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  4. iPSC Disease Models: CRISPR-Edited and Stable Transfected

Our in vitro disease model research platform provides genetically edited iPSCs that retain pluripotency and can help facilitate the creation of isogenic cell line disease models.

Induced pluripotent stem cells (iPSCs) provide an ideal in vitro platform for studying disease mechanisms and developing cell therapy approaches, as they can be derived from cells with a singular genetic background and tailored to replicate specific disease phenotypes.

When combined with gene editing techniques, iPSCs can be used to explore the mechanisms of disease, and develop effective new drugs and cell therapies. Cyagen’s iPSC disease model research platform has mature gene editing technologies and stem cell culture systems; it has overcome many difficulties related to iPSC cultivation, genetic modification, and monoclonalization.

Additionally, we provide one-stop preclinical CRO capabilities for developing new drugs and cell therapies, including phenotype analysis, construction and testing of in vitro models for various disease application scenarios, and even drug efficacy (pharmacokinetics/pharmacodynamic) evaluations. You are welcome to contact us at 800-921-8930 or email to animal-service@cyagen.com for free project consultations.

Type Project Delivery Standard Quality Controls (QCs) Turnaround Order
CRISPR Gene Edited Cell Lines (iPSCs) Knockout (KO) 1 monoclonal heterozygous cell line, 2 tubes (10^6/tube), experiment report PCR and sequencing, immunofluorescence 8-12 weeks
Point mutation (PM) 12-18 weeks
Knock-in (KI) 12-18 weeks
Transfected Stable Cell Lines (iPSC) Transfection stable Knockdown expression Transfected stable cell lines, 2 tubes/strain (10^6/tube), including control cell lines, experiment report qPCR, immunofluorescence Cell pool: 9-11 weeks;
Monoclonal: 13-15 weeks
Stable overexpression strain Cell pool: 8-10 weeks + gene synthesis;
Monoclonal: 12-14 weeks + gene synthesis
*Additional QCs & detection services are based on customer needs: karyotyping, off-target analysis, flow cytometry, Western Blot (WB), luciferase, proliferation, apoptosis, cell cycle, migration/invasion, etc. can be provided upon request.
Challenges Cyagen’s solutions
Construction of iPS Cell Colonies: Harsh culture conditions can lead to cells losing pluripotency and differentiating during the editing process. We have 16 years’ of experience in stem cell culture and a mature iPSC editing and culture system. With the culture system from Cyagen Biosciences, we can cultivate ideal iPS cell colonies that are compact internally, uniform in size, and have clear edges.
iPSC Gene Editing: Large genetic differences between iPSCs from different individuals and tissue sources, and the high level of difficulty in performing precise genetic modifications. Cyagen has a well established and stable gene editing platform, with over ten thousand gene editing projects under our belt and rich experience in iPSC projects. We have the following advantages: 1.Smart-CRISPR™ Cell Gene Editing System: It can scientifically design highly efficient gRNAs with low off-target risk. 2.Our proprietary α-donor carrier homology-directed repair (HDR) system: HDR efficiency can reach up to 50%, much higher than the market average, allowing for footprint-free repair. 3.Optimal transfection conditions: By optimizing the transfection conditions, our transfection efficiency is over 50% and iPS cell viability can reach 80%. We choose RNP delivery systems to enhance gRNA cleavage efficiency and transfected cell viability, reducing off-target effects and achieving KO efficiency of up to 90%.
Single Clone Formation: Complicated preparation of stable monoclonal iPSC culture and low cloning rates. With our unique single-cell screening technology, the monoclonal formation rate can reach >30%, and enough positive clones can be obtained through one round of screening.
Design Your Cell Gene Knockout Strategy Instantly With our Smart-CRISPR™

