Currently I am trying to create a CRISPR line for a c terminal Eos fusion. My PAM sequence with a score of 90 in the design tool is 80 bp downstream of the stop codon. Is it fine to design my giude RNA for this site to acheive the Cterminal fusion.
Its not a good option. Rather you can try to find a PAM sequence before the stop codon and design the sgRNA and the homology arms accordingly. You can refer Figure2 of the paper(http://www.nature.com/srep/2015/150305/srep08841/full/srep08841.html), wherein they have attempted the same. The figure clearly addresses your question. All the best.
Is not a good idea. you need used a sequence before your stop codon, and also, in the same reading frame. You can use the bleuheron tools (http://blueheronbio.com). It's a really good tool to find your PAM sequence. Regards
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