• Stem Cell Genome Editing

RACE™ High-throughput iPSC Genome Editing Service

Rapid Automated Cell Line Editing (RACE™) in iPSCs! Engineer predictive cell line models in the more physiologically relevant iPSC lines. After >11 years’ genome editing and stem cell expertise, & having engineered 500+ unique cell line models, Applied StemCell offers you the best CRISPR-iPSC service with:

  • Up to 60% faster turnaround times than traditional protocols
  • High success rate (>98%)
  • Your patient iPSC lines or our master iPSCs
  • Automated & efficient CRISPR & single cell cloning protocols
  • Pluripotency maintained throughout genome editing

 Any type of modification to suit your needs: Complex and mainstream genetic modifications. And, one of the few providers for integrated upstream iPSC generation & downstream differentiation services.

 

Products and Services
Support Materials
Application Notes

Towards Generating Allogenic/ Immunocompatible iPSCs

Read the 2019 Nature Experimental & Molecular Medicine paper which cites Applied StemCell’s CRISPR-iPSC Genome Editing service to generate HLA-B gene knockout (KO) in a human iPSC line. We successfully generated HLA-B knockout in these cell lines which continued to express HLA-A, while maintaining their pluripotent stem cell-like characteristics. The authors observed that the HLA-B-KO cell lines exhibited less immunogenicity as compared to the isogenic, parental control line in HLA-targeted complement-dependent cytotoxicity assays.

We can help you genome engineer iPSC lines with multiplexed inactivation and activation of immunogenic genes to advance development of your non-immunogenic, universal/ master iPSC lines for next generation stem cell-based cell replacement therapies.

Technical Details

CRISPR-engineered induced pluripotent stem cells (iPSCs) and their differentiation to many different cell lineages-of-choice provide a very valuable, unlimited and consistent source of physiologically relevant and predictive cell line models for understanding biological mechanisms, disease pathology, and developing cell-replacement therapies. The parental iPSC line can be used as an isogenic control for quality research and reliable interpretation of results. This is a huge advantage over using primary cells which are: (1) hard-to-source; (2) have large genetic variability; (3) and batch-to-batch variability in quality of cells.

While both the iPSC and CRISPR technologies have advanced considerably in recent years, handling of iPSCs throughout the genome engineering process requires a special skill set for accurate genetic modification while maintaining the health and pluripotency of the cell lines.

That is where Applied StemCell’s (ASC) special expertise lies! We were one of the first CRISPR-iPSC custom service providers, and over the years we have optimized and evolved our iPSC service offerings to become of the premier CRISPR-iPSC service providers.

In fact, Applied StemCell is a recognized leader in stem cell and genome editing technologies, and is a member of the National Institute of Standards and Technology (NIST) Genome Editing Consortium.

Why Choose ASC’s CRISPR-iPSC Services?

technical-service-stemcell-genomeediting-graph

  • Faster turnaround times: Up to 60% faster than conventional processes
  • High success rate: >98% projects completed to customer’s specifications
  • Automated processes for consistency and high throughput scalability
  • Single cell cloning (clonal isolation) offered as standard service milestone to provide homogenous population with desired genotype
  • Pluripotency maintained throughout genome editing process using high-end cell culture reagents and protocols

All our protocols use our several years of expertise in stem cell genome editing and CRISPR, and are optimized for higher efficiency and accuracy.

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%

We can engineer/ correct mutations in your control/ disease iPSC lines or choose from one of our well-characterized master iPSC lines derived from cord blood (male: ASE-9109; female: ASE-9110) or fibroblasts (male: ASE-9211; female: ASE-9209) with proven CRISPR gene editing and differentiation potential.

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

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.

START-to-finish Stem Cell Services

schematic-service-stemcell-workflow

We offer a fully customizable one-stop-shop experience! In addition to our CRISPR-iPSC platform, we are one of the few companies that also offer custom upstream services such as iPSC reprogramming and characterization, and downstream differentiation to various cell lineages (neural lineage, T cells, cardiomyocytes, hepatocytes, and more), cell line validation, as well as early-stage preclinical drug screening and toxicity testing. 

