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FAQ
What is the core principle of gene knock-in technology?
Why choose EDITGENE, and what are EDITGENE’s main advantages in gene knock-in technology?
What is the difference between a stable cell line and a transient cell line?
Why conduct gene overexpression?
What is gene overexpression?
How does EDITGENE ensure the purity and stability of cells during monoclonal screening?
What unique advantages does EDITGENE offer for monoclonal screening services?
How do I choose suitable cells for library screening?
1.It should align with the research objectives.
2.The genes targeted by the sgRNA library should correspond to the cell's lineage.
3.The cells should be capable of stable passaging.
4.The transfection efficiency should be high.
5.Avoid primary cells whenever possible. Primary cells cannot be stably passaged and may experience significant cell death during the library screening process, which can hinder experiment completion. If primary cells must be used for library screening, mitigating this risk can be achieved by lowering cell coverage and choosing a library with fewer gRNAs to minimize the cell pool size and shorten the experimental duration.
How do I choose between a whole-genome or subgenomic CRISPR library?
What is the difference between a single-plasmid system and a dual-plasmid system for library vectors?
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.
What issues should be considered when culturing cells for gene delivery?
How to choose the appropriate gene delivery method?
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.
How long can CRISPR-related reagents and Cas proteins be stored?
1.The RPA isothermal amplification kit can be stored at -20°C for long-term storage.
2.Target plasmids can be stored at -20°C for long-term use.
3.Cas proteins are sensitive to repeated freeze-thaw cycles; it is recommended to aliquot into multiple tubes and store at -80°C, retrieving them as needed for experiments. For short-term use, they can be stored at -20°C.
4.crRNA is prone to degradation and should be stored at -80°C if not used in the short term.
5. Probes, being double-stranded DNA, are relatively stable and can be stored at -20°C.
Can both dsDNA and ssDNA targets activate the trans-cleaving activity of Cas12a? Which has higher efficiency?
How to Improve the Detection Sensitivity of Cas Enzymes?
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.