iPSC Disease Models: CRISPR-Edited and Stable Transfected

Type	Project	Delivery Standard	Quality Controls (QCs)	Turnaround
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

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

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.

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.

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.

Marker

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:

AGGCAGAAACAGAAAAGAATGAAATATTCCGCCGGCAT--TGGAAGCGGAGCCACGAACTTCTCTCTGTTAAAGCAAGCA

KI band 3' sequence:

CGCCGGGATCACTCTCGGCATGGACGAGCTGTACAAGTAA--AAGACTCTTGGCCTCTCCAGAGACGCCCCTTTCCTCGTCC

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.

Smart-CRISPR^™Cell Gene Editing System

Figure 1: Design of EGFP knock-in scheme

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

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).

Figure 1: Strategy for deletion of hTNFAIP8L2

Figure 2: iPSC-hTNFAIP8L2-KO sequencing detection

Figure 3: iPSC-hTNFAIP8L2-KO pluripotency detection

iPSC Disease Models: CRISPR-Edited and Stable Transfected

Smart-CRISPR™Cell Gene Editing System

Figure 1: Design of EGFP knock-in scheme

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

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).

Figure 1: Strategy for deletion of hTNFAIP8L2

Figure 2: iPSC-hTNFAIP8L2-KO sequencing detection

Figure 3: iPSC-hTNFAIP8L2-KO pluripotency detection

Smart-CRISPR^™Cell Gene Editing System