Gene Knockout Services

CRISPR/Cas9-mediated gene knockout is a core technology in functional genomics and disease model construction. Alfa Cytology offers efficient gene knockout services based on CRISPR/Cas9, enabling complete and stable disruption of target genes across a wide range of cell types. Leveraging our optimized delivery systems, rigorous monoclonal screening strategies, and comprehensive validation at both genomic and protein levels, we deliver high-quality, reproducible gene knockout models to support diverse applications from basic research to drug discovery.

Introduction to Gene Knockout

Gene knockout is a gene editing technology that achieves permanent disruption of target genes by introducing insertions or deletions (indels) into the coding sequence or functional domains, thereby inactivating gene expression or abolishing protein function. This technology is one of the core tools for studying gene function, dissecting signaling pathways, and constructing disease models. In the field of oncology, gene knockout technology plays an irreplaceable role. By knocking out tumor suppressor genes such as TP53, PTEN, and RB1, or negative regulators of oncogenes, researchers can establish cell lines or mouse models that recapitulate key genetic events during tumor initiation and progression, providing critical insights into the core molecular mechanisms driving oncogenesis. Furthermore, whole-genome CRISPR knockout screening enables systematic identification of genes that regulate tumor proliferation, metastasis, immune evasion, and drug resistance, offering robust support for the discovery and validation of novel therapeutic targets. This technology is also widely employed to generate drug-resistant cell models by knocking out genes associated with drug sensitivity, facilitating the elucidation of resistance mechanisms and the screening of combination therapeutic strategies to overcome resistance. Additionally, in the context of cancer immunotherapy, knockout of immune checkpoint genes (e.g., PDCD1, CTLA4) or negative regulatory factors that modulate T cell function can significantly enhance the anti-tumor activity of T cells, laying the foundation for the development of next-generation immunocellular therapeutic products.

Fig. 1 Establishment of clonal TP53/ARID1A knockout human gastric organoid lines. (Lo YH, et al.; 2021)

Our Services

Leveraging our deep expertise in CRISPR gene editing and cell model construction, Alfa Cytology provides end-to-end gene knockout services tailored to meet your specific research needs. Our comprehensive service portfolio covers a wide range of options to accommodate diverse research scenarios and project requirements.

Standard Gene Knockout Services. Our standard service delivers a complete gene knockout solution applicable to a broad range of conventional cell lines (HEK293, HCT116, A375, HeLa, etc.), representing an ideal choice for most basic research and functional validation projects. Services include:

• Efficient sgRNA design targeting your gene of interest, balancing editing efficiency and specificity.
• Optimized transfection or transduction protocols to ensure efficient CRISPR/Cas9 delivery.
• Monoclonal cell screening and expansion to ensure homozygosity and stability of gene knockout.
• Genomic-level validation (Sanger sequencing or T7EI assay) and protein-level validation (Western blot).
• Comprehensive knockout validation report, including sgRNA sequences, sequencing results, and protein expression analysis.

Cell Line Gene Knockout Services

We offer comprehensive solutions ranging from single gene knockout to multiplex gene knockout, large-fragment deletion, and customized knockout, covering conventional cell lines as well as challenging cell types including primary cells, stem cells, and suspension cells.

Mouse Model Gene Knockout Services

Based on CRISPR/Cas9 technology, we provide comprehensive mouse model construction services ranging from single gene knockout to multiplex gene knockout, large-fragment deletion, and conditional knockout, supporting in vivo functional studies and disease model development.

Conditional knockout mice require crossing with tissue-specific Cre mouse strains to achieve gene deletion in target tissues. We provide supporting technical services including Cre mouse strain recommendations, breeding scheme design, and genotyping.

Deliverables

• Experimental design and sgRNA sequence documentation.
• Knockout cell line (including at least 2 independent monoclonal clones) or knockout mouse model.
• Genomic-level validation data (sequencing chromatograms/electrophoresis gels).
• Protein-level validation data (Western blot).
• Comprehensive knockout validation report.
• For cell lines: Cryopreserved cell stocks and quality control report.
• For mouse models: Genotyping data and breeding recommendations.

Applications of Gene Knockout Services

Workflow of Gene Workflow of Gene Knockout Services

Experimental Design and Consultation

Work closely with the client to identify the target gene, cell type, and project requirements. Design and evaluate multiple sgRNA sequences, and select the optimal editing strategy.

Delivery and Editing

Deliver CRISPR/Cas9 components into target cells using optimized transfection or transduction protocols. For RNP delivery, pre-assemble Cas9 protein with sgRNA into ribonucleoprotein complexes and introduce directly into cells.

Monoclonal Screening

Obtain monoclonal cells via limiting dilution or single-cell sorting, and perform preliminary genotypic screening of positive clones using PCR or T7EI assay.

Monoclonal Expansion and Banking

Expand the preliminarily screened positive monoclonal cells, and cryopreserve aliquots to establish a master cell bank, ensuring an adequate supply of low-passage cell reserves for subsequent validation and final delivery.

Knockout Validation

Confirm indel mutations at the genomic level using Sanger sequencing or T7EI assay; verify protein expression depletion at the protein level via Western blot, and provide a comprehensive validation report.

Service Highlights

• High Editing Efficiency: Optimized sgRNA design and delivery systems ensure high knockout efficiency.
• Rigorous Clonal Selection: Standardized monoclonal selection and validation workflows ensure homozygosity and stability.
• Comprehensive Validation System: Dual validation at both genomic and protein levels ensures the reliability of knockout outcomes.
• Broad Cell Coverage: Supports a wide range of cell types, including conventional cell lines, primary cells, and stem cells.
• Cell Line & Animal Model Dual Capability: Supports gene knockout in both cell lines and mouse models, enabling seamless transition from in vitro to in vivo studies.

Stable, homozygous gene knockout models are fundamental tools for functional genomics and disease research. Alfa Cytology's gene knockout services integrate high-efficiency editing technology, rigorous selection strategies, and comprehensive validation systems to deliver high-quality, reproducible knockout models that support your entire research spectrum, from basic mechanistic studies to drug discovery. Contact us today to learn how our gene knockout services can meet your specific research needs.

For research use only.