Selecting a suitable gene delivery system requires a comprehensive assessment based on specific experimental conditions, research objectives, and cell types. Quantitatively comparing various systems in terms of delivery efficiency, cytotoxicity, and stability is an important step in determining the choice.
Viral delivery systems are suitable for experiments that require high delivery efficiency and sustained gene expression, especially when cells can tolerate higher levels of cytotoxicity and immune responses. If lower cytotoxicity and immune response, along with ease of use and cost-effectiveness, are priorities, then a liposome-based gene delivery system should be chosen. For high delivery efficiency that involves delivering large DNA fragments, and if the user can accept a higher operational complexity, a gene gun delivery system is an optional method. If high delivery efficiency is needed while maintaining relative simplicity and no special equipment is required, then the electroporation delivery system may be a suitable choice.

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.

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.

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.

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.

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.

The main difference lies in the duration and stability of gene expression: Transient cell line – The target gene is expressed temporarily in cells, typically lasting hours to days, and is suitable for short-term experiments. Stable cell line – The target gene is stably integrated into the cell genome, allowing long-term expression, suitable for extended research and production.

1.Design an efficient crRNA sequence. Proper design and structure prediction using online resources can help select suitable crRNA to achieve good trans-cleavage activity of the Cas enzyme.
2.Choose an appropriate signal reporter substrate. Research indicates that using a 15 nt single-stranded DNA (ssDNA) as a reporter substrate maximizes the cleavage reaction rate of Cas12a, significantly enhancing the reaction rate compared to the commonly used 5-nt ssDNA.
3.Optimize reaction conditions and buffers. Adjusting the CRISPR reaction parameters, such as the ratio of Cas enzyme to crRNA, the concentration of the Cas enzyme, and the reaction temperature, can improve detection performance to some extent.

What is the difference between a single-plasmid system and a dual-plasmid system for library vectors? A single-plasmid system can achieve gene editing with one transfection, making construction relatively simple, but the larger plasmid size can lead to lower infection efficiency. In a dual-plasmid system, two vectors are used, each carrying either the Cas9 or sgRNA expression cassette. A stable Cas9 cell line is first constructed, and then the sgRNA library is transfected into this cell line. This approach has several advantages:
1.Increased Editing Efficiency: The independent and stable expression of Cas9 protein and sgRNA on different vectors enhances editing efficiency.
2.Flexibility: Vectors can be designed and constructed flexibly based on experimental needs, such as loading two sgRNA expression cassettes into one vector.
3.Increased Viral Titer: By splitting into two plasmids, the load on each plasmid is reduced, facilitating viral packaging and increasing yield and titer.
4.Increased Stability: Independently constructing a stable Cas9 cell line ensures that the Cas9 expression levels and editing efficiency in each cell are approximately the same, enhancing experimental accuracy.

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.