gRNA all ON-target sites in genome

Is there any tool where you can put the sequence of your sgRNA and predict all the ON target sites in the genome. In case the guide might also bind to other sequences as well. It is most unlikely but if it can happen how to can predict the site.

I knockout one GOI in cancer cells, I see the effect after the functional test like proliferation and migration etc. but can't see the deletion after sequencing.

Any thoughts??


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Gold Gorgonover 3 years ago

Normally, On-Target defines the target sequence, if it refers to possible off-target targets (another sequence in which they could cut their gRNAs), you can use https://cm.jefferson.edu/Off-Spotter/

Blue Harpyover 3 years ago

For predicting gRNA binding sites I would recommend CCTop (https://crispr.cos.uni-heidelberg.de/) which will show you all ON and OFF target sites in the genome. As far as knocking out your GOI, it is not uncommon for a CRISPR cut to cause a large deletion (a couple kb) that can take out primer sites. Try designing some primers 1 kb up and downstream of your target site (or even better, a few primer sites that are increasingly far apart) and see if you get a smaller-than-expected bands.

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