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closeOrientation of the stop codon cassette oligo?
Posted by JamesGagnon on 25 Nov 2014 at 17:37 GMT
a reader writes:
My question is regarding the stop oligo cassette insert orientation. In you Supp. example the stop oligo is inserted 5' to 3' while the homology arms on either side are in the 3' to 5' orientation. Are the homology arm orientations dependent on whether you pick a top strand (5' to 3') or bottom strand CRISPR from CHOPCHOP and the orientation of the arms are the reverse complement to your pick? If that's the case does the orientation of the stop oligo cassette not matter since in the example it reads in the opposite orientation to the homology arms or does the cassette orientation need to match the the homology arm orientation?
my response:
I think your confusion may stem from the fact that the target site for ctgfa (GGGGCGGCACTTCAGGG^CAGTGG) is actually on the reverse strand. Cas9 will make a DSB at the ^ indicated above, and then the stops oligo is actually 5' to 3' with the gene - homology arms included. We haven't tested whether it helps to have the target site and oligo targeted to different strands, it's just how it worked out in this example.
Also, even in the unlikely case that the stop codon cassette is inserted backwards, there are stops in those three frames as well.
James Gagnon