I am using the pSp-Cas9(BB)2A-Puro system for creating a knockout cell line. My question is after the drug selection of the transfected cells, is it advisable to pool together all the colonies or to grow them individually and then screen for the absence of protein of interest. What does your experience says? I am working with HeLa cells (aneuploidy is likely).What is the chance that all the existing alleles are deleted? TIA.
Hi,
The answer to this question depends on a lot of different factors.
To mention a few, if the protein is involved proliferation/survival, copy number etc. Make sure, you are using PX459 latest version as its Puro gene was mutated in the older version.
You can use sorter or use serial dilution to get single cell clones. I got 9/24 k/o +ve clone in HeLa by serial dilution without selection. I have tested the same in other cell lines but since I didn't select for transfection positive cells, I used to get lower efficiency in difficult to transfect cell lines. (The reason I didn't select using puro was that Cas9 is reported to give secondary mutations so longer it is in the cells, the higher are the chances of getting secondary mutations). Now ,I use PX458 (GFP) and use sorter to get single cell clones.
Hopefully this helps.
Abhishek
Hi,
imwould prefer the clones. The efficiency can be high or low. This dependents on many things and the specific sgRNA sequence is one of them. Often I try 3 different sequences and at least one works really good
- Badges
- Science method