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Drawing on extensive expertise in gene editing, Creative Biogene has developed a comprehensive, efficient, and reliable platform for generating stable gene knockout (KO) cell lines using cutting-edge CRISPR/Cas9 technology. Our customizable service supports a wide range of mammalian cell lines, including both conventional and challenging-to-transfect varieties, delivering functionally validated, well-characterized, and long-term stable knockout cell models to research institutions and pharmaceutical companies worldwide.
Stable knockout cell line generation with CRISPR/Cas9 uses targeted DNA breaks guided by sgRNA. Repair via non-homologous end joining (NHEJ) introduces insertions or deletions that disrupt gene function permanently. Creative Biogene optimizes sgRNA design, delivery, and clone selection to produce high-specificity knockout lines with minimal off-target effects. This efficient, scalable method supports diverse research and therapeutic needs.
Creative Biogene's platform integrates high-throughput gRNA screening systems, optimized ribonucleoprotein (RNP) delivery methods, and streamlined clonal screening workflows. We offer comprehensive editing services, including single-gene knockouts, multi-gene knockouts, and targeted fragment deletions. Our end-to-end pipeline encompasses gRNA design, Cas9 delivery, positive clone screening, genotype verification, and functional validation, achieving an optimal balance between editing efficiency and long-term stability to ensure high success rates, consistent outcomes, and accelerated project timelines.
| Service Type | Description | Quality Control | Deliverables | Timeline |
| Standard Single-Gene KO | CRISPR/Cas9-mediated biallelic knockout generating stable monoclonal or pooled cell lines | Sanger sequencing, Western blot protein validation | Sanger-verified homozygous KO clones + optional negative control pool | Pools: 3–6 weeks |
| Multi-Gene KO | Simultaneous or sequential knockout of multiple genes with optimized multi-gRNA design | Multi-locus Sanger sequencing, key protein validation | KO clones with complete target allele disruption + optional control pool | Pools: 4–8 weeks Clones: 12–16 weeks |
| Targeted Fragment Deletion | Precise deletion of regulatory regions, conserved domains, or promoters while maintaining reading frame integrity | PCR verification of deletion, qPCR expression analysis | 1–2 clones with precise deletions + untreated control pool | 8–12 weeks |
Optimized Guide RNA Development
Our design criteria prioritize optimal GC content, comprehensive off-target prediction, strategic PAM distribution, and secondary structure assessment to select gRNA sets with maximum targeting efficiency and minimal off-target potential, ensuring superior specificity and editing performance.
High-Efficiency RNP Delivery System
Unlike plasmid or viral delivery methods, direct transfection of Cas9 protein complexed with synthetic sgRNA (RNP) enables rapid, transient genome editing without dependence on transcription or expression systems. This approach reduces DNA integration risks while enhancing editing efficiency, with preliminary cutting efficiency assessable within 5–6 days, significantly accelerating project timelines.
90+ Cell Line Support
Our platform supports over 90 mammalian cell lines, including HEK293, HeLa, CHO, A549, U2OS, iPSCs, primary fibroblasts, and specialized cell types. We customize electroporation parameters, culture conditions, selection protocols, and cloning strategies for each cell type, with all lines confirmed mycoplasma-free to ensure experimental reliability.
Basic Research
Gene function analysis, signaling pathway elucidation, epigenetic regulation studies, protein interaction network mapping, transcription factor characterization, and promoter validation.
Medical Research
Tumorigenesis mechanism studies, disease factor validation, genetic disease model development, functional gene screening, and CRISPR screening platform establishment.
Drug Development
Target validation, mechanism of action studies, high-throughput screening model development, toxicity assessment, and personalized medicine research.
Our services follow a structured five-phase approach, managed by senior technical teams and dedicated project managers to ensure seamless communication and timely delivery:
01
Target gene analyzed, 3+ optimized gRNAs synthesized. Cell type assessed, culture expanded, transfection readiness confirmed.
gRNA Design
03
Up to 100+ single clones isolated. Genotype confirmed via PCR and Sanger sequencing.
Cloning & Genotyping
05
Validated clones cryopreserved. Shipped with controls, sequence data, full documentation, and protocols.
Cryostorage & Delivery
Transfection & Screening
CRISPR RNP delivered via refined electroporation. Positive cells enriched by selection or FACS. Editing verified by PCR and sequencing.
02
Knockout Validation
Selected clones were evaluated for editing accuracy, genomic integrity, viability, sterility, and stability.
04
Enhance your research capabilities with our additional customizable analyses:
Case Study 1
To support the development and validation of the FORCETM platform for targeted oligonucleotide delivery in neuromuscular disorders, Creative Biogene provided homozygous TfR1 knockout HeLa cells (TfR1⁻/⁻). These cells were employed in internalization assays to elucidate the mechanism of TfR1-mediated uptake and evaluate the specificity and efficiency of FORCE-conjugated oligonucleotides. By comparing TfR1⁻/⁻ and wild-type HeLa cells, researchers demonstrated that the FORCE platform enables efficient intracellular delivery via the transferrin receptor pathway, supporting its translational potential for treating diseases such as myotonic dystrophy type 1 (DM1).
Figure 1. TfR1 is essential for targeted oligonucleotide uptake. Fluorescent imaging and flow cytometry demonstrate significantly reduced uptake in knockout cells, validating the importance of receptor expression in nucleic acid delivery.
Case Study 2
The TMEM165 knockout (KO) HeLa cell line, developed by Creative Biogene, supports advanced research into lysosomal calcium (Ca2+) channel functions and mechanisms. TMEM165 functions as a key lysosomal Ca2+ importer, and its knockout provides a valuable model for studying calcium signaling pathways and neurodegenerative diseases. This robust and well-characterized cell line is cultured under optimized conditions, ideal for gene function studies and drug discovery applications.
Figure 2. Human LCI (TMEM165) mediates lysosomal Ca²⁺ import linked to lysosomal pH decrease. In WT HeLa cells, ATP induces a drop in lysosomal pH and a rise in Ca²⁺ levels, whereas TMEM165 KO cells show no such changes.
Whether you're conducting exploratory research or require robust, validated models for defined targets, Creative Biogene serves as your trusted gene editing partner. We deliver tailored knockout solutions with comprehensive support—from initial design through final validation—enabling high-quality, rapid, and risk-minimized scientific advancement. Contact us today for a complimentary consultation and customized project design.
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