Knockout Cell Line

Gene knockout technology enables the precise removal or inactivation of specific genes. Developing knockout cell models is instrumental in elucidating gene functions within organisms and has extensive applications in biomedical research, drug target discovery, and other fields. Current gene-editing platforms include CRISPR/Cas9, TALENs, and ZFNs, with CRISPR/Cas9 being the tool of choice due to its high efficiency and ease of use. During the knockout process, a guide RNA (gRNA) complementary to the target DNA sequence directs the Cas9 nuclease to specifically recognize and cleave the target site, resulting in double-strand breaks (DSBs) that induce gene disruption.

Service Details

Cell Line Various cell types including tumor cells, epithelial cells, and stem cells.Click to view the full list of cell lines
Service Single-gene knockout/multi-genes knockout/frameshift mutation/large fragment deletions
Deliverables Gene knockout monoclonal cells: ≥1 clone (2 vials per clone, 1 × 10^6 cells per vial)
Time/Quote   Consult online for details
At EDITGENE, we harness our proprietary EditX™ gene editing platform and an optimized CRISPR/Cas9 system to deliver precise knockout strategies customized for specific genes, cell types, and experimental needs. This ensures the efficient creation of gene knockout cell models that meet exact research objectives.

Knockout Service Advantages

Efficient sgRNA Design
With thousands of projects, EDITGENE's proprietary sgRNA design ensures exceptional gene editing results
High-Performance Cas9 Protein
EDITGENE provides patented, high-activity Cas9 protein that significantly boosts editing efficiency
Efficient Cell Transfection
With well-established systems, EDITGENE offers lentiviral, plasmid, RNP transfection, and more
Worry-Free Cell Screening
3D printing enables precise, efficient selection of positive monoclonal clones by EDITGENE

Knockout Cell Line Services

We design customized knockout strategies tailored to client needs and gene characteristics.
Single-gene Knockout Effective knockout of a single target gene.
Multi-genes Knockout Simultaneous knockout of multiple genes.
Frameshift Mutation sgRNA design targeting the coding region to induce indels that cause frameshift mutations by altering the reading frame, ensuring the indels are not in multiples of three.
Large Fragment Deletion Strategic sgRNA design to enable the deletion of substantial gene segments.

CRISPR Knockout Service Workflow

Gene Knockout Cell Service Case

Based on our customers' needs, EDITGENE carefully considers the specific characteristics of target genes and cells to design precise gene knockout protocols.
 
● Successfully achieved a small fragment knockout of Gene A in Huh6 cells
 
1. Knockout Strategy
In Huh6 cells, two sgRNAs were designed to target the coding region of Gene A, achieving a fragment knockout .
 
 
 
2. Editing Efficiency
① The editing efficiency of sgRNA-1 was 100% (approximately 98% effective).
 
 
 
② The editing efficiency of sgRNA-2 was 68% (approximately 65% effective).
 
 
 
 
3. Sequencing Validation
We successfully achieved the knockout of Gene A, as mutations occurred at the sgRNA target site in the monoclonal cell line, resulting in a 73 bp deletion. This deletion caused a frameshift mutation, leading to premature termination of gene encoding .
 

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

Genetic Reference Book

ATM gene knockout in NGP and CHP-134 NB cell lines
Article Title: ATM depletion induces proteasomal degradation of FANCD2 and sensitizes neuroblastoma cells to PARP inhibitors

Neuroblastoma (NB) is a common pediatric solid tumor characterized by high clinical and prognostic heterogeneity. Despite multiple treatment strategies, tumors in high-risk NB patients exhibit resistance to standard therapies and may progress to metastasis. ATM gene is involved in DNA damage response, and heterozygous deletions or hemizygous mutations of the ATM gene located on chromosome 11q are mutually exclusive in NB tumors. While ATM knockdown has been shown to promote tumor formation in NB cell lines in vitro and in vivo, the connection between ATM and tumor formation or cancer invasiveness remains unclear.
Researchers used CRISPR/Cas9 technology to knockout the ATM gene in NGP and CHP-134 NB cell lines, analyzing cell proliferation and colony formation capabilities, and protein expression related to DNA repair pathways through Western blot. In ATM-knockout cells, stable transfection of FANCD2 expression plasmids was used to overexpress FANCD2. Immunofluorescence microscopy was employed to determine protein expression. The results showed that ATM depletion leads to a decrease in FANCD2 protein levels, and ATM-knockout cells are more sensitive to the PARP inhibitor. Reintroduction of FANCD2 in ATM-knockout cells restored cell proliferation capacity. ATM knockout cells and its relevant findings reveal the role of ATM haploinsufficiency in neuroblastoma and illustrate how ATM inactivation enhances NB cell sensitivity to PARP inhibitor, which is significant for treating high-risk NB patients with ATM gene dosage and cancer progression issues.