Smart-CRISPRCell Gene Editing System

Comprehensive Gene Editing Technology
We can develop genetically modified cell lines and rodent models of the highest complexity for in vitro/in vivo research, with tens of thousands of successful gene editing projects accumulated.
Optimized iPSC Gene Editing System
We have improved upon all nearly all aspects of iPSC reprogramming: the upgraded delivery vector provides up to 50% HDR efficiency, over 50% transfection efficiency, transfection viability more than 80%, and RNP delivery achieving up to 90% KO efficiency.
Proprietary Smart-CRISPR™ Technology
Cyagen has combined our artificial intelligence (AI)-driven AlphaKnockout Gene Editing Expert System and CRISPR/Cas9 technology to develop the upgraded Smart-CRISPR™ Cell Gene Editing System. Smart-CRISPR™ is more efficient in gene cleavage than CRISPR/Cas9 technology alone and can easily accomplish multiple knockout strategies in cell lines — such as frameshift mutation, fragment knockout, and multiple gene knockout — with an editing efficiency of up to 90% to provide scientific solutions to problems such as protein-positive residues.
Fast Delivery
Transfected stable cell line strain delivery within 8 weeks, gene knockout delivery within 8 weeks, and gene knock-in delivery within 12 weeks.
Professional Project Management
Our team aims to respond within 24 hours for project plan inquiries, providing clients with regular updates on project progress, technical support from PhD teams, comprehensive delivery reports, and the offering ability to deliver different clones (homozygous, heterozygous, and control) cells as required.
① Establishment of iPSC-EGFP-KI cell model
② Establishment of iPSC-hTNFAIP8L2-KO Cell Model

Using CRISPR/Cas9 gene-editing technology, an EGFP fluorescent marker (700 bp) was inserted into a gene in iPSCs. As shown in the figure, the sgRNA was designed upstream and downstream of exon E2, and the Donor sequence, which contained the EGFP, was also designed. The sgRNA and Donor were transfected into the iPSCs using the RNP method, and the EGFP sequence was inserted into the latter part of the exon E2 sequence through the homology-directed repair (HDR) pathway.

Figure 1: Design of EGFP knock-in scheme

After identification with PCR and sequencing, it was determined that the heterozygous iPSC cell line with EGFP inserted after the exon E2 sequence was obtained. After cell culture, the karyotyping was performed using G-banded chromosome analysis (G-banding), showing that the chromosome number was normal with 46 chromosomes and no obvious structural abnormalities. Through immunofluorescence staining, the expression of the three pluripotency (stemness-associated) marker genes NANOG, OCT4, and SOX2 was detected and positive signals were observed, indicating that the gene edited knock-in cells have pluripotency.

WT:2430bp; KI:3213bp

Note: The primer design strategy is to amplify the entire EGFP sequence and part of the upstream and downstream genomic sequence. The band pattern without EGFP insertion is 2430 bp, and the successful EGFP insertion should result in a band at 3213 bp. Results show that 8 single clones (numbered 1 to 8) have bands at both 2430 bp and 3213 bp, indicating that these clones have successfully inserted EGFP and are heterozygous clones.

Figure 2: Result of iPS-EGFP KI agarose gel electrophoresis identification

KI band 5' sequence:
KI band 3' sequence:

Note: The KI band 5' sequence displays the sequence of the 5' end of the EGFP insertion fragment and part of the upstream genomic sequence; the KI band 3' sequence is the 3' end of the EGFP insertion fragment and part of the downstream genomic sequence. The sequencing analysis of the KI band 5' sequence and the KI band 3' sequence both match with EGFP and have overlapping peaks, proving that the clone is a heterozygous clone of the EGFP insertion fragment.

Figure 3: Result of iPS-EGFP KI sequencing

Figure 4: Karyotype analysis of iPS-EGFP (Chromosome analysis was performed on the cells after cell culture using G-banding, showing that the number of chromosomes is 46, which is normal, and the chromosome structure has no obvious abnormality.)

Figure 5: iPS-EGFP Immunofluorescence Staining for Pluripotency Markers (Immunofluorescence staining was performed to detect NANOG, OCT4, and SOX2, three pluripotency marker genes, and positive signals were detected for all three markers, indicating that the transfected cells possess pluripotency).

The Human TNFAIP8L2 gene was knocked out in iPSCs using CRISPR/Cas9 gene editing technology. Two sgRNAs were set in exon 2 of TNFAIP8L2 to delete 300bp of exon 2 by small fragment deletion method. The homozygous IPS cells with TNFAIP8L2 gene knockout were confirmed by PCR and sequencing. The pluripotency of the knockout cells was further confirmed by detecting positive signals of the pluripotency markers NANOG, OCT4 and SOX2 through immunofluorescence staining.

Figure 1: Strategy for deletion of hTNFAIP8L2

Figure 2: iPSC-hTNFAIP8L2-KO sequencing detection

Figure 3: iPSC-hTNFAIP8L2-KO pluripotency detection