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iPSC Genome Editing

Custom CRISPR & TARGATT™
Cell Engineering

Homozygous or Heterozygous

Single Cell Cloning

Downstream iPSC Differentiation

Applied StemCell’s (ASC) gene editing platforms offer cutting-edge solutions for precise and efficient genome modification. Leveraging advanced technologies such as CRISPR/Cas9, MAD7, base editing, and ASC’s proprietary TARGATT™™ technology, we are revolutionizing genetic research and therapeutic development. Our comprehensive suite of tools and services ensures high accuracy and versatility in gene editing, facilitating the creation of customized cell lines for a wide array of applications.

Genetic modifications include but are not limited to:

Gene knockout

  • Frame shift
  • Gene disruption
  • Site-specific large fragment deletion

Gene insertion

  • Large or small transgene insertion
  • Reporter / tag knock-in

SNPs

  • Point mutations

Overexpression

  • Conditional or Inducible gene expression
  • Gene overexpression

Gene fusion

  • Translocation
  • Inversion
Ask an Expert

ASC’s Comprehensive, Next-Generation Gene Editing Technologies


While the Nobel-prize winning CRISPR/Cas9 technology has revolutionized gene editing in iPSCs there are still some limitations to the technology. ASC is unique in that we have several proprietary gene editing technologies as an alternative to CRISPR to expand the scope of modalities we can engineer in iPSCs.

Applied StemCell, TARGATT™™

TARGATT™™ is a site-specific genome editing technology that enables single-copy large DNA fragment insertion at a preselected safe harbor locus.  At ASC, we integrated our TARGATT™ system into iPSCs and developed the TARGATT™ Master iPSC Line for site-specific knock-in. The master line contains an “attP” landing pad in the H11 safe harbor locus. When used with an “attB” gene of interest (GOI) vector and integrase expression, the GOI is inserted at the safe harbor locus, which is located in an intergenic region. The knock-in cell line is ideal for gene overexpression, reporter/tag insertion, conditional expression cell line models, and isogenic cell line generation projects. High efficiency, Large transgene insertion (up to 20kb).

  • Unidirectional integration, Site-specific, Stable knock-in
  • Single copy gene integration into safe harbor locus
  • Gene expression from an active, intergenic locus
  • Control “Master” cell line generated from our well-characterized iPSC lines with proven differentiation capability
  • No disruption of internal genes
  • Custom TARGATT™™ Cell Line Generation – our team can integrate the TARGATT™™ system into the cell line of your choice

SSELECT Sselect™ uses integrase variants, obtained by directed evolution, and enables one-step insertion of large size DNA (up to 20kb) into a safe harbor genomic locus via a SSELECT Sselect™ integrase. It is applicable to all autologous, allogeneic, and genomic therapeutic modalities.

 

Accubase

AccuBase technology is developed by Base Therapeutics. The base editor enzyme is a synthetic protein. Within this synthetic AccuBase protein, the deaminase domain is not in contact with non-targeting dsDNA. Guided by sgRNA, the AccuBase protein associates with the target double-stranded DNA, triggering structural alterations within the AccuBase protein. This results in the exposure of the deaminase domain, facilitating base editing. Accubase editing has very high safety profile and low off-target events compared to other CRISPR / base editing technologies.

 

MAD7

Similar to Cpf1 but not Cas9, Mad7 naturally employs a single RNA species to guide it to the target DNA sequence and it creates DNA DSB with sticky ends rather than blunt ends. Mad7 displays a preference for a 5′-TTTN-3′ or 5’ –CTTN-3’ PAM site rather than 5′-NGG-3′, which is preferred by Cas9. Inscripta owns the IP of MAD7.

Add on our custom iPSC Differentiation services to generate isogenic cell line models for drug screening, cell-based assay development, and other applications.

Further along your drug discovery pipeline? We have cGMP compliant iPSC gene editing and associated services for your needs.

Why Choose ASC for Your iPSC Gene Editing Projects?

We have worked with researchers worldwide and have engineered 1,800 unique cell line models. As one of the earliest CRISPR/Cas9 genome editing services providers, ASC has the experience and optimized protocols for Rapid Automated Cell Line Editing (RACE™) in induced pluripotent stem cells. ASC’s experts can produce any complex or mainstream genetic modification in your healthy or diseased iPSCs for basic research, disease modeling, tissue engineering, iPSC-based medicine, or cell-based therapeutic research.

