I received a review comment,

'how I can validate that the phenotype I observed is not due to multiple mutations (or possibly, off-target effect) in a CRISPR-KO cell line'

We performed several experiments with CRISPR-KO cell line.

In both CONTROL & EXPERIMENTAL group, we samely used CRISPR-KO cell line. (Only difference was to add saline or chemical)

As we used same cell line in both condition, I think possible mutation and/or off-target effect might have not affected, but reviewers have argues on this, which I think need to be responded well.

However, I have not much good idea to validate this.

I found some in silico anlysis (https://crispr.cos.uni-heidelberg.de/), but more is needed.

Would you recommed the proper way to validate multiple mutations and/or off-target effect in CRISPR generated KO cell line ?

Only idea I came up with is whole exome sequencing... but I think it is not a good idea, as a cell line would contain various mutations, and bioinformatic analysis would be not easy...

I look forward for helpful comments. Thanks!