Hi Everyone!
I am currently designing primers to validate my CRISPR-KO mammalian cell line using TIDE. The primer is designed about 250bp upstream of the PAM site. Then, Sanger sequencing is run using the primer on DNA extracted from the KO cell line. The TIDE software then deconvolutes the indels in the mixed KO population to tell you the efficiency of the CRISPR on the gene of interest.
TIDE software: https://tide.nki.nl/
In order to make the primer sequence specific to a region of genomic DNA (an intron) upstream of my PAM site (in an exon), I think that I should make the primer sequence longer. What is the max length you would make a Sanger primer to obtain specificity? I am using BLASTn to check the primer sequence against the human genome. Even once I have a specific primer, how can I validate that it does not have off-target binding sites in the human genome? Is there an experimental validation that should be run first? Or, will the purity of my Sanger read upstream of the cut site show me that I have only sequenced the target? What are other recommendations for TIDE sequencing primers?
Thanks!
You don't need any specific consideration beyond what you will use for any primer set. Just check for specificity by running an in-silico PCR on the UCSC browser (make sure you select genome and not transcripts as template) or with any other tool you use anyway.
Make sure to also sequence a wt amplicon since TIDE use it as a reference.
You may also want to check up ICE (https://ice.synthego.com/#/), it works on the same idea as TIDE but have some additional features.
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