  • Fast turnaround time: As early as 6-8 weeks when you select one of the ASC control lines or 2-3 months when you send in your iPSCs
  • Cutting-edge Technologies: CRISPR, TARGATT™™, MAD7, Accubase gene editing technologies
  • Wide-range of genetic modifications: Gene knockout, knock-in, point mutations, gene fusion, conditional and inducible gene expression and overexpression models and more
  • High success rate: >98% projects completed to customer’s specifications
  • Genetically modify healthy or diseased iPSCs; control lines are available
  • Single-cell cloning (clonal isolation)
  • Customizable deliverables: Choice of Homozygous or Heterozygous mutations; footprint-free  genome editing (ideal for GMP applications
  • Pluripotency maintained throughout genome editing process using high-end cell culture reagents and protocols
  • GMP iPSC Gene Editing Available (link to GMP manufacturing page: this page needs to re-write with more info on GMP iPSC reprogramming, gene editing and differentiateion)

ASC is a one-stop shop for all your iPSC service needs. Along with two genome editing technologies, we are one of the few providers of integrated upstream iPSC generation, downstream differentiation, and assay development services. Contact info@appliedstemcell.com to learn more.

  • Case Studies

    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

    • 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.

MTA Agreement

Material Transfer Agreement

IMPORTANT! PLEASE READ CAREFULLY BEFORE SUBMITTING AN ORDER. THIS IS A CONTRACT.

This Material Transfer Agreement (“MTA”) is between you (“Purchaser”) and the Applied StemCell, Inc. a California company, having its principal place of business at 521 Cottonwood Drive Suite 111, Milpitas, CA 95035 USA (“ASC”). Purchaser must have an approved, current ASC account to place an order. This MTA is effective for a period of five (5) years as of the last date of execution by the parties and governs the purchase and use of all ASC Materials under the terms and conditions set forth below.

TERMS AND CONDITIONS

Definitions

“ASC Material(s)” means materials acquired from ASC as documented on an ASC Sales Order, such as iPS Cell lines.

"ASC Sales Order" means an order submitted for ASC Materials in a form and format as determined by ASC from time to time. "Biological Material(s)" means ASC Materials, Progeny, Unmodified Derivatives and Modifications, either individually or jointly. "Commercial Use" means the sale, license, lease, export, transfer or other distribution of the Biological Materials to a person or entity not party to this MTA for financial gain or other commercial purposes and/or the use of the Biological Material: (a) to provide a service to a person or entity not party to this MTA for financial gain; (b) to produce or manufacture products for general sale or products for use in the manufacture of products ultimately intended for general sale (c) in connection with ADME (Absorption, Distribution, Metabolism and Excretion) testing; (d) in connection with drug potency or toxicity testing (e) in connection with proficiency testing service(s), including but not limited to, providing the service of determining laboratory performance by means of comparing and evaluating calibrations or tests on the same or similar items or materials in accordance with predetermined conditions; or (f) for research conducted under an agreement wherein a for-profit entity receives a right whether actual or contingent to the results of the research. Commercial Use specifically does not include Industry Sponsored Academic Research.

“Contributor(s)” means an organization(s) and/or individual(s) providing original material to ASC for deposit.

"Industry Sponsored Academic Research" means research sponsored by a for-profit organization carried out at a non-profit organization and by the non-profit organization’s employees. "Investigator" means the Purchaser’s principal scientist or researcher using the Biological Material(s). "Modification(s)" mean substances created by Purchaser which contain and/or incorporate a significant or substantial portion of ASC Material. "Progeny" means an unmodified descendant from the ASC Materials, such as virus from virus, cell from cell, or organism from organism. "Purchaser(s)" means the organization purchasing and receiving ASC Material pursuant to this MTA. "Unmodified Derivative(s)" mean substances created by Purchaser that constitute an unmodified functional sub-unit or product not changed in form or character and expressed by the ASC Material provided by ASC. Unmodified Derivatives include, but are not limited to, subclones of unmodified cell lines, purified or fractionated subsets of materials provided by ASC, proteins expressed by DNA/RNA supplied by ASC, or monoclonal antibodies secreted by a hybridoma cell line.

Scope of Use

Subject to the terms of this MTA, Purchaser’s Investigator may make and use the Biological Materials provided to Purchaser by ASC for research purposes only in Purchaser’s Investigator’s laboratory only. The Biological Materials are not intended for use in humans. Purchaser agrees that Biological Materials designated as biosafety level 2 or 3 constitute known pathogens and that other Biological Materials not so designated may be pathogenic under certain conditions. Purchaser assumes all risk and responsibility in connection with the receipt, handling, storage, disposal, transfer and Purchaser’s use of the Biological Materials including without limitation taking all appropriate safety and handling precautions to minimize health or environmental risk. Purchaser agrees that any activity undertaken with the Biological Materials will be conducted in compliance with all applicable guidelines, laws and regulations, and that Purchaser will obtain all permits, licenses or other approvals required by any governmental authority in connection with purchaser’s receipt, handling, storage, disposal, transfer and use of the Biological Materials.

