3' Ribozyme design
Because the last few nucleotides of the guide RNA are relatively invariable (only depending on the chimera guide RNA design and the CRISPR system used), there are more choices regarding which 3' ribozyme can be used. Essentially any self-cleaving ribozymes could be used as long as the end of the guide RNA meets the sequence requirement upstream the cleavage site.

Hammerhead ribozymes could again be used if the guide RNA ends with "UH".



In the design above, only 4 nucleotides in the end of the hammerhead region need to be modified according to the guide RNA sequence. The same sequence could be used as long as the same chimerical guide RNA is used regardless of the target DNA sequence.


Alternatively, HDV ribozymes are also useful, as HDV ribozymes have no requirement on the sequence upstream of the cleavage site. The same sequence could be used for any guide RNA constructs.