Stem Cell Genome Editing
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, a bottleneck when using primary cells. An added advantage of iPSCs can be induced to differentiate into specific tissue lineage(s), therefore providing a consistent source of physiologically relevant cell line models for research/ screening purposes.
ASC is one of the earliest “licensed” provider of both CRISPR and iPS technology-based services. With our extensive expertise in both technologies, we our uniquely placed to provide the best CRISPR/ Cas9-based iPSC disease modeling service (> 98% success rate), as well as downstream differentiation and characterization services. We have optimized transfection and culture conditions for iPSCs that provide the healthiest and most robust iPSCs on the market, genetically modified to your specifications.
Unique advantages of ASC's CRISPR-iPSC engineering services:
- Engineered hundreds of iPSC lines with >98% success rate
- Genome editing in iPSCs from healthy and disease donors
- Fully customizable deliverables:
- Heterozygous/ homozygous mutations
- Engineer with or without silent mutations
- Footprint-free, and feeder-free transfection & iPSC culture protocols (>80% transfection efficiency)
- Three control iPSC lines available: cord blood (male: ASE-9109; female: ASE-9110) and male fibroblasts (ASE-9203)
- Suitable for CRISPR-Cas9 genome editing
- Ideal isogenic control lines for engineered and differentiated iPSCs
- Professional project management with biweekly milestone reports and final report
- 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
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
To avail our acclaimed custom iPSC engineering services:
- You provide us with either your source cells or iPSCs, or we can start with our own iPSCs. Applied StemCell will genetically modify the iPSCs according to your specifications using CRISPR/ Cas9.
- Please 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.
iPS Cell Genome Editing
|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, Inc. also offers CRISPR Cas9 modifications of other cell lines or complete kits to do it yourself. We also offer reprogramming of patient fibroblasts or blood cells to create patient-specific iPSCs, further differentiation services, and phonetype 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).
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).
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.
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).
Figure 2: Sequencing result of corrected clone (CTG > CCG). Red arrow indicates the nucleotide correction from a CTG to CCG.
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).
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.
Applied StemCell's published and cited articles:
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.