Purchaser shall not distribute, sell, lend or otherwise transfer to a person or entity not party to this MTA the Biological Material, as defined above, for any reason, without ASC’s prior written agreement.

Any Commercial Use of the Biological Material is strictly prohibited without ASC’s prior written consent. Purchaser acknowledges and agrees that Purchaser’s use of certain Biological Material may require a license from a person or entity not party to this MTA, or be subject to restrictions that may be imposed by a person or entity not party to this MTA (“Third Party Terms”). To the extent of ASC’s knowledge of the existence of any such applicable rights or restrictions, ASC will take reasonable steps to identify the same, either in ASC’s catalog of ASC Materials and/or through ASC’s customer service representatives, and to the extent they are in the possession of ASC, ASC shall make information regarding such Third Party Terms reasonably available for review by Purchaser upon request. Purchaser expressly acknowledges that if there is a conflict between this MTA and the Third Party Terms, the Third Party Terms shall govern. Use of the Biological Materials may be subject to the intellectual property rights of a person or entity not party to this MTA, the existence of which rights may or may not be identified in the ASC catalog or website, and ASC makes no representation or warranty regarding the existence or the validity of such rights. Purchaser shall have the sole responsibility for obtaining any intellectual property licenses necessitated by its possession and use of the Biological Materials.

The use permitted under this MTA for Industry Sponsored Academic Research extends only to the academic research carried out at the non-profit organization and the non-profit organization’s employees. Any non-profit Purchaser using the Biological Materials in connection with Industry Sponsored Academic Research agrees to notify the industrial sponsor that any use of the Biological Materials by the industry sponsor will require a separate license from ASC and/or its Contributors and that ASC and/or its Contributors are under no obligation hereunder to license any Biological Materials to any such industry sponsor.

Warranty; Warranty Disclaimer

ASC warrants that (a) cells and microorganisms included in the ASC Material shall be viable upon initiation of culture for a period of thirty (30) days after shipment thereof from ASC and (b) any ASC Material other than cells and microorganisms shall meet the specifications on the applicable ASC Material product information sheet, certificate of analysis, and/or catalog description until the expiration date on the applicable ASC Material’s product label (such thirty (30) day period, or period until the expiration date, referred to herein as the “Warranty Period”). Purchaser’s exclusive remedy, and ASC’s sole liability, for breach of the warranties set forth in this paragraph is for ASC to, at ASC’s sole option, either (i) refund the fee paid to ASC for such ASC Material (exclusive of shipping and handling charges), or (ii) replace the ASC Material. The warranties set forth in this paragraph apply only if Purchaser handles and stores the ASC Material as described in the applicable ASC Material product information sheet. To obtain the exclusive remedy, Purchaser must report the lack of viability or non-conformation to specifications to ASC’s Technical Service Department within the applicable Warranty Period. Any expiration date specified on the ASC Material shipment documentation states the expected remaining useful life, but does not constitute a warranty or extend any applicable Warranty Period. Except as expressly provided above, the ASC Material and any technical information and assistance provided by ASC are provided as-is, without warranties of any kind, express or implied, including but not limited to any implied warranties of merchantability, fitness for a particular purpose, typicality, safety, accuracy and/or non-infringement.

Purchaser acknowledges that the ASC Material and any technical information and assistance provided by ASC are developed and provided exclusively for research purposes, and Purchaser agrees to use the same at its sole risk. Purchaser agrees that ASC and its Contributors will not be liable for any loss, claim or demand made by Purchaser, or made against Purchaser by any other party, due to or arising from the use of the ASC Material by Purchaser, except to the extent permitted by law when caused by the gross negligence or willful misconduct of ASC.

Limitation of Liability

In no event shall ASC or its Contributors be liable for any use of the Biological Material by Purchaser, for any loss, claim, damage, or liability, of whatever kind or nature, which may arise from or in connection with this MTA or the use, handling, storage, or disposal of the Biological Material. ASC’s liability to Purchaser for any claim related to or arising from this MTA or the Biological Material, whether in contract, warranty, tort, or otherwise, shall be limited to the amount paid by Purchaser for the Biological Material. In no event shall ASC be liable for any indirect, special, incidental, consequential, or punitive damages, including without limitation, loss of profits or loss of use, even if ASC has been advised of the possibility of such damages. The limitations of liability set forth in this MTA shall survive termination or expiration of this MTA for any reason.