I'm trying to obtain genetic KO in my cell line of interest using Lentiviral-mediated CRISPR-Cas9 delivery system. I would like to test more than one unpubblished sgRNA targetting a couple of genes of interest. Previous KO efficiency in our lab have been validated through T7 assay or TIDE sequencing. Which would you reccomend to use? Aa far as you know, are there other strategies more suitable or robust that are routinely used to validate CRISPR-Cas9 editing?
Thanks in advance!
I generally like to use Sanger sequencing followed by TIDE or ICE (https://ice.synthego.com/#/). The ICE tool allows batch analysis.
Alternatively, you could go for amplicon sequencing
I generally like to use Sanger sequencing followed by TIDE or ICE (https://ice.synthego.com/#/). The ICE tool allows batch analysis.
Alternatively, you could go for amplicon sequencing
I agree with Ward. You might find this paper helpful: https://www.nature.com/articles/s41598-018-19441-8
I'd also add that flow cytometry, if you can stain for your target, will tell you if your edit was truly loss of function.
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