Gene Knock-in Services

Precise gene knock-in is an essential technology for studying gene function, constructing disease models, and developing cell therapy products. Alfa Cytology offers gene knock-in services based on CRISPR/Cas9 and proprietary HDR enhancement technologies, enabling precise integration of exogenous sequences at target gene loci. Leveraging our optimized HDR enhancement protocols and rigorous clonal screening workflows, we deliver high-quality, precise gene knock-in models to support a broad spectrum of applications, from basic research to drug discovery and cell therapy development.

Introduction to Gene Knock-in 

Gene knock-in is a gene editing technology that precisely integrates exogenous genes or specific sequences into target loci of the host cell genome, based on homology-directed repair (HDR) or non-homologous end joining (NHEJ) repair mechanisms. This technology enables site-directed replacement, insertion, or modification of gene sequences, and serves as a powerful tool for studying gene regulation, protein function, and constructing disease models. In the field of oncology, gene knock-in technology plays an irreplaceable role. By introducing specific point mutations into tumor-associated genes such as EGFR, KRAS, and TP53, researchers can establish cell lines or mouse models carrying clinically relevant driver mutations to recapitulate common oncogenic mutation backgrounds observed in human tumors, providing an ideal platform for investigating tumorigenic mechanisms, driver gene functions, and drug responses. Furthermore, by inserting fluorescent or reporter genes into tumor suppressor genes, real-time monitoring of gene expression dynamics during tumor development can be achieved, facilitating studies on the tumor microenvironment and metastatic mechanisms. This technology is also widely employed to generate cell models harboring drug-resistant mutations (e.g., EGFR T790M, BCR-ABL T315I) for evaluating the sensitivity of next-generation targeted therapeutics and screening novel strategies to overcome drug resistance. Additionally, knock-in of luciferase reporter genes into tumor cell lines enables the establishment of tumor models suitable for high-throughput drug screening and in vivo imaging-based tracking, significantly accelerating the preclinical evaluation of anticancer agents.

Fig. 1 DSBs repair-mediated knock-in strategies. (Chen X, et al.; 2024)

Our Services

Leveraging deep expertise in CRISPR gene editing and precision genome modification, Alfa Cytology provides end-to-end gene Knock-in services tailored to your specific research needs. Our comprehensive service portfolio encompasses multiple options to accommodate diverse research scenarios and project requirements.

Standard Gene Knock-in Service. Our standard service delivers a complete gene knock-in solution suitable for a broad range of knock-in applications in conventional cell lines (HEK293, HCT116, A375, etc.), representing an ideal choice for most basic research and cell model construction projects. Services include:

• Efficient sgRNA design targeting the locus of interest and donor vector construction.
• Optimized transfection protocols and HDR enhancement strategies to improve knock-in efficiency.
• Single-cell cloning and expansion to ensure homozygous and stable knock-in.
• Genomic-level validation (Sanger sequencing, PCR, and Southern blot) to confirm precise integration.
• Protein-level validation (Western blot or fluorescence imaging) to confirm exogenous protein expression.
• Comprehensive knock-in validation report.

Cell Line Gene Knock-in Services

We provide comprehensive solutions ranging from standard knock-in to point mutation knock-in, reporter gene knock-in, and custom knock-in, covering conventional cell lines as well as challenging cell types including primary cells and stem cells.

For challenging cell types: For primary cells, stem cells, and other cell types with limited editing efficiency, we provide delivery system optimization (RNP electroporation, lentivirus, etc.) and HDR enhancement strategies to achieve efficient knock-in while maintaining cell viability.

Mouse Model Gene Knock-in Services

Based on CRISPR/Cas9 technology, we provide comprehensive mouse model construction services ranging from point mutation knock-in to reporter gene knock-in and conditional knock-in, supporting in vivo functional studies and disease model development.

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

Deliverables

• Experimental design and donor vector construction documentation
• sgRNA sequences and validation data
• Knock-in cell line (including at least 2 independent monoclonal clones) or knock-in mouse model
• Genomic-level validation data (Sanger sequencing / PCR / Southern blot)
• Protein-level validation data (Western blot / fluorescence imaging)
• Comprehensive knock-in validation report
• For cell lines: Cryopreserved cell stocks and quality control report
• For mouse models: Genotyping data and breeding recommendations

Applications of Gene Knock-in Services

Workflow of Gene Knock-in Services

Experimental Design and Consultation

Work closely with the client to determine the knock-in strategy (point mutation / reporter gene / conditional allele), target locus, cell type, and donor vector design.

Donor Construction and Delivery

Construct a donor vector containing homology arms and the exogenous sequence, or synthesize an ssODN donor. Deliver CRISPR/Cas9 components and the donor template into cells using optimized transfection protocols, and incorporate HDR enhancement technologies to improve knock-in efficiency.

Monoclonal Screening

Obtain monoclonal cells via limiting dilution or single-cell sorting, expand the clones, and screen for genotype-positive clones. Confirm precise knock-in using PCR and Sanger sequencing.

Knock-in Validation

Confirm the precision of the knock-in locus at the genomic level using Sanger sequencing or Southern blot; verify exogenous protein expression at the protein level via Western blot or fluorescence imaging, and provide a comprehensive validation report.

Service Highlights

• Proprietary HDR Enhancement Technology: Optimized HDR enhancement strategies significantly improve knock-in efficiency and reduce the proportion of NHEJ-mediated imprecise repair.
• Versatile Application Scenarios: Supports multiple knock-in types, including point mutation, reporter gene, and conditional allele knock-in.
• Cell Line & Animal Model Dual Capability: Supports gene knock-in in both cell lines and mouse models, enabling seamless transition from in vitro to in vivo studies.
• Comprehensive Validation System: Multi-level validation at both genomic and protein levels ensures the accuracy and reliability of knock-in outcomes.

Precise gene knock-in models are essential tools for gene function studies, disease model generation, and cell therapy development. Alfa Cytology's gene knock-in services combine highly efficient HDR enhancement technology, rigorous selection strategies, and comprehensive validation systems to deliver high-quality, precisely engineered knock-in models that support your entire research journey, from basic discovery to translational applications. Please don't hesitate to contact us to learn how our gene knock-in services can meet your specific research needs.

For research use only.