Hi there
I am trying to design guide RNAs for cutting the yeast 2 micron plasmid using CRISPR. Using Chopchop, I got a 23 nt sequence which was suitable for gRNA design.
When I blast the 23 nt sequence against the S. cerevisiae genome to check for any off-site cutting, I get multiple results of 100% query coverage, see the attached picture. However, when I click these results to investigate the alignment further, the alignments are all 14 nt or shorter. It seems that BLAST is finding multiple short alignments which cumulatively cover the entire query sequence - however, none of the individual alignments match the entire query sequence, which is what I'm interested in.
Why do I not just get the highest query cover result as being 14/23 nt=60.86%? And is there any option I can toggle to get these values?
Thanks! :-)
I am currently experiencing very similar problems.
And I also have another question, so I am trying to design gRNA for my targets. When I use both chopchop and crispor they provide me with sequences that they call target sequence (TS), this sequence contains the PAM sequence which according to NEB and IDT should be located on the non-target sequence (NTS). So I think there might be some confusion with the nomenclature here?
Then went I put these two sequences in the BLAST (the TS and NTS, separately). The TS doenst have any other hits on the query cover but when I blast the complementary NTS I have a lot of missmatches with 100% query cover on other targets.
Do you know anything about this? Or somewhere I can find more information?
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