EDITGENE CO., LTD
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FAQ
What is the core principle of gene knock-in technology?
What is the difference between a stable cell line and a transient cell line?
Why conduct gene overexpression?
Why choose EDITGENE to establish stable overexpression cell lines?
What is monoclonal screening, and why is it so important in gene editing research?
How does EDITGENE ensure the purity and stability of cells during monoclonal screening?
What unique advantages does EDITGENE offer for monoclonal screening services?
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.
How do I choose between a whole-genome or subgenomic CRISPR library?
What issues should be considered when culturing cells for gene delivery?
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.
What are the differences between Cas9, Cas12, and Cas13?
· Cas12 is activated to cleave ssDNA trans-cleaving after binding with guide RNA and target DNA.
· Cas13 is activated to cleave ssRNA trans-cleaving after binding with guide RNA and target RNA.
· Cas9 has not been reported to exhibit trans-cleaving activity.
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.
How to Design crRNA?
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.