Currently, sgRNAs are usually transcribed by type Ⅲ promoters in CRISPR/Cas9 system, which makes it difficult to achieve tissue specific and time specific expression. Cytotoxicity is an additional concern for constitutive transcription of small RNAs. In this paper, we designed an intron-based approach for sgRNA expression by type Ⅱ promoters, which uses ribozyme switches to facilitate sgRNA release from the spliced intron. We designed three combinations of "ribozyme sgRNA ribozyme" at both ends of sgRNAs to release them through the self-cleavage of ribozymes. The results indicated that the design of HHRz-sgRNA-HDVRz could correctly release sgRNA from the intron, and the released sgRNAs could achieve gene editing in human cells. Our finding demonstrated that an intron-based sgRNA expression cassette was compatible with type Ⅱ promoters and ribozyme switches could be used to facilitate sgRNAs release for gene editing.
