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Editx Technology Co., Ltd
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FAQ
How is Prime Editing 7 (PE7) different from traditional CRISPR/Cas9 technology?
Traditional CRISPR/Cas9 technology achieves gene editing by introducing double-strand breaks at the target DNA site and then using the cell’s homologous recombination repair mechanism. This approach carries multiple risks, such as lower editing efficiency, reduced homozygous mutation rates, and random insertions or deletions. Prime Editing, however, does not require double-strand breaks. With its Cas9n-RT editing enzyme system and pegRNA, Prime Editing achieves more accurate and safer gene editing with reduced off-target effects.
Are all types of genes suitable for KO cell lines?
Not all genes are suitable for knockout. Some gene knockouts may result in cell death or severe dysfunction, particularly for essential genes. In such cases, conditional knockouts or gene knockdowns (e.g., RNAi) may be used instead.
What is the difference between KO cell lines and gene knockout animal models?
KO cell lines are used for in vitro experiments, suitable for high-throughput screening and cellular studies, while gene knockout animal models are used for in vivo experiments to study gene functions within an entire organism and its interaction with the environment.
Why choose EDITGENE?
EDITGENE provides access to a comprehensive library of over 4,500 high-quality knockout (KO) cell lines, enabling researchers to save valuable time. Our custom gene knockout services are highly efficient, boasting a high positive rate while minimizing off-target effects. Clients also benefit from personalized, one-on-one support from a team of PhD experts from globally renowned institutions, ensuring top-tier service and results.
Are KO cell lines applicable to all cell types?
KO cell lines can be applied to various cell types, including cancer cells, stem cells, and primary cells, but different cell types may have varying sensitivities to gene editing, and may vary among different cell types. In certain cell types, achieving gene knockout may require optimization of transfection conditions and selection of appropriate gene-editing tools.
How to Design crRNA?
1.The design process can follow these steps:
1.Identify the target gene sequence.
2.Specify the Cas protein being used. Different Cas proteins require corresponding PAM (Protospacer Adjacent Motif) sequences; for instance, Cas12a needs the "TTTV" PAM sequence for target recognition.
3.Select the crRNA targeting region. Choose a 20 nt nucleotide sequence on the target gene that is adjacent to the PAM site and pairs with the complementary strand of the crRNA.
4.Combine the selected 20 nt target sequence (variable part) with the scaffold sequence (fixed part) to design the crRNA sequence.
5.Use online tools such as CRISPR design tools (e.g., CRISPOR, Benchling, etc.) to assist in designing crRNA. These tools can predict the efficiency and specificity of the sgRNA, helping to avoid potential off-target effects.
6.After completing the design, the synthetic crRNA sequence can be ordered from a synthetic biology company.
1.Identify the target gene sequence.
2.Specify the Cas protein being used. Different Cas proteins require corresponding PAM (Protospacer Adjacent Motif) sequences; for instance, Cas12a needs the "TTTV" PAM sequence for target recognition.
3.Select the crRNA targeting region. Choose a 20 nt nucleotide sequence on the target gene that is adjacent to the PAM site and pairs with the complementary strand of the crRNA.
4.Combine the selected 20 nt target sequence (variable part) with the scaffold sequence (fixed part) to design the crRNA sequence.
5.Use online tools such as CRISPR design tools (e.g., CRISPOR, Benchling, etc.) to assist in designing crRNA. These tools can predict the efficiency and specificity of the sgRNA, helping to avoid potential off-target effects.
6.After completing the design, the synthetic crRNA sequence can be ordered from a synthetic biology company.
What is a KO cell line?
KO (Knockout) cell line is a cell line where a specific gene has been completely removed or rendered non-functional through gene editing technologies such as CRISPR-Cas9. These cell lines are critical for understanding gene functions and disease mechanisms.
Why conduct gene overexpression?
Gene overexpression aids in studying the function of specific genes, revealing their role within the organism. It is also commonly used in drug screening, vaccine development, and protein production. For example, by overexpressing a therapeutic protein, researchers can evaluate its efficacy in disease models.
How do I choose between a whole-genome or subgenomic CRISPR library?
CRISPR libraries can be divided into whole-genome libraries and subgenomic libraries. If the goal is to perform screenings across the entire genome, a whole-genome library is the best choice. Such libraries typically contain sgRNAs targeting the entire genome. If the research focus is specific, such as targeting only particular gene families or specific signaling pathways, a subgenomic library can be chosen to reduce unnecessary screening workload and costs.
How to choose the appropriate vector type?
When selecting a vector, consider the purpose of the experiment and the type of host cells. For example, plasmid vectors are commonly used for gene expression or amplification in bacteria, while viral vectors are more suitable for gene transfer in mammalian cells. Additionally, the vector's promoter, replicon, and antibiotic selection markers should be chosen based on specific requirements.
What is monoclonal screening, and why is it so important in gene editing research?
Monoclonal screening is the process of isolating a single clone from a mixed pool of cells and expanding that clone into a cell line. Monoclonal screening ensures that the cell lines used originate from a single cell, guaranteeing a high degree of genetic background consistency. After cells are gene-edited or genetically modified, the genetic background differences among the cells in the initial cell pool can be significant, making subsequent experimental results inaccurate. By using monoclonal screening, researchers can obtain cell populations with consistent genetic backgrounds and stable gene edits, allowing for stable and accurate monitoring of phenotypic changes.
Why do researchers use KO cell lines?
Researchers use KO cell lines to investigate gene functions by observing the effects of gene deletion on cellular behavior. This helps in understanding the role of genes in various processes like cell growth, metabolism, and signal transduction. KO cell lines are vital for studying diseases like cancer, genetic disorders, and neurodegenerative diseases.
Why choose lentivirus as a gene delivery tool?
Lentiviruses have high transduction efficiency and the ability to maintain stable gene expression over the long term, making them particularly suitable for cell types that are difficult to transfect. Furthermore, lentiviruses can integrate exogenous genes into the host genome, ensuring prolonged gene expression.
