iPSC Genome Editing
Custom CRISPR & TARGATT™
Cell Engineering
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:
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.
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 cell gene therapy modalities, including gene therapy, autologous and allogeneic cell therapy.
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, regenerative medicine, or cell-based therapy 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)
Figure 1: Knock-in strategy for 1bp insertion in the AAVS1 locus of the ASE-9211 Master Cell Line.
Genotyping Clone #6
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)
Figure 3: Knock-in strategy for 150bp insertion at the AAVS1 locus of the Master iPSC Line.
Genotyping Positive Clone #21
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
Figure 5. Sequence chromatogram of iPSC line with 1 bp deletion (AAVS1-1bp DEL; bottom) compared to wild type (WT; top).
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
Figure 7. AAVS1 wild type (WT) sequence showing gRNA cut sites and position of 1007 bp (~1000 bp) deletion (sequence in red).
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.
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.
Support Materials
Brochures/ Flyers:
Posters:
Webinar:
Stem Cell Services (iPSC/ESC)
iPSC Genome Editing Rapid Automated Cell Line Editing (RACE™)Publications
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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.
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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).
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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.
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Comley, J. (2016). CRISPR/Cas9 – transforming gene editing in drug discovery labs. Drug Discovery Weekly. Fall 2016; 33-48.