Cas9 Stable Cell Line - HEK293
Cat.No. : CSC-RO0152 Host Cell: HEK293
Size: >1x10^6 cells/vial Validation: T7 Endonuclease I assay
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Cell Line Information
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Datasheet
| Cat. No. | CSC-RO0152 |
| Product Type | Cas9 overexpression stable cell line |
| Introduction | Clustered regularly interspaced palindromic repeats (CRISPR)/Cas9 is a gene-editing technology that contains two essential components: a guide RNA (gRNA) to match a target gene, and the Cas9 (CRISPR-associated protein 9) endonuclease which causes a double-stranded DNA break, allowing modifications to the genome via nonhomologous end joining (NHEJ) or homology-directed repair (HDR). |
| Cell Line Information | HEK293-Cas9 cell line is engineered to stably overexpress Cas9 nuclease. The Cas9 nuclease in HEK293-Cas9 cell line has been functionally validated using T7 Endonuclease I assay. In combination with separately transfected sgRNAs, HEK293-Cas9 cell line can be used to efficiently generate targeted genomic modifications including gene knockout, gene knockin, gene mutagenesis, gene tagging etc. It is also an ideal cell line model for sgRNA screening and validation, either individually or in pools. |
| Target Gene | Cas9 |
| Host Cell | HEK293 |
| Applications | 1) CRISPR genome editing, such as gene knockout (KO), gene knockin (KI), gene mutagenesis, gene tagging etc. 2) High-throughput sgRNA screening and validation |
| Quality Control | 1) T7E1 assay 2) Mycoplasma detection |
| Size Form | One vial of frozen cells, typically >1x10^6 cells/vial |
| Shipping | Dry ice |
| Storage | Liquid nitrogen |
| Mycoplasma | Negative |
| Format | One frozen vial containing millions of cells |
| Storage | Liquid nitrogen |
| Safety Considerations |
The following safety precautions should be observed. 1. Use pipette aids to prevent ingestion and keep aerosols down to a minimum. 2. No eating, drinking or smoking while handling the stable line. 3. Wash hands after handling the stable line and before leaving the lab. 4. Decontaminate work surface with disinfectant or 70% ethanol before and after working with stable cells. 5. All waste should be considered hazardous. 6. Dispose of all liquid waste after each experiment and treat with bleach. |
| Ship | Dry ice |
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Background
Case Study
Applications
Cas9 stable cell line A cell line called HEK293 has been genetically modified to express the Cas9 protein, which is essential to the CRISPR-Cas9 genome editing mechanism. The CRISPR (Clustered Regularly Interspaced Short Palindromic Repeats) system in bacteria is the source of the Cas9 gene, which has been modified for application in mammalian cells. The HEK293 cell's genome has integrated the Cas9 gene sequence, enabling constitutive and stable Cas9 protein expression.
Using guide RNAs (gRNAs) as a guide, the Cas9 protein functions as a molecular scissors that can cut DNA precisely at specified target sequences. This feature enables researchers to explore gene function, regulatory components, or disease causes by introducing specific mutations, insertions, or deletions into the HEK293 cell genome. A popular cell line produced from human embryonic kidney cells is HEK293 cells. They grow robustly and are simple to culture in vitro using conventional cell culture methods. In culture plates or flasks, HEK293 cells cling to one another and proliferate as a monolayer. To ensure their viability and proliferation, they are regularly cultivated in growth media supplemented with fetal bovine serum and antibiotics.
The goal of the study was to improve the specificity of the CRISPR/Cas9 system for human treatments, namely in stem cell modification. They evaluated the effectiveness of current Cas9 high fidelity variants in therapeutically relevant contexts and found decreased on-target activity. They discovered a variation, HiFi Cas9 (R691A), through a bacterial screen that maintained high on-target activity with reduced off-target effects. Effective gene targeting in human CD34+ hematopoietic stem cells and primary T-cells was made possible by HiFi Cas9, including the repair of the mutation that causes sickle cell disease. This development could lead to precise genome editing for both medicinal and scientific purposes.
Figure 1. The researchers used GUIDE-seq to empirically identify off-target locations after electroporating gRNA into HEK293 cells that expressed modest amounts of either HiFi Cas9 or WT. R691A HiFi Cas9 showed a notable decrease in off-target regions while preserving a high level of on-target editing. HiFi Cas9 demonstrated an advantage in minimizing off-target events while maintaining on-target efficiency through quantitative evaluation using multiplex PCR. HiFi Cas9 consistently outperformed other high-fidelity Cas9 mutants without sacrificing on-target editing efficiency, achieving 99% or more on-target events in comparison to WT Cas9. (Vakulskas CA, et al., 2018)
Using Creative Biogene's Cas9 Stable Cell Line-HEK293 cell line can improve the above experiments to a certain extent and increase efficiency. These cell lines are optimized to provide stable Cas9 expression, reducing variability and complexity in experiments and improving the accuracy and consistency of gene editing.
1. CRISPR-Cas9 Genome Editing: The Cas9 stable cell line edits its genome precisely by using CRISPR-Cas9 technology.
2. Guide RNA (gRNA) Functionality: Cas9 protein is guided by guide RNAs to particular DNA regions, enabling targeted cleavage of DNA.
3. Gene Function Exploration: Using Cas9, researchers can introduce mutations, insertions, or deletions to alter the way genes function in HEK293 cells.
4. Regulatory Component Analysis: Cas9 stable cells make it easier to investigate the genome's regulatory elements.
5. Disease Cause Investigation: Using HEK293 cells, researchers can employ specific mutations to explore the role of genes in disease pathology.
For research use only. Not intended for any clinical use.
