Hello!
I am planning to KO a gene that seems critical to the cell's viability by CRISPR/Cas9.
The question is, how can I detect the dead cells that are truly dead by KO by Cas9 when the gene is indispensable for maintaining its life?
In my opinion, the assay that just identify dead/live cells cannot explain the KO of the gene.
Should I relatively measure their death rate with control cells by MTT assay or other methods? Or do you have another recommendations?
Thank you for in advance. Have a nice day.
Ps. In my lab, I am the one who starts the gene editing. I cannot expect help by my colleagues.
Lactate Dehydrogenase (LDH) Release Assay and Propidium Iodide (PI) Staining are widely used technique for cytotoxicity
I think you should use the T7 endonuclease assay to visualize the relative proportion of knockout and wildtype genomes in the culture over time.
If your hypothesis is correct, you should see knockout genomes appearing soon after transfection (hours if delivered as RNP). Later, as the affected cells die off, the knockout genomes should disappear again, until you have only wild types left after some days or weeks.
Le Chang Sir your answer is reasonable. I would incorporate Tet-On system.
John Schloendorn Sir, your answer is easy to apply in Lab-scale. I will probably apply your recommended T7E1 assay before NGS. Thank you
Sujan Bishwakarma Sir, your recommended assays seem reasonable. I will apply to my experiment. Thank you.
- Badges
- Science topic
- Similar topics
- Biological Science
- Molecular Cell Biology
- Culture Cells
- Cell Line