Hi everyone,
My gene of interest is expressed in my cell line of interest from a WT and a mutated allele. These 2 alleles have only one base pair difference (missense subsitution). I would like to inactivate the WT allele so that my cell line express the protein only from the mutated allele. I guess I can do that with CRISPR-Cas9. Is it possible to target only the WT allele when there is so little difference between the 2 alleles ? How do I check that I have what I want ?
Thank !
Hi Delphine,
one base difference in CRISPR designing is very thin, since typically CRISPR oligos are 20p length. you should design your test with your variant sequence (http://crispor.tefor.net) and see if your WT allele is in the off targets list.
fred
SNP-derived PAM is an ideal approach for such purposes.
It means that the PAM sequence of a chosen Cas is present only at the target allele. Thus, the "non-target" allele remains intact since the Cas is not able to bind it.
You may further read DG Courtney et al (2016) for the principles of this approach.
Feel free to contact me for further help with designing your experiment. I will soon publish a web tool for designing such allele-specific targeting.
Roy
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