Trans-cleaving techniques have been most enthusiastically embraced in the development of therapy for genetic diseases, particularly in the correction of monogenic recessive mutations at the messenger RNA level. However, easy degradation and poor catalytic activity in vivo remain significant obstacles to trans-cleaving of the hammerhead ribozyme. Herein, we found a novel scaffold RNA that stabilizes the ribozyme structure in trans-cleaving and promotes the knockdown efficiency of the hammerhead ribozyme in specific regions of living cells. We can give the trans-cleaving hammerhead ribozyme the ability to knock down specific genes in specific cell regions by changing different scaffolds. Therefore, our study proves the potential usefulness of the RNA knockdown strategy with high-specific trans-cleaving hammerhead ribozyme as a therapeutic approach in gene therapy.