Stable Knockout Cell Line Generation

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.

Advanced Gene Editing Solutions

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.

Custom Stable Knockout Cell Line Generation Services

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 weeksClones: 10–14 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

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 weeksClones: 10–14 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

Key Technical Advantages

Service Applications

Stable Knockout Cell Line Generation Project Workflow

Our services follow a structured five-phase approach, managed by senior technical teams and dedicated project managers to ensure seamless communication and timely delivery:

Custom Analysis & Validation Support

Client Case Studies & Research Outcomes

Phenotypic analysis revealed L-sorbose-dependent changes in fitness and/or virulence. The indicated strains were tested in biological replicates (n ≥ 3).

Genomic and functional characterization of the L‑sorbose phosphotransferase system in high-risk Escherichia coli lineages

Paper 2026

mSystems | DOI

Background
This study explores the L-sorbose phosphotransferase system (sor-operon) as a pathogen-enriched marker in high-risk Escherichia coli lineages. Genomic analysis of over 22,000 strains revealed the operon in >95% of pathogenic STs but rarely in commensal ST10. Functional validation in ST131 and ST648 showed L-sorbose-induced expression that conferred competitive advantage over ST10. Multi-omics identified co-regulated pathways including tryptophan and purine metabolism, flagellar assembly, and capsule production, linking sugar utilization to fitness and virulence traits. This research underscores the value of combining genomics with functional assays to discover pathogen-specific targets against multidrug-resistant clones.
Key Findings
Through comparative genomics of 22,267 E. coli genomes, the sor-operon encoding the L-sorbose phosphotransferase system was identified as a marker present in >95% of pathogenic sequence types (ST38, ST131, ST405, ST410, ST648) but in <0.1% of commensal ST10. Functional studies in ST131 (PBIO729) and ST648 (PBIO730) demonstrated sor-operon induction by L-sorbose, providing a competitive fitness advantage against ST10 during co-culture. Transcriptomic and proteomic analysis uncovered upregulation of tryptophan and purine biosynthesis, flagellar genes, and kps capsule polysaccharide operons exclusively in pathogenic strains. Knockout mutants lacking sorE, which encodes sorbose-1-phosphate reductase, lost competitive advantage, displayed reduced motility, and decreased serum survival in the presence of sorbose. Conversely, introducing the sor-operon into commensal strain PBIO365 enabled L-sorbose utilization and conferred increased competitiveness and heightened virulence in Galleria mellonella larvae, consistent with the operon's role in enhancing bacterial fitness and pathogenic potential in high-risk clones.
Contribution
Creative Biogene generated marker-less mutant strains PBIO729ΔsorAM, PBIO729ΔsorE, and PBIO365:sor via homologous recombination, enabling functional validation of the sor-operon.

Induction of ampC RNA and protein occurs upon imipenem treatment.

AmpC induction by imipenem in Pseudomonas aeruginosa occurs in the absence of OprD and impacts imipenem/relebactam susceptibility

Paper 2024

Microbiology Spectrum | DOI

Background
This study investigates how imipenem/relebactam restores susceptibility in Pseudomonas aeruginosa isolates that lack functional OprD and do not overproduce AmpC. Using 17 clinical isolates and laboratory strains including oprD and opdP mutants, the authors found that imipenem can enter cells independent of OprD, inducing chromosomal bla_ampC expression. Induction of AmpC provides a target for relebactam, explaining lowered imipenem minimal inhibitory concentrations. The findings suggest alternative porins facilitate imipenem uptake and highlight the importance of AmpC induction in imipenem/relebactam susceptibility, offering insights for treating carbapenem-resistant P. aeruginosa infections.
Key Findings
1. Imipenem can enter Pseudomonas aeruginosa cells in the absence of OprD, as demonstrated by ampC induction in 17 clinical isolates and oprD transposon/CRISPR mutants. 2. All 20 tested isolates exhibited bla_ampC transcript induction upon sublethal imipenem exposure; 18 lacked detectable OprD protein. 3. Relebactam lowered imipenem MICs in 64.7% of clinical isolates, even when OprD was absent, by inhibiting imipenem-induced AmpC. 4. OpdP may contribute to imipenem uptake but is not essential; one isolate lacking both OprD and OpdP still showed ampC induction. 5. Transcriptomic analysis revealed common upregulation of creD and ampC under imipenem challenge, with downregulation of denitrification and flagellar operons. 6. AmpC overexpression alone (derepressed mutants) did not correlate with relebactam efficacy, indicating other mechanisms contribute to resistance.
Contribution
Creative Biogene constructed the OprD CRISPR knockout strain (OprDKOCR) used to validate phenotypic and genotypic results, confirming imipenem activity and AmpC induction in the absence of OprD.

Interaction between fragment 6 of L and M protein is modulated by Y1631 and V153 in L and M proteins, respectively.

Interaction Between the Matrix Protein and the Polymerase Complex of Respiratory Syncytial Virus

Paper 2024

Viruses | DOI

Background
This study investigates interactions between respiratory syncytial virus (RSV) matrix protein and polymerase L protein, revealing a novel association. Comparing two L inhibitors, PC786 uniquely induced mutation V153A in M in addition to Y1631H in L. Protein fragment analysis shows M directly interacts with L residues 1392–1735, the region where PC786 binds, and disrupting this interaction alters M nuclear localization and delays viral assembly. The findings propose that targeting the L–M interface may represent a new antiviral strategy.
Key Findings
Both PC786 and PC751 potently inhibited RSV-induced cytopathic effect in HEp-2 cells and polymerase activity in minigenome assays, with PC786 showing greater potency in differentiated bronchial epithelium. Resistance selection produced Y1631H in the L protein for both inhibitors, but only PC786 caused V153A in the M protein. Recombinant protein and live-cell assays demonstrated direct binding between M protein and the L-protein fragment spanning amino acids 1392–1735, corresponding to the proposed PC786 binding region. PC786 treatment promoted nuclear accumulation of M protein and delayed F-protein localization to budding filaments. An RSV mutant carrying both L and M mutations (A2-786) exhibited delayed assembly and filament formation compared to wild-type or L-only mutant viruses. Thus, disrupting the newly identified L–M interaction, potentially by antivirals, may impair RSV assembly and budding.
Contribution
Creative Biogene supplied key reagents: RSV minigenome luciferase reporter plasmid, wild-type M expression vector, mutant L expression vector, and engineered the Y1631H L mutation, enabling polymerase and binding analyses.

