• Cell and Gene Therapy Bioservice
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    Cell and Gene Therapy Bioservice

CRISPR/Cas9 Genome Editing in Stem Cells

ASC is one of the first “licensed” service providers of CRISPR/Cas9 and iPSC technologies, and provides the best iPSC disease modeling service on the market.

  • >98% success rate in genetically engineering hundred of iPSC lines

  • iPSC genome editing using healthy/ diseased donors

  • Customizable deliverables: Heterozygous/ homozygous mutations; Point mutations with/ without silent mutations

  • Footprint-free, feeder-free transfection and iPSC culture protocols

  • Use your iPSC lines or any of our fully characterized control iPSC lines derived from cord blood (male: ASE-9109; female: ASE-9110) or male fibroblasts (ASE-9203) that are amenable to CRISPR gene editing

  • Isogenic control lines for reliable experiments

  • Full range of downstream iPSC differentiation and characterization services

Support Materials



To view the following posters, please click on the URL below. /supporting/materials/whitepaper


Engineering of Isogenic Cell Lines Using the CRISPR/Cas9 Technology, and Precise Characterization of Low Allelic Frequency FFPE Cell Line Blocks for Use as Molecular Reference Standards (November 2017)

TARGATT™ and CRISPR/Cas9 modified induced pluripotent stem cells (iPSCs) for in vitro genetic disease modeling (December 2015)

Technical Details

Induced pluripotent stem cells (iPSCs) derived from healthy patients or those with injury or disease provide an unlimited, isogenic source of cell line models, especially since they are amenable to genome engineering using CRISPR/Cas9 and other technologies. An added advantage of using iPSCs is the ability to induce differentiation into specific tissue lineage(s), thereby providing a consistent source of physiologically relevant cell line models for research and screening purposes.


  • 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

A variety of mutations can be engineered:

  • Gene knockout: gene disruption or site-specific large fragment knockout (> 10kb)

  • Gene insertion: reporter gene insertion, small fragment insertions, point mutations

  • Gene correction or replacement

  • Inducible gene expression/ gene overexpression models

iPS Cell Genome Editing - iPSC Disease Modeling

Service details:

  • You need to provide your source cells, iPSCs, or you can start with Applied StemCell’s own iPSCs. Applied StemCell will genetically modify the iPSCs according to your specifications using CRISPR/Cas9 technology.

  • Provide 1 x 106 cells of your iPSC or choose a cell line from ASC's iPSC catalog.

  • Source cells can be fibroblasts, PBMCs, or other biosamples. 

Service Time
1. Cell Line Validation  
Cell recovery, culturing and expansion
2-3 weeks
Mycoplasma test 1 week
Drug kill curve evaluation 1 week
Sequencing targeted region 1 week
Transfection optimization 2 weeks
2. Targeting DNA Vector Construction and Validation  
gRNA design and construction, off target analysis and SNP check, gRNA cloning (up to 4 gRNAs)
1 week
gRNA in vitro validation (up to 4 gRNAs) 2-3 weeks
Donor oligo design and construction 2-3 weeks
3. Transfection of CRISPR/ Cas9 Constructs  
Cell transfection / electroporation
1 week
Drug selection 1 week
NHEJ in pooled, transfected cells 1 week
Cell cloning and expansion 2-4 weeks
4. Cell Confirmation and Expansion  
Genetic screening by PCR and sequencing (up to 200 clones)
2-3 weeks
Positive clone expansion and confirmation by PCR and sequencing (1-2 clones) 2-4 weeks
Cell Cryopreservation 1 week
  16-26 weeks

Applied StemCell offers CRISPR/ Cas9 modifications of other cell lines or complete kits to do it yourself. Other services we offer include reprogramming of patient fibroblasts or blood cells to create patient-specific iPSCs, further differentiation of cells, and phenotype analysis.

