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Location：Home > Application > How to Construct a KO Cell Line
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How to Construct a CRISPR Knockout (KO) Cell Line: A Practical Guide for Researchers
The CRISPR/Cas9 system has revolutionized functional genomics by enabling targeted, efficient gene disruption. By introducing loss-of-function mutations in specific genes, researchers can generate knockout (KO) cell lines to investigate gene function, elucidate protein roles, or assess reagent specificity. While the technology is powerful, generating high-quality, clonal KO cell lines remains a technically demanding process that requires careful planning, expertise, and optimization.
This guide outlines the five essential steps in the construction of a KO cell line and introduces Ubigene's CRISPR-U™ platform, a cutting-edge solution that greatly enhances editing efficiency and reliability across a wide range of cell lines.
1. Design and construction of gRNA and vectors
2. Cell transfection/infection
3. Enrichment of edited cells
4. Single-cell cloning and expansion
5. Validation of knockout efficiency
Each of these stages involves critical decisions and specialized techniques. The success of your KO cell line depends on optimizing each step.
The gRNA directs the Cas9 nuclease to the genomic locus of interest, where a double-strand break is induced. For effective genome editing, the design of the gRNA must consider factors such as target sequence specificity, on-target efficiency, and potential off-target risks.
Formats of gRNA delivery include:
1. Single-guide RNA (sgRNA)
2. Dual crRNA:tracrRNA system
3. In vitro–transcribed RNA
4. Plasmid vectors
5. Lentiviral vectors
Plasmid and lentivirus are the most commonly used delivery systems. While lentiviral transduction is advantageous for hard-to-transfect cells, it may increase the risk of off-target integration. Using multiple sgRNAs targeting different regions of the gene can significantly improve KO success rates, particularly in the early enrichment phase.
✅ Ubigene’s Red Cotton™ CRISPR Gene Editing Designer
To simplify gRNA selection, Ubigene offers the Red Cotton™ CRISPR Gene Editing Designer, which provides three optimized KO strategies based on Ubigene’s proprietary design algorithm and a curated dataset of over 800 cell lines. In addition, Ubigene maintains a plasmid bank of over 10,000 validated gRNAs, enabling rapid access to tested reagents—each available for just $80.
Transfection is a critical bottleneck in CRISPR editing workflows. Each cell type requires distinct optimization of transfection reagents, timing, and conditions. Particularly challenging are stem cells, primary cells, and hematopoietic lineages, which are often more sensitive and less permissive to DNA uptake.
✅ Ubigene’s Expertise in Transfection Optimization
Ubigene has developed optimized transfection protocols for 300+ cell lines, backed by an extensive parameter database and hands-on experience. This dramatically improves gene delivery efficiency and reduces the trial-and-error time for researchers.
After transfection, enriching for edited cells and isolating clonal populations are key to ensuring homogeneity and reproducibility.
Common methods:
1. Limiting dilution: Cost-effective and less cytotoxic, but may yield lower clonal success.
2. FACS (Fluorescence-Activated Cell Sorting): Highly precise in isolating double-positive or marker-tagged cells.
However, some cell lines are not amenable to single-cell expansion, and only a few clones may survive. Optimizing culture conditions during this stage is crucial to avoid cell death and data loss.
✅ Ubigene’s Clonal Isolation Solutions
Ubigene has robust experience in generating single-cell clones from difficult cell types. Preliminary viability and growth tests are performed to determine the most suitable isolation method for each project.
Comprehensive validation ensures that the gene of interest is truly knocked out and suitable for downstream applications.
Genomic-level validation:
1. Sanger sequencing
2. Next-generation sequencing (NGS)
3. qPCR
Proteomic-level validation:
1. Western blotting
2. Mass spectrometry
Functional validation:
1. Immunocytochemistry (ICC)
2. Immunohistochemistry (IHC)
3. Flow cytometry (FACS)
✅ Ubigene’s Triple Validation Guarantee
All Ubigene-generated KO cell lines undergo validation using PCR, Sanger sequencing, ensuring knockout at DNA levels.
Ubigene's proprietary CRISPR-U™ system is designed for precise and efficient genome editing in mammalian cells. By combining:
1. Cell-line–specific transfection protocols
2. Cotton™ CRISPR Gene Editing Designer
3. Optimized Cell Pool screening
4. High-throughput genotyping
5. Advanced single-cell expansion strategies
CRISPR-U™ technology can increase editing efficiency by 10–20x compared to conventional methods. With 5000+ in-stock KO cell lines, Ubigene provides the fastest and most reliable KO cell line services for researchers worldwide—ready for immediate use in disease modeling, drug discovery, and functional genomics.
Whether you're starting a new project or looking to outsource complex gene editing, Ubigene’s expert team is here to help.
Contact us today to get a custom proposal>>Reference:
Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9. Curr Protoc Mol Biol. 2019.
