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Creative Biogene leverages the precision of CRISPR/Cas9 technology combined with the homologous recombination (HDR) repair pathway to offer a comprehensive Stable Gene Knock-in Cell Line Construction Service. This service enables accurate insertion ranging from single-nucleotide mutations to large therapeutic gene fragments.
Stable gene knock-in depends on CRISPR/Cas9-induced double-strand DNA breaks repaired precisely via the HDR pathway, rather than the error-prone, random insertions characteristic of non-homologous end joining (NHEJ). By designing donor DNA templates with homologous arms flanking the target site, we guide efficient and accurate integration of target gene sequences or marker elements at designated loci.
The diversity of knock-in applications dictates variation in donor template structure, delivery methods, and selection strategies. Creative Biogene currently supports the following mainstream knock-in types:
| Type | Application Area | Insert Content Examples | Key Features |
| Point Mutation | SNP validation, genetic disease modeling | Single-nucleotide substitution | High precision |
| Reporter Gene Knock-in | Transcription activity, drug response monitoring, Protein tracking, affinity purification, and localization imaging | Luciferase, β-gal, RFP, His, Myc | Quantifiable |
| Safe Harbor Knock-in | Therapeutic gene expression, CAR construction | AAVS1, ROSA26, CCR5 loci | Safe and stable |
This versatility addresses broad needs in academic research, drug screening, and preclinical studies, with notable advantages in immune cell engineering and stem cell platform development.
Our service supports a wide array of cell types, including but not limited to:
Unlike traditional knock-in methods, Creative Biogene implements deep optimization across critical steps, including donor design, delivery systems, cut site selection, and fragment standardization:
Dynamic Homology Arm Design
Standard 1500 bp arms are offered with adaptive length adjustment based on insert size, cell type, and target gene features, maximizing integration efficiency while minimizing off-target effects.
Precise Insertion Site Selection
Cut sites are preferentially chosen within 10 bp of the intended insertion locus to maintain HDR efficiency, avoiding positions beyond 100 bp that drastically reduce success rates. Insertions can be seamlessly placed within coding regions, 5'/3' UTRs, or upstream promoters to preserve endogenous expression regulation.
Multi-Modal Delivery Platforms
Delivery methods include lentivirus (LV), adeno-associated virus (AAV), lipid nanoparticles (LNP), electroporation, and microinjection. For hard-to-transfect cells like iPSCs and primary T cells, we combine micro-electroporation with enhanced transduction systems to significantly boost editing success.
To guarantee successful project delivery, we adhere to a fully traceable, end-to-end process focused on data-driven results and delivery standards:
01
Assess target site and cell compatibility, define knock-in fragment and tag, design sgRNA and donor constructs, and select optimal Cas9 format (mRNA, protein, or plasmid) to maximize HDR efficiency.
Project Design
03
Optimize transfection conditions based on cell type, monitor delivery efficiency, and enrich positive cells through fluorescence or functional selection.
Cell Transfection
05
Provide frozen cell vials, comprehensive validation reports, clone screening records, and thawing instructions. Optional services include RNA sequencing and functional validation reports.
Delivery & Reporting
Vector Construction & Validation
Construct donor templates, verify with restriction mapping and sequencing; validate sgRNA activity in vitro to ensure high editing efficiency with minimal off-target risk.
02
Clone Screening
Confirm knock-in at nucleic acid and protein levels via ddPCR, RT-qPCR, Western blot; isolate and expand monoclonal lines to establish stable populations.
04
Clients provide gene sequences with corresponding NCBI database IDs and specify host cell types along with culture conditions. The final deliverable is a monoclonal gene knock-in cell line, accompanied by a Certificate of Analysis (COA) and sequencing validation. The entire process typically spans 12 to 16 weeks.
Optional Add-on Services
Creative Biogene strictly follows ISO quality management standards, utilizing automated equipment and standardized record-keeping to ensure traceability, reproducibility, and batch consistency for every knock-in cell line project. All deliverables include genotypic verification and cell status documentation, with optional customized testing services and methodological documentation available, enabling a truly one-stop, full-cycle solution from project initiation to final delivery.
Case Study 1
A luciferase knock-in SKOV3 cell line was used to evaluate TAT-HA2-based siRNA delivery systems. The multicomponent platform showed enhanced cytoplasmic delivery and gene silencing (up to 55% suppression), offering a sensitive, low-toxicity model for functional RNAi screening. Custom knock-in models from Creative Biogene enable precise assessment of delivery efficiency in RNA-based therapeutic development.
Figure 1. TAT-HA2-based siRNA complexes achieved significant luciferase knockdown with minimal toxicity in SKOV3 knock-in cells, outperforming standard transfection agents across various doses.
Case Study 2
Creative Biogene has successfully developed the CT26-KRASG12C-luciferase knock-in murine colorectal cancer cell line, engineered to precisely introduce the KRASG12C mutation and stably express GFP and luciferase for in vivo tumor tracking. This cell line was used to establish a syngeneic liver tumor model in the evaluation of BBO-8520, a dual inhibitor targeting both ON and OFF states of KRASG12C. The model enables real-time monitoring of tumor progression and supports preclinical assessment of KRAS-targeted therapies, including combination strategies with immunotherapy, making it a valuable tool for translational cancer research.
Figure 2. Starting from a noncovalent KRASG12D inhibitor, compound 1, the series was evolved by adding a covalent warhead and improving cell potency and ADME properties. The final compound, BBO-8520, demonstrates selective covalent binding to active KRASG12C and disrupts its interaction with RAF1, confirmed by SPR, PPI assays, and mass spectrometry.
Whether you are pursuing precise gene insertion for functional studies or developing stable models for therapeutic applications, Creative Biogene is your reliable partner for knock-in cell line construction. We provide fully customized solutions with end-to-end support—from donor design and delivery optimization to thorough clone validation, ensuring accurate, efficient, and reproducible knock-in outcomes. Reach out today for a free consultation and tailored project planning.
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