• iPSC Disease Modeling with CRISPR
    Genome Editing
    • Single Cell Cloning, Homozygous or Heterozygous
    • Downstream iPSC Differentiation
      iPSC Disease Modeling with CRISPR<br/>Genome Editing

    High-throughput iPSC Genome Editing Service

    Applied StemCell (ASC) has provided stem cell and genome editing services for over 12 years, and we have worked with researchers all across the globe to engineer over 500 unique cell line models. As one of the earliest providers of CRISPR/Cas9 genome editing services, ASC has the experience and optimized protocols for Rapid Automated Cell Line Editing (RACE™) in induced pluripotent stem cells (iPSCs)!

    With our well-established high-throughput protocols, ASC's experts can produce any complex or mainstream genetic modification in your healthy or diseased iPSCs for your basic research, disease modeling, tissue engineering, regenerative medicine, or cell-based therapy research. Leverage our affordable, customizable iPSC genome editing service to obtain your genetically modified iPSCs in just a few weeks.

    technical-service-stemcell-genomeediting-graph

    • High success rate: >98% projects completed to customer’s specifications

    • ASC can genetically modify your healthy or diseased iPSCs; control lines are available

    • Single cell cloning (clonal isolation) 

    • Homozygous or Heterozygous 

    • Automated processes for consistency and high throughput scalability

    • Pluripotency maintained throughout genome editing process using high-end cell culture reagents and protocols

    • Fast turnaround time: 6-8 weeks when you select one of the ASC control lines, 3-4 months when you send in your iPSCs

    ASC is a one-stop-shop for all your iPSC service needs. We are one of the few providers of integrated upstream iPSC generation & downstream differentiation and assay development servicesIf you are looking to engineer iPSCs in a GMP setting, we invite you to read more about our new GMP-grade iPSC service offerings

    Applied StemCell's iPSC Genome Editing Service Compared to Other Providers

      ASC Other Providers
    Final Deliverables Isogenic Clones with Detailed QC Mostly pool
    Choice of Zygosity Flexible Fixed Standard
    Types of Gene Modifications Flexible Fixed Standard
    Starting iPSC Lines Customer’s, ASC’s Master Lines Mostly Customer’s only
    Donor Genetic Background Normal, Disease-specific Limited to Healthy
    QC for Final Product Standard, Flexible Mostly Standard
    Downstream Assays Available Mostly Limited
    Timeline for Projects 2-3 months 5-6 months
    Products and Services
    Technical Details

    Automated High-throughput Protocols for the Genetic Modification of Your iPSC Line of Choice

    We can engineer your control and disease iPSC lines or choose from one of our well-characterized master iPSC lines derived from fibroblasts (male: ASE-9211; female: ASE-9209), cord blood, or PBMCs, which have proven CRISPR gene editing and differentiation potential.

    Faster Timelines with Automated High-throughput Protocols

    Project type

    Conventional Protocols

    ASC’s Optimized High-throughput Protocols

    Improvement in Delivery Times

    Knockout (KO)

    12-20 weeks

    6-8 weeks

    60%

    Point Mutation

    (Single Nucleotide Polymorphism or Variant)

    12-20 weeks

    6-8 weeks

    60%

    Knock-in

    (Reporters/ Tags, Large Transgenes)

    12-24 weeks

    10-15 weeks

    20-30%

     


    A Variety of Other Modifications: Standard & Complex

    Correct/engineer mutations or introduce a variety of genetic modifications in iPSCs:

    • Gene knockout: gene disruption or site-specific large fragment knockout (>10kb)
    • Gene insertion: reporter gene/ tag insertion, small fragment insertions, SNV/ point mutations
    • Inducible gene expression/ gene overexpression models
    • Gene fusion (translocation, inversion, etc)

    Don't limit yourself to only the standard modifications. Ask us about: Multiplexed genome editing; conditional knock-in, gene fusion, and other models that you would like for your projects.


    And…. We offer Customized Deliverables

    • Choice of heterozygous or homozygous mutations
    • Footprint-free genome editing – Ex. Single nucleotide variant (SNV; point mutation) engineering without silent mutations for regulatory compliance
    • Specific genetic or safe harbor locus
    Case Studies

    Applied StemCell (ASC) became a member of the National Institute of Standards and Technology (NIST) Genome Editing Consortium in 2018. Its “Master” iPSC Line is being utilized to develop measurements and standards for genetically engineered cells that can be potentially be used by gene and cell therapy, NGS, biotechnology, and diagnostic companies as well as other key industry players as benchmark data.

    ASC is a leading induced pluripotent stem cell (iPSC) service provider that has over 12 years of expertise in iPSC generation, characterization, precise genome editing, differentiation, and analysis in both cells and animals. With our well-established iPSC platform, we have shared our expertise and completed a series of CRISPR-iPSC genome editing projects for the NIST program.  ASC designed and executed various insertion and deletion projects using our well-characterized Master iPSC Line, ASE-9211. Our scientists have confirmed the desired mutations and delivered the genetically modified iPSCs, and we expect to see additional characterization data from the other members in the near future.

    NIST Projects:  

    ASC provided its proprietary iPS cell line, ASE-9211 as the starting material for all projects. Single cell cloning, screening, and genotyping by PCR and sequencing was completed by ASC.

