Cas9, as well as all the necessary reagents to produce high yields of sgRNAs, for electroporation into hiPSCs. It also includes components from the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit (discussed above) for performing single-cell cloning post-editing. Following clonal expansion, hiPSCs can continue to be cultured using the Cellartis DEF-CS 500 Culture System (Cat. # Y30010).

The Cellartis iPSC CRISPR/Cas9 Gesicle and Single-Cell Cloning System (Cat. # 632642) is a complete system that allows efficient gene editing of hiPSCs via gesicle delivery of Cas9/sgRNA complexes followed by single-cell cloning and expansion into 48-well plates. Gesicles are a non-toxic and efficient alternative to electroporation that do not rely on costly equipment or consumables. This system provides all the necessary reagents to produce cell-derived nanovesicles, called gesicles, that deliver Cas9 and sgRNA to hiPSCs. It also includes components from the Cellartis iPSC Single-Cell Cloning DEF-CS Culture Media Kit (discussed above) for performing single-cell cloning post-editing. Following clonal expansion, hiPSCs can continue to be cultured using the Cellartis DEF-CS 500 Culture System (Cat. # Y30010).

  At-A-Glance   Documents   Images & Data   Resources

Features

  • Highly efficient gene editing in hiPSCsuse either electroporation or gesicles to deliver Cas9 protein and sgRNA with no genomic integration and reduced off-target effects
  • Superior single-cell survivaledited hiPSCs exhibit high (typically ~50%) survival when seeded as single cells in a 96-well plate
  • Maintenance of pluripotency after editing and during single-cell cloninghiPSCs maintain high levels (>90%) of pluripotency markers Oct-4, TRA-1-60, and SSEA-4
  • Stable karyotype from start to finish—maintain a normal and stable karyotype throughout editing, single-cell cloning, and expansion
  • Flexibility to perform gene editing experiments your way—choose from complete kits that provide editing and single-cell cloning solutions using either electroporation-based (Cat. # 632643) or gesicle-based (Cat. # 632642) CRISPR/Cas9 techniques, or utilize the culture media kit (Cat. # Y30021) to perform efficient single-cell cloning following your own editing experiments
  • Continuous use of DEF-CS technology throughout gene editing/single-cell cloning experiments—use the Cellartis DEF-CS 500 Culture System (Cat. # Y30010) before and during gene editing experiments, then during scale-up after single-cell cloning experiments

Applications

  • CRISPR/Cas9-mediated gene editing of hiPSCs
  • Single-cell cloning of hiPSCs
  • Disease modeling

References

Asplund, A. et al. One Standardized Differentiation Procedure Robustly Generates Homogenous Hepatocyte Cultures Displaying Metabolic Diversity from a Large Panel of Human Pluripotent Stem Cells. Stem Cell Rev. Reports 12, 90–104 (2016).

Jinek, M. et al. A Programmable Dual-RNA-Guided DNA Endonuclease in Adaptive Bacterial Immunity. Science 337, (2012).

Kim, S., Kim, D., Cho, S. W., Kim, J. & Kim, J.-S. Highly efficient RNA-guided genome editing in human cells via delivery of purified Cas9 ribonucleoproteins. Genome Res. 24, 1012–9 (2014).

Sander, J.-D. & Joung, J.-K. CRISPR-Cas9 systems for genomic editing, regulation and targeting. Nat. Biotechnol. 32, 347–55 (2014).

Additional Product Information

Please see the product's Certificate of Analysis for information about storage conditions, product components, and technical specifications. Please see the Kit Components List to determine kit components. Certificates of Analysis and Kit Components Lists are located under the Documents tab.


 
 
Products
New Cat. # Product Contents Size Price License Units Select
Y30021 Cellartis® iPSC Single-Cell Cloning DEF-CS™ Culture Media Kit 1 Kit $555.00 License Statements
632642 Cellartis® iPSC CRISPR/Cas9 Gesicle and Single-Cell Cloning System 1 System $1,748.00 License Statements
632643 Cellartis® iPSC rCas9 Electroporation and Single-Cell Cloning System 1 System $1,689.00 License Statements
 

 

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