I am new to plant biology, looking for a gRNA and a Cas9 expressing construct to transform dicot plants. pRGE31 hosts a gRNA scaffold sequence but can't find a restriction site upstream of the scaffold to insert gRNA oligos. Not sure how to add a guide RNA seq upstream of the gRNA scaffold. Any help would be appreciated. Thanks
You use BsaI cloning for that plasmid (order oligos that form compatible end to the BsaI digested plasmid when hybridized)
Amy Johnson
Thanks, I'll check it out.Hanna Alalam I believe this plasmid is a modified version of pRGE with the Bsa1 sites removed. ' ggagaccgaggtctcg ' This is the sequence found between the end of U6 promoter and upstream of gRNA scaffold.
The plasmid on addgene with that name carries these sites. Also the sequence that you sent has two bsaI sites that are inverted relative to each other which is what you should have. You might not being seeing them because you are only looking a unique cutters if for example you are using snap gene (turn on unique and dual cutter and you will be able to see the sites).
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