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Knock-In Cell Line
Gene knock-in (KI) is a precise genome editing technique that involves the targeted insertion of exogenous DNA fragments into specific genomic loci, enabling these sequences to fulfill designated biological functions. This method is instrumental in conferring new functionalities to cells or organisms, or in restoring the normal function of defective genes. It plays a critical role in investigating gene function and regulatory mechanisms, constructing disease models, and facilitating advancements in gene therapy.
Principle of HES-KI
HES-KI employs CRISPR editing in the exonic regions of essential genes alongside a specially designed transgenic knock-in template. This strategy ensures that a correct knock-in restores the function of the essential gene while simultaneously integrating the target transgene.
If the knock-in is unsuccessful, NHEJ-mediated insertions or deletions (indels) inactivate the essential gene, rendering the cell non-viable. This naturally selects for cells with successful knock-in events.
Applications of HES-KI
Gene Therapy: Ensures the stable integration of therapeutic genes in cell-based treatments.
Stem Cell Engineering: Demonstrates high-efficiency transgene knock-in in induced pluripotent stem cells (iPSCs).
Functional Genomics: Facilitates the screening of functional domains or regulatory elements of essential genes.
Disease Modeling: Enables the construction of highly pure gene-modified cell lines, such as tumor mutation models.
| Item | HES-KI | Traditional KI Methods |
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| Efficiency | High | Low |
| Cost | Low | High |
| Specificity | High | Low |
| Flexibility | Extensive | Limited |
服务详情
| Cell Types |
Various cell types including tumor cells and stem cells, etc. Click to view the Comprehensive Cell List |
|---|---|
| Services | Fluorescent protein knock-in / Tag protein knock-in |
| Deliverables | Gene knock-in monoclonal cell line: 1 clone (2 vials, 1×10^6 cells per vial) |
| Turnaround/Price |
 Consult online for details |
EDITGENE has developed an innovative and highly efficient gene knock-in technology leveraging an upgraded CRISPR/Cas9 system. With over a decade of gene editing expertise, EDITGENE has optimized gRNA and homology arm design strategies, delivering higher positive rates and broader knock-in site flexibility.
EDI-Service Advantages
High Efficiency
HES-KI achieves a 68% knock-in efficiency across various cell types, substantially outperforming existing techniques
Broad Compatibility
The technology is compatible with both AAV and non-viral DNA templates, reducing dependence on AAV vectors and lowering both cost and manufacturing complexity
By editing essential genes, HES-KI facilitates the natural selection of successfully edited cells, thereby minimizing the risk of off-target insertions
Multiplex Gene Editing
HES-KI enables the simultaneous knock-in of multiple CAR genes, allowing for concurrent targeting of several tumor antigens and reducing the likelihood of tumor escape
Service Types
Customized knock-in strategies can be designed according to client requirements, incorporating specific gene and cell characteristics.
| Fluorescent Protein Knock-in | -EGFP, Luc, mCherry, and more. |
|---|---|
| Tag Protein Knock-in | -Flag, HA, Myc, HiBiT, and others. |
| Precise insertion of specific DNA fragments into targeted genomic loci. | / |
| Targeted knock-in of specific DNA fragments into genomic safe harbor regions. | / |
Workflow
Application Case
According to customer needs, EDITGENE comprehensively considers the conditions of target genes and cells and designs targeted gene knock-in solutions.
● Insert EGFP at the C-terminus of the GAPDH gene in K562 cells
1. Design
2. Results
1）Using HES-KI technology, EGFP was knocked into GAPDH in K562 cells. The knock-in efficiency of polyclonal clones reached 37% without resistance screening
2）Sanger sequencing confirmed the precise insertion of EGFP at the C-terminus of the K562 GAPDH gene
3）There was no significant difference in the doubling time between K562 EGFP-KI cells and WT cells, and EGFP insertion did not affect the growth of K562 cells
4）There were no significant differences in EGFP mRNA expression levels among various K562 EGFP-KI monoclonal cell lines, demonstrating good uniformity across monoclonal populations
5）Images of K562 EGFP-KI cells
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K562 EGFP-KI Polyclonal cells |
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K562 EGFP-KI monoclonal cells |
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●Insert EGFP at the C-terminus of the GAPDH gene in 293T and CHO-K1 cells
1. Design
2. Results
1）Using HES-KI technology, the knock-in efficiency of EGFP in 293T polyclonal cells reached 68%, and in CHO-K1 polyclonal cells, the knock-in efficiency of EGFP reached 55%
2）Sanger sequencing confirmed the precise insertion of EGFP at the C-terminus of the GAPDH gene
293T：
CHO-K1：
3）Images of EGFP-KI polyclonal cells：
| 293T | CHO-K1 | ||
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Advantage and Characteristic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
Optimazied Strategy
We have create a unique sgRNA Design Logic
参考文献
Enhancing CRISPR-mediated homology-directed repair (HDR) efficiency through cell cycle synchronization
Article Title: Modulation of cell cycle increases CRISPR-mediated homology-directed DNA repair
This study explores a method to enhance CRISPR-mediated HDR efficiency by synchronizing the cell cycle. Using small molecules to modulate the cell cycle, researchers achieved a 1.2- to 1.5-fold increase in knock-in efficiency across various cell lines. The study also demonstrated this approach's application in animal embryos, significantly increasing knock-in frequency in pig embryos. This technique improves knock-in success by guiding cells to an HDR-favorable cycle stage, offering a new optimization strategy for CRISPR gene editing.
Knock-in strategy based on CLASH technology
Article Title: CLASH enables large-scale parallel knock-in for cell engineering
The CLASH (Cas9-Linked Adaptor Synthesis for Homology-directed repair) technology enables efficient large-scale gene knock-in for cell engineering. This method combines the Cas9 protein and adaptor synthesis, allowing parallel knock-in across various cell types. By providing specific adaptors during the DNA repair process, it significantly enhances homology-directed repair (HDR) efficiency, thereby increasing knock-in success rates. This technology shows great potential in cell engineering and gene editing, especially for complex bioengineering applications requiring multi-gene modifications.
FAQ
What is the core principle of gene knock-in technology?
Gene knock-in technology involves inserting an exogenous gene sequence into a specific location within the genome for gene function studies or disease treatment. Edigene utilizes advanced gene editing tools, such as the CRISPR/Cas9 system, to guide nucleases to cut the target DNA, and employs homology-directed repair or non-homologous end joining to accurately insert the gene at the desired location, achieving efficient and precise gene knock-in.
What role does gene knock-in play in drug development?
Gene knock-in plays a crucial role in drug development. It is used in target validation by introducing specific genes into cell lines or animal models to confirm drug target efficacy. It also aids in establishing disease models, testing drug efficacy and safety in these models, and supporting drug screening through high-throughput screening in knock-in cell lines to identify potential drug candidates. Additionally, gene knock-in helps uncover drug mechanisms, optimize drug structure, and improve dosing strategies, expediting drug development while enhancing efficacy and safety.
Why choose EDITGENE, and what are EDITGENE’s main advantages in gene knock-in technology?
EDITGENE’s advantages in gene knock-in technology include:
Guaranteed results: With 10 years of CRISPR gene editing experience and a team of PhDs from world-renowned institutions offering one-on-one support.
High precision: EDITGENE’s optimized tools reduce off-target effects, enhancing editing accuracy.
High efficiency: EDITGENE’s technology platform improves knock-in success rates, accelerating experimental progress.
Customized service: Tailored knock-in solutions to meet specific research or therapeutic goals.
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