Figure 2: Restoring blocked guide RNA activity with an embedded hammerhead ribozyme in human cells.

(a) Schematic representation of the hammerhead ribozyme-embedded guide RNA and the expected activities in the presence of Cas9; (b) Genome-editing activities of hammerhead ribozyme-embedded guide RNAs in HEK293-GFP cells. The blocking sequence that is complementary to the spacer was fused to the guide RNA through a hammerhead ribozyme (HHR-bsgRNA). The length of the blocking sequence was varied from 10 to 17 nt. In HHR-bsgRNA variants, the hammerhead ribozyme is active and the blocking sequence is removed in situ once the guide RNA is transcribed, whereas in dHHR-bsgRNA variants, the hammerhead ribozyme is inactive and the blocking sequence remains appended to the guide RNA. The full activity of HHR-bsgRNA is shown by directly expressing the processed HHR-bsgRNA without the blocking sequence (pHHR-sgRNA). Canonical sgRNA (WT) served as the positive control and cells that were transfected with the Cas9 expression plasmid but no guide RNA plasmid were used as a negative control. The GFP fluorescence loss was quantified by comparing the mean cell fluorescence in transfected cells to that in cells treated with lipids only. Values and error bars reflect mean GFP fluorescence loss and the s.d. of three biological replicates.