Hi everyone,
I am trying to knock out a non-coding region downstream of my gene of interest. I looked at a publication which did exactly this but in a different species.
They seemed to use two sgRNAs flanking the region of interest and didn't mention any additional components to the knockout in the method section. They show almost complete knockout in their paper (using PCR, the bad after KO is 500bp shorter than in the mock)
My question is; is this a common approach? Just using two sgRNAs a few hundred bps apart without any HDR template and hope that the NHEJ leads to a loss of the region between the two sgRNAs?
Yes, this is very common method for making deletion/KO using a pair of sgRNAs. The efficiency is length dependent and also requires each sgRNA to be efficient. Check out the publication bellow https://www.jbc.org/article/S0021-9258(20)47516-7/fulltext The repair by NHEJ will not be perfect with some random indels, and occasionally the deletion may spread further away from the predicted cut site. You could add an HDR template if you need a very precise deletion with specific length.
Hi Sigrun S.
The answer is yes, you two guide RNAs will perfectly delete the in between region, this region ranges from few hundred base pair to several Kbs, In our hand we delete up to 7 kb fragment in vivo (mice) with only two sgRNAs, also I would recommend to design one of your guide on the sense strand and the second on antisense to maximize the efficiency, in all conditions you can not avoid collateral off-targets
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