Professional technology integration to support the entire R&D process

Gene knockout (KO) cell lines are engineered cellular models in which the function of a specific target gene has been completely ablated using advanced genome editing technologies. This powerful tool allows researchers to investigate gene function, validate drug targets, and model genetic diseases by establishing a direct causal link between gene loss and phenotypic outcome. Utilizing state-of-the-art platforms like CRISPR/Cas9, our service provides reliable, high-efficiency generation of knockout cell lines to accelerate your research in basic science, drug discovery, and therapeutic development.

Gene knockout can be achieved through several established methodologies. While homologous recombination and RNA interference (RNAi) have historical significance, CRISPR/Cas9-mediated genome editing is now the gold standard for creating permanent, specific gene knockouts due to its unparalleled precision and efficiency.

The CRISPR-Cas9 Knockout Principle

The CRISPR-Cas9 system utilizes a designed single-guide RNA (sgRNA) to direct the Cas9 nuclease to a precise location within your target gene, inducing a double-strand break (DSB). The cell primarily repairs this break via the non-homologous end joining (NHEJ) pathway. NHEJ is an error-prone process that often introduces small insertions or deletions (indels) at the break site. These mutations frequently disrupt the reading frame or create premature stop codons, leading to complete loss of functional protein expression and a successful gene knockout.

We guide your project from concept to validated cell line with a rigorous, optimized workflow:

Strategy & sgRNA Design: Our experts analyze your target gene to design and select highly efficient, specific sgRNAs, minimizing off-target effects.

Vector Construction & Delivery: We clone sgRNAs into optimized CRISPR vectors and introduce them into your chosen cell line via advanced methods like electroporation or viral transduction.

Editing & Screening: Post-delivery, cells are screened for indels using PCR-based genotyping (T7E1, Surveyor assay) or Sanger sequencing to identify a successfully edited polyclonal population.

Monoclonal Cell Line Generation: We perform single-cell cloning via limiting dilution or FACS sorting to isolate pure, genetically uniform clonal populations.

Rigorous Validation: Each clonal line undergoes multi-level validation, including:

• Genomic DNA Sequencing: Confirms the precise indel mutation and biallelic disruption.
• mRNA Analysis (qRT-PCR): Verifies the loss of target transcript.
• Protein Analysis (Western Blot, IHC): Confirms the absence of target protein expression.
• Functional Validation: Tailored assays to demonstrate the expected phenotypic change.

Knockout cell lines are indispensable tools across the life sciences:

• Basic Research & Gene Function: Conduct loss-of-function studies to decipher gene roles in cell proliferation, apoptosis, migration, metabolism, and signaling pathways.
• Disease Mechanism & Modeling: Create precise in vitro models of genetic diseases or cancer by knocking out oncogenes or tumor suppressor genes to study pathogenesis and drug resistance.
• Drug Discovery & Target Validation: Validate the essentiality of a potential drug target—if knockout phenocopies drug inhibition, it strongly supports target feasibility. KO cells are also vital for phenotypic screening and biomarker discovery.
• Advanced Therapeutics: Engineer cells for CAR-T and CAR-NK therapies by knocking out genes like *PD-1* (to enhance potency) or TCR (to improve safety for allogeneic use).

• Expertise & Experience: Deep knowledge in cell biology and a proven track record across diverse cell types and genes.
• Advanced Platform: Utilization of optimized CRISPR-Cas9 systems and delivery methods for maximum efficiency.
• End-to-End Solution: Full-service support from design to delivery of fully characterized, ready-to-use monoclonal cell lines.
• Fast & Reliable Workflow: Established, efficient processes to accelerate your project timeline.
• Stringent QC: Comprehensive validation at every step ensures high-quality, publication-ready results.

Gene knockout cell line technology is a foundational pillar of modern biology, enabling definitive causal inference from genotype to phenotype. Whether your goal is to unravel a fundamental biological mechanism, validate a promising drug target, or engineer the next generation of cell therapies, a precisely engineered knockout model is the critical first step.

Contact us today to discuss your specific gene knockout project and receive a detailed proposal. Let our expertise in precision genome editing become the cornerstone of your next discovery.

Contact Email: sales@reqbio.com