Case Studies

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

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

The gRNA candidates for each gene were selected based on the proximity to the knock-in sites and off-target profiles. Each gRNA was cloned into a Cas9 gRNA expression vector, sequence verified, and transfected into a model cell line. They were subsequently harvested for PCR amplification. An in vitro SURVEYOR assay was performed on the PCR product (Figure 1). The gRNAs that produced the desired NHEJ frequencies were then used for the transfection studies (Figure 2). 

Gene A
 Case Studies Figure 1 - IPSC Disease Modeling
Figure 1. SURVEYOR assay for gRNA activity in model (K562) cells. A donor vector was designed to provide the intended modifications via homologous recombination mechanism. The 5’ and 3’ homology arms, together with T2A-GFP-Lox-Puro-Pox fragment were subcloned into a linearized backbone vector, and sequence verified. The gRNA plasmid, donor plasmid, and Cas9 plasmid were transfected into Applied StemCell's episomal human iPSC line. Single cell colonies were screened by genotyping (Figure 2). 
The gRNA plasmid, donor plasmid and Cas9 plasmid were transfected into Applied StemCell's episomal human iPSC line (Cat# ASE-9202). Single cell colonies were screened for genotyping (Figure 2).
Case Studies Figure 2 - iPSC Disease Modeling
Figure 2: 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).

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 found in a mutant allele of a gene in patient derived iPSCs. The point mutation was corrected using CRISPR/cas9 by co-transfection with Cas9/gRNA vectors and a single stranded oligodeoxynucleotides donor (ssODN) into the iPSCs. 

The gRNAs were designed and tested based on the proximity to the mutation seen in the mutant allele and a NHEJ frequency using an in vitro SURVEYOR* assay (Figure 1). The ssODN was designed to replace the mutation with wildtype sequence using homology directed repair. Off-target analysis was also performed for each gRNA.

Case Studies 2 Figure 1 - iPSC Disease Modeling
Figure 1. gRNA in vitro validation. Red arrows indicate positive bands generated by Cas9/gRNAs. Green arrows indicate two background bands in either unmodified iPSC or wildtype HEK293 cells. 

Single clones were genotyped with genomic DNA PCR and then sequenced. One corrected clone was identified and confirmed by sequencing (Figure 2 and 3).

Case Studies 2 Figure 2 - iPSC Disease Modeling
Figure 2:  Sequencing result of corrected clone (CTG > CCG). Red arrow indicates the nucleotide correction from a CTG to CCG.
Case Studies 2 Figure 3 - iPSC Disease Modeling
Figure 3: Alignment of corrected clone sequence (CTG > CCG). Point mutation is the top sequence while the corrected variance is the bottom sequence. Two silent mutations were introduced by donor ssODN (CGC > CGT, GGG to GAG).

Case Study #3: Frameshift Knockout Mutation in a Human Induced Pluripotent Stem Cell Line 

Goal: The goal of this project was to generate a frameshift knockout mutation in Applied StemCell’s human iPS cell line using CRISPR/Cas9 to deliver knockout iPSC clones. 

Applied StemCell’s iPSC line was used for this study (catalog # ASE-9202). The gRNAs were designed to target the early exon (exon 1) to generate the frameshift and were validated similar to case #1 (data not shown). After transfection, the iPSCs were screened and cultured. Isolated individual colonies were picked and allowed to expand.

The genotyping strategy employed gel analysis of PCR amplification products from the exon 1 region straddling the gRNA cut site (See Figure 1). For verification of a frameshift mutation in the gene of interest, clones producing 551 bp PCR product was indicative of a 22 bp deletion of exon and were subjected to sequence analysis (Figure 2).

Case Studies 3 Figure 1 - iPSC Disease Modeling

Figure 1. Genotyping analysis of KO iPSC clones. PCR amplification products straddling the gRNA cut site in exon 1 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).

Case Studies 3 Figure 2 - iPSC Disease Modeling

Figure 2:  Sequence confirmation of KO clone.   a)  Sequence confirmation of clone #6. The vertical line in the histogram indicates the new sequence generated by the 22 bp deletion. b) Sequence alignment between wildtype and clone #6.      

  • 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?

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