How to Construct a CRISPR Knockout (KO) Cell Line: A Practical Guide for Researchers
The CRISPR/Cas9 system has revolutionized functional genomics by enabling targeted, efficient gene disruption. By introducing loss-of-function mutations in specific genes, researchers can generate knockout (KO) cell lines to investigate gene function, elucidate protein roles, or assess reagent specificity. While the technology is powerful, generating high-quality, clonal KO cell lines remains a technically demanding process that requires careful planning, expertise, and optimization.
This guide outlines the five essential steps in the construction of a KO cell line and introduces Ubigene's CRISPR-U™ platform, a cutting-edge solution that greatly enhances editing efficiency and reliability across a wide range of cell lines.
1. Design and construction of gRNA and vectors
2. Cell transfection/infection
3. Enrichment of edited cells
4. Single-cell cloning and expansion
5. Validation of knockout efficiency
Each of these stages involves critical decisions and specialized techniques. The success of your KO cell line depends on optimizing each step.
The gRNA directs the Cas9 nuclease to the genomic locus of interest, where a double-strand break is induced. For effective genome editing, the design of the gRNA must consider factors such as target sequence specificity, on-target efficiency, and potential off-target risks.
Formats of gRNA delivery include:
1. Single-guide RNA (sgRNA)
2. Dual crRNA:tracrRNA system
3. In vitro–transcribed RNA
4. Plasmid vectors
5. Lentiviral vectors
Plasmid and lentivirus are the most commonly used delivery systems. While lentiviral transduction is advantageous for hard-to-transfect cells, it may increase the risk of off-target integration. Using multiple sgRNAs targeting different regions of the gene can significantly improve KO success rates, particularly in the early enrichment phase.
✅ Ubigene’s Red Cotton™ CRISPR Gene Editing Designer
To simplify gRNA selection, Ubigene offers the Red Cotton™ CRISPR Gene Editing Designer, which provides three optimized KO strategies based on Ubigene’s proprietary design algorithm and a curated dataset of over 800 cell lines. In addition, Ubigene maintains a plasmid bank of over 10,000 validated gRNAs, enabling rapid access to tested reagents—each available for just $80.
Transfection is a critical bottleneck in CRISPR editing workflows. Each cell type requires distinct optimization of transfection reagents, timing, and conditions. Particularly challenging are stem cells, primary cells, and hematopoietic lineages, which are often more sensitive and less permissive to DNA uptake.
✅ Ubigene’s Expertise in Transfection Optimization
Ubigene has developed optimized transfection protocols for 300+ cell lines, backed by an extensive parameter database and hands-on experience. This dramatically improves gene delivery efficiency and reduces the trial-and-error time for researchers.
After transfection, enriching for edited cells and isolating clonal populations are key to ensuring homogeneity and reproducibility.
Common methods:
1. Limiting dilution: Cost-effective and less cytotoxic, but may yield lower clonal success.
2. FACS (Fluorescence-Activated Cell Sorting): Highly precise in isolating double-positive or marker-tagged cells.
However, some cell lines are not amenable to single-cell expansion, and only a few clones may survive. Optimizing culture conditions during this stage is crucial to avoid cell death and data loss.
✅ Ubigene’s Clonal Isolation Solutions
Ubigene has robust experience in generating single-cell clones from difficult cell types. Preliminary viability and growth tests are performed to determine the most suitable isolation method for each project.
Comprehensive validation ensures that the gene of interest is truly knocked out and suitable for downstream applications.
Genomic-level validation:
1. Sanger sequencing
2. Next-generation sequencing (NGS)
3. qPCR
Proteomic-level validation:
1. Western blotting
2. Mass spectrometry
Functional validation:
1. Immunocytochemistry (ICC)
2. Immunohistochemistry (IHC)
3. Flow cytometry (FACS)
✅ Ubigene’s Triple Validation Guarantee
All Ubigene-generated KO cell lines undergo validation using PCR, Sanger sequencing, ensuring knockout at DNA levels.
Ubigene's proprietary CRISPR-U™ system is designed for precise and efficient genome editing in mammalian cells. By combining:
1. Cell-line–specific transfection protocols
2. Cotton™ CRISPR Gene Editing Designer
3. Optimized Cell Pool screening
4. High-throughput genotyping
5. Advanced single-cell expansion strategies
CRISPR-U™ technology can increase editing efficiency by 10–20x compared to conventional methods. With 5000+ in-stock KO cell lines, Ubigene provides the fastest and most reliable KO cell line services for researchers worldwide—ready for immediate use in disease modeling, drug discovery, and functional genomics.
Whether you're starting a new project or looking to outsource complex gene editing, Ubigene’s expert team is here to help.
Contact us today to get a custom proposal>>Reference:
Generating Single Cell–Derived Knockout Clones in Mammalian Cells with CRISPR/Cas9. Curr Protoc Mol Biol. 2019.
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