ADAR1p90 represses PKR-eIF2伪 activation through cytoplasmic dsRNA sequestration.

Intron 13 retention expands the ADAR1 isoform repertoire to rewire PKR signaling and promote tumorigenesis

Paper 2026

bioRxiv | DOI

Background
This study reveals that intron 13 retention (I13R) expands the ADAR1 isoform repertoire. A conserved weak 5' splice site permits I13R, which is negatively autoregulated by ADAR1 via antagonizing hnRNPA1 binding. I13R transcripts are sensitive to nonsense-mediated decay yet generate two uncharacterized truncated isoforms, p90 and p130, lacking the C-terminal deaminase region. p90 is cytoplasmic and suppresses PKR/eIF2α activation by sequestering immunogenic dsRNA substrates. In colorectal cancer, p90 levels are elevated and correlate with hnRNPA1 expression; p90-overexpressing xenografts show accelerated tumor growth compared with p110.
Key Findings
RNA-seq and minigene assays establish that ADAR1p110 suppresses retention of a conserved weak 5' splice site intron 13 in its own pre-mRNA in an editing-independent but dsRNA-binding-dependent manner, competing with hnRNPA1 for binding to intron 13. I13R transcripts are NMD-sensitive but escape decay sufficiently to generate p90 and p130, both lacking the C-terminal deaminase region and catalytically inactive; p90 acts as a dominant negative on canonical A-to-I editing. p90 localizes to the cytoplasm and represses PKR and eIF2α phosphorylation more strongly than p110 by sequestering immunogenic dsRNA substrates, as demonstrated with a BRI3BP dsRNA probe. In matched CRC specimens, p90 levels are elevated and correlate with hnRNPA1. Xenografts expressing p90 grow larger/faster and show reduced eIF2α activation, supporting an oncogenic role via PKR-eIF2α repression.
Contribution
Creative Biogene generated the HEK293T 3'SS mutant cell line harboring an A>G mutation at the ADAR1 intron 13 splice acceptor (chr1:154585346), which was essential for validating endogenous ADAR1p90 expression. This CRISPR-engineered cell model enabled the study to confirm I13R-derived p90/p130 isoforms and their cytoplasmic, PKR-repressive functions.

Cellular levels physiologically relevant metals quantified by ICP-MS in aerobically grown WT and ΔdgcO Pcc.

An O2-sensing diguanylate cyclase broadly affects the aerobic transcriptome in the phytopathogen Pectobacterium carotovorum

Paper 2023

Frontiers in Microbiology | DOI

Background
Pectobacterium carotovorum causes bacterial soft rot, with virulence influenced by oxygen. DgcO, a globin-coupled sensor with diguanylate cyclase activity, produces c-di-GMP upon O2 binding. This study combined RNA sequencing, differential expression, protein pulldown, growth assays, and ICP-MS to define DgcO-dependent regulation. Deletion of dgcO altered 372 transcripts only under aerobic conditions, including metal transporters, motility, secretion, quorum sensing, translation, and anaerobic metabolism genes. ICP-MS further showed lower cellular Co, Cu, Mg, Mn, Mo, and Zn in ΔdgcO, linking an O2-sensing diguanylate cyclase to metal homeostasis in this phytopathogen.
Key Findings
RNA sequencing showed that deleting dgcO in Pectobacterium carotovorum affected 372 transcripts (~9% of genome) only during aerobic growth, with most changes subtle; 258 transcripts decreased and 114 increased. Affected functions included T6SS, flagellar and virulence genes, metal homeostasis/transport, chemotaxis, carbohydrate metabolism, T3SS/T6SS, PCWDEs, quorum sensing, translation, and anaerobic citrate/glycerol metabolism. c-di-GMP quantification revealed no significant aerobic difference between WT and ΔdgcO, but anaerobically WT had higher c-di-GMP; both strains increased c-di-GMP anaerobically. Growth assays showed ΔdgcO had a longer lag entering anaerobiosis in M9+lettuce lysate and lower final OD upon return to aerobic conditions. ICP-MS found significantly lower Co, Cu, Mg, Mn, Mo, and Zn in ΔdgcO, while iron was unchanged. Pulldown identified interactions with CheY, LuxS, and translation elongation factors. These results indicate DgcO broadly regulates aerobic transcriptome and metal homeostasis, likely via localized c-di-GMP signaling.
Contribution
Creative Biogene constructed the PccDgcO deletion strain (ΔdgcO) used throughout this study. They PCR-amplified dgcO, cloned it into the pLP12 suicide vector, transformed E. coli, conjugated the deletion construct into Pectobacterium carotovorum, and verified the knockout. This provided the critical ΔdgcO mutant enabling transcriptomic, c-di-GMP, phenotypic, and metal-homeostasis analyses.

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).

Fluorescent microscopy and flow cytometry showing reduced FAB02 uptake in TfR1 knockout HeLa cells compared to wild-type.

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.

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.

Lysosomal Ca²⁺ and pH response to ATP in WT and TMEM165 knockout HeLa cells, highlighting TMEM165's role in calcium import.

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.