Guidelines for ribozyme-gRNA-ribozyme (RGR) cassette design
To produce guide RNA with no extra sequence, one self-cleaving ribozyme is attached to EACH end of the guide RNA sequence. The cleavage site of the 5' ribozyme should be immediately before the first nucleotide of the guide RNA (also the first nucleotide of the target sequence), and the cleavage site of the 3' ribozyme should be immediately after the last nucleotide of the guide RNA.
Because self-cleaving ribozymes have different requirements on the sequences upstream or downstream of the cleavage site, different classes of ribozymes could be utilized on either end of the guide RNA. Click the following links for more information: