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
To view the following posters, please click on the URL below. /supporting/materials/whitepaper
- WPC 2017 (#1) - Combined CRISPR - TARGATT™ Gene Editing Generates High Efficiency Knock-out, Point Mutation and Large Fragment Knock-in in Human iPS Cells
- ISSCR 2017 - CRISPR/CAS9 Platform Facilitates Efficient and Precise Disease Modeling in Human Induced Pluripotent Cell Line
- AACR 2017 - Using CRISPR/Cas9 to generate isogenic cell lines and reference standards for applications in cancer diagnostics
- Discovery on Target 2016 - Genome Editing in Human iPSCs with CRISPR and TARGATT™ to Generate Cellular Disease Models
- World Preclincal Congress 2016 – CRISPR-TARGATT™ gene editing in iPSC
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)
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
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.
|1. Cell Line Validation|
Cell recovery, culturing and expansion
|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)
|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
|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)
|Positive clone expansion and confirmation by PCR and sequencing (1-2 clones)||2-4 weeks|
|Cell Cryopreservation||1 week|
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 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).
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.
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 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).
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).
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 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.