    CRISPR Knock-In Projects

    Project 1:

    Goal: Knock-in of 1 bp at the AAVS1 locus using the ASE-9211 Master iSPC Line by CRISPR/Cas9 technology

    Knock-In Strategy for AAVS1 (1bp insertion)

    Knock-In Strategy for AAVS1 (1bp insertion)

    Figure 1: Knock-in strategy for 1bp insertion in the AAVS1 locus of the ASE-9211 Master Cell Line.

     

    Genotyping Clone #6

    Genotyping Clone #6 - 1bp CRISPR

    Figure 2: Sequencing chromatogram of iPSC line with 1bp insertion in the AAVS1 locus (top: Clone #6) compared to the Parent line, ASE-9211 (bottom).


    Project 2:

    Goal: Knock-in of 150bp at the AAVS1 locus using the ASE-9211 Master iPSC Line by CRISPR/Cas9 technology

    Knock-In Strategy for AAVS1 (150bp insertion)

    Knock-In Strategy for AAVS1 (150bp insertion)

    Figure 3: Knock-in strategy for 150bp insertion at the AAVS1 locus of the Master iPSC Line.

    Genotyping Positive Clone #21

    Genotyping Positive Clone #21 - 150 bp CRISPR

    Figure 4:  Sequencing chromatogram showing the ~150bp insertion at AAVS1 locus.


    CRISPR Knockout Projects

    Project 3:

    Goal: 1bp deletion in the AAVS1 locus using the ASE-9211 Master Cell Line by CRISPR/Cas9 technology

    CRISPR  iPSC line with 1 bp deletion

    Figure 5. Sequence chromatogram of iPSC line with 1 bp deletion (AAVS1-1bp DEL; bottom) compared to wild type (WT; top).

    1 bp deletion iPSC - CRISPR

    Figure 6. Sequence alignment between the 1 bp deletion iPSC line (AAVS1-1bp DEL; bottom) and wild type (WT; top).


    Project 4:

    Goal: 1000bp Deletion in the AAVS1 locus using the ASE-9211 Master Cell Line by CRISPR/Cas9 technology

    Cut sites - CRISPR iPSC

    Figure 7. AAVS1 wild type (WT) sequence showing gRNA cut sites and position of 1007 bp (~1000 bp) deletion (sequence in red).

    ~1000 bp deletion sites (CRISPR iPSC)

    Figure 8. AAVS1 WT chromatogram showing sites of ~1000 bp deletion (sequence in red). Top: Sequence for 5’ deletion site; Bottom: Sequence for 3’ deletion site.

    ~1000 bp deletion in the AAVS1 locus - CRISPR iPSC

    Figure 9. Sequence chromatogram of iPSC line with ~1000 bp deletion in the AAVS1 locus.

    Only a few NIST projects are listed, if you would like to learn more, contact us today.

    Publications
    • Ilic, D. (2019). Latest developments in the field of stem cell research and regenerative medicine compiled from publicly available information and press releases from nonacademic institutions in October 2018. Regenerative medicine, 14(2), 85-92.
    • Simkin, D., Searl, T. J., Piyevsky, B. N., Forrest, M., Williams, L. A., Joshi, V., ... & Penzes, P. (2019). Impaired M-current in KCNQ2 Encephalopathy Evokes Dyshomeostatic Modulation of Excitability. bioRxiv, 538371. https://doi.org/10.1101/538371
    • Jang, Y., Choi, J., Park, N., Kang, J., Kim, M., Kim, Y., & Ju, J. H. (2019). Development of immunocompatible pluripotent stem cells via CRISPR-based human leukocyte antigen engineering. Experimental & Molecular Medicine, 51(1), 3.
    • Lizarraga, S. B., Maguire, A. M., Ma, L., van Dyck, L. I., Wu, Q., Nagda, D., ... & Cowen, M. H. (2018). Human neurons from Christianson syndrome iPSCs reveal allele-specific responses to rescue strategies. bioRxiv, 444232.
    • Tanaka, H., Kondo, K., Chen, X., Homma, H., Tagawa, K., Kerever, A., ... & Fujita, K. (2018). The intellectual disability gene PQBP1 rescues Alzheimer’s disease pathology. Molecular Psychiatry, 1.
    • Selvan N., George, S., Serajee, F. J., Shaw, M., Hobson, L., Kalscheuer, V. M., ... & Schwartz, C. E. (2018). O-GlcNAc transferase missense mutations linked to X-linked intellectual disability deregulate genes involved in cell fate determination and signaling. Journal of Biological Chemistry, jbc-RA118.

    • Chai, S., Wan, X., Ramirez-Navarro, A., Tesar, P. J., Kaufman, E. S., Ficker, E., ... & Deschênes, I. (2018). Physiological genomics identifies genetic modifiers of long QT syndrome type 2 severity. The Journal of clinical investigation, 128(3).

    • Seigel, G. M., et al. (2014). Comparative Analysis of ABCG2+ Stem-Like Retinoblastoma Cells and Induced Pluripotent Stem Cells as Three-Dimensional Aggregates. Investigative Ophthalmology & Visual Science, 55(13), 3068-3068.

    • Comley, J. (2016). CRISPR/Cas9 - transforming gene editing in drug discovery labs. Drug Discovery Weekly. Fall 2016; 33-48.

    FAQs
    Is the gene editing process feeder independent?
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    An Applied StemCell technical expert is happy to help, contact us today!

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