Article Title: BRCA1 regulates HMGA2 levels in the Swan71 trophoblast cell line

In early placental development, tumor suppressor genes and oncogenes work together to regulate cell proliferation and differentiation. BRCA1 is a well-known tumor suppressor gene that forms a complex with ZNF350 and CtIP to bind to the promoter region of the HMGA2 gene, preventing its transcription. This regulation has been studied in cancer cells but less so in placental cells.
Researchers used the CRISPR-Cas9 system to knockout the BRCA1 gene in the Swan71 cell line, generating BRCA1-knockout cells. Lentiviral particles with miR-182 overexpression were used to overexpress miR-182 in Swan71 cells. The results showed that BRCA1-knockout cells had significantly higher HMGA2 mRNA and protein levels compared to wild-type cells. miR-182 overexpression led to a decrease in BRCA1 protein levels and an increase in HMGA2 protein levels. BRCA Knockout Cell Lines and its relevant findings demonstrate that BRCA1 plays an important role in regulating HMGA2 levels in trophoblast cells and may be involved in placental development and function by influencing apoptosis, providing new insights into BRCA1's role in placental development.

Article Title: GPER Mediates a Feedforward FGF2/FGFR1 Paracrine Activation Coupling CAFs to Cancer Cells toward Breast Tumor Progression

The fibroblast growth factor (FGF)-fibroblast growth factor receptor (FGFR) signaling axis is a key mediator of interactions between the tumor stroma and cancer cells. FGFR1 activation through translocation, point mutations, or gene amplification can lead to cancer progression. In addition, G-protein-coupled estrogen receptor (GPER, GPR30) has been identified as a receptor mediating estrogen's role in various pathophysiological conditions.
To investigate how GPER mediates communication between cancer-associated fibroblasts (CAFs) and breast cancer cells through the FGF2/FGFR1 signaling axis, researchers used CRISPR/Cas9 gene editing to knockout FGFR1 in the MDA-MB-231 breast cancer cell line. Conditioned media (CM) from estrogen-stimulated CAFs induced the expression of connective tissue growth factor (CTGF) in FGFR1 wild-type (WT) MDA-MB-231 cells and promoted migration and invasion via the FGFR1-ERK1/2-AKT signaling pathway. However, this effect was significantly reduced or abolished in FGFR1-knockout cells. FGFR1 knockout cells have revealed a novel role of GPER in regulating FGF2 expression within the tumor microenvironment. The study confirmed that FGFR1 gene amplification is closely associated with overall survival rates in breast cancer patients, suggesting that FGFR1 could serve as a potential therapeutic target in breast cancer treatment. Furthermore, it elucidates the paracrine activation between cancer-associated fibroblasts (CAFs) and breast cancer cells, providing a theoretical foundation for the development of new therapeutic strategies.

Article Title: Bromodomain protein BRD4 promotes cell proliferation in skin squamous cell carcinoma

Recent studies show that the incidence of skin squamous cell carcinoma (SCC) is rapidly increasing, with this and other non-melanoma skin cancers leading to numerous deaths annually. Over 20% of the global population may develop skin cancer during their lifetime. BRD4 has been proposed as a potential oncogenic protein, but its role in skin SCC remains understudied.
Researchers used CRISPR/Cas9 to directly knockout the BRD4 gene in SCC cells. BRD4-knockout or silenced cells showed a significant reduction in proliferation, and the expression of oncogenes closely related to cell proliferation (e.g., cyclin D1, Bcl-2, MYC) was significantly decreased. In vivo experiments showed that BRD4 knockout significantly inhibited tumor growth in A431 cells in SCID mice. BRD4 knockout cells and its relevant findings suggest that BRD4 could be a therapeutic target in skin squamous cell carcinoma.

FAQ

Are all types of genes suitable for KO cell lines?
Not all genes are suitable for knockout. Some gene knockouts may result in cell death or severe dysfunction, particularly for essential genes. In such cases, conditional knockouts or gene knockdowns (e.g., RNAi) may be used instead.
KO cell lines are used for in vitro experiments, suitable for high-throughput screening and cellular studies, while gene knockout animal models are used for in vivo experiments to study gene functions within an entire organism and its interaction with the environment.
EDITGENE provides access to a comprehensive library of over 4,500 high-quality knockout (KO) cell lines, enabling researchers to save valuable time. Our custom gene knockout services are highly efficient, boasting a high positive rate while minimizing off-target effects. Clients also benefit from personalized, one-on-one support from a team of PhD experts from globally renowned institutions, ensuring top-tier service and results.
KO cell lines can be applied to various cell types, including cancer cells, stem cells, and primary cells, but different cell types may have varying sensitivities to gene editing, and may vary among different cell types. In certain cell types, achieving gene knockout may require optimization of transfection conditions and selection of appropriate gene-editing tools.
KO (Knockout) cell line is a cell line where a specific gene has been completely removed or rendered non-functional through gene editing technologies such as CRISPR-Cas9. These cell lines are critical for understanding gene functions and disease mechanisms.
Researchers use KO cell lines to investigate gene functions by observing the effects of gene deletion on cellular behavior. This helps in understanding the role of genes in various processes like cell growth, metabolism, and signal transduction. KO cell lines are vital for studying diseases like cancer, genetic disorders, and neurodegenerative diseases.
EDGENE

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