Ribozymes based self-cleavage sgRNA and its application in gene editing

Abstract:

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.