Applications

  • Physiologically relevant disease models for hard-to-model diseases (Ex. ALS, muscular dystrophy, Parkinson's disease, Alzheimer's)
  • Differentiate to study mutations in different tissue lineages with an isogenic panel of cell line models
  • Ideal for target drug discovery, drug and toxicity screening

Service Details

  • Provide 1 x 10^6 cells of your iPSC lines or choose a cell line from ASC's well-characterized master cell lines: derived from cord blood (male: ASE-9109; female: ASE-9110) or fibroblasts (male: ASE-9211; female: ASE-9209).
  • Source cells can be fibroblasts, PBMCs, or other biosamples. 

 

Case Studies

Case Study #1: GFP Reporter Knock-in in iPSCs

Goal: The purpose of this project was to genetically introduce a GFP reporter tag into control human iPSCs at a specified locus "A".      

Two gRNA candidates were selected based on the proximity to the knock-in sites and off-target profiles, and functionally validated in a model cell line. The gRNAs that produced the desired NHEJ frequencies were then used for the transfection into the control human iPSC line. Single cell colonies were screened by genotyping

 
Case Studies Figure 2 - iPSC Disease Modeling
Figure 1. PCR genotyping screening of GFP knock-in at locus "A".  Three sets of PCR primers for each knock-in line were designed to amplify PCR fragments flanking left homology region (5 arm), right homology region (3 arm), and reporter gene insertion region (M) (to identify homozygous clones). Both Clone#1 and 2 were homozygous as attested by absence of PCR band (when compared to WT) and further by Sanger Sequencing (not shown).

Case Study #2: Point Mutation Correction of a Mutant Allele in a Human Induced Pluripotent Stem Cell Line

Goal: The goal of this project was to correct a point mutation (single nucleotide polymorphism; SNP) found in a mutant allele of the gene-of-interest in a patient-derived iPSC line.

The point mutation was corrected by co-transfection of CRISPR reagents: Cas9, validated gRNA, and a single stranded oligodeoxynucleotide donor (ssODN) into the iPSCs. The gRNAs were designed based on the proximity to the mutation seen in the mutant allele and functionally validated for optimal NHEJ frequency.  The ssODN was designed to replace the mutation with wildtype sequence using homology directed repair (HDR).  Off-target analysis was also performed for each gRNA (not shown). After transfection, single clones were isolated and genotyped to confirm desired mutation correction. One corrected clone was identified and confirmed by sequencing (Figure 2A and 2B).

A. casestudy-servcice-stemcell-genomeediting-2a-2b
 
 
B.casestudy-service-stemcell-genomeediting-2b

Figure 2. (A) Sequencing chromatogram of corrected clone (CTG > CCG). Red arrow indicates the nucleotide correction from a CTG to CCG. (B) Sequence alignment of corrected clone (bottom) against the parental SNP sequence (top). Two silent mutations were introduced by donor ssODN (CGC > CGT, GGG to GAG).

Case Study #3: Frameshift Knockout Mutation in a Control Human iPSC Line 

Goal: To generate a frameshift knockout (KO) mutation in a control human iPSC line using CRISPR/Cas9 and non-homologous end joining (NHEJ). 

Out of two gRNAs designed to target an early exon to generate the desired frameshift mutation, one gRNA was selected based on functional validation for optimal NHEJ frequency. After transfection and clonal selection, individual colonies were screened for desired frameshift mutation by PCR using primer sets for the region flanking the gRNA cut site (Figure 3A) and then confirmed by Sanger sequencing (Figure 3B).

A. casestudy-service-stemcell-genomeediting-3a

B.casestudy-service-stemcell-genomeediting-3b

Figure 3. (A) PCR genotyping of KO single cell iPSC clones. PCR amplification products for the region flanking the gRNA cut site were run on a 2% agarose gel. The presence of a 551 bp PCR product identified clone #6 as an iPSC line with a 22bp deletion. Lanes containing the 573 bp PCR product do not have deletion and are similar to wildtype (wt); (B) a) Sequence chromatogram: The vertical line in the histogram indicates the new sequence generated by the 22 bp deletion; b).Sequence alignment between KO clone (#6; top) and wildtype clone (wt; bottom).

Additional Case Studies:

 

 
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 investigation128(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 Science55(13), 3068-3068.

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

Have Questions?

An Applied StemCell technical expert is happy to help, contact us today!