Background

Almost all of the reported cases of CRISPR-mediated gene editing used promoters of small nuclear RNAs such as the U6 and U3 snRNA promoters to drive the production of gRNAs in vivo (Cong et al., 2013; Mali et al., 2013). However, the U6 and U3 promoters have several major limitations: 1) They are constitutively active and not tunable; 2) They lack cell/tissue specificities; 3) They have not been well defined in many organisms; 4) U6 requires a G and U3 requires an A for transcription initiation, thus limiting target selections; 5) They are not suitable for in vitro transcriptions because of the lack of commercial RNA polymerase III. Unfortunately, RNA polymerase II promoters, which constitute the majority of the characterized promoters, cannot be directly used for gRNA production in vivo because of the following reasons: 1) The primary transcripts of RNA polymerase II promoters undergo extensive processing such as 5’-end capping, 3’-end polyadenylation, and splicing out of the introns. Some of the modifications may render the designed gRNA non-functional. 2) The mature RNA molecules are transported into cytosol; thus they are physically separated from the intended targets that are located in the nucleus. That is why production of gRNA in vivo using U6 and U3 snRNA promoters has been the dominant method (Gao and Zhao, 2014; Yoshioka et al., 2015). In this protocol, we use a ribozyme-based strategy to overcome the aforementioned limitations of RNA polymerase III promoters, enabling gRNA production from any promoters and in any organisms. We design an artificial gene named RGR (Ribozyme-gRNA-Ribozyme) that, once transcribed, generates an RNA molecule with ribozyme sequences flanking both ends of the designed gRNA (Gao and Zhao, 2014). We show that the primary transcripts of RGR undergo self-catalyzed cleavage to precisely release the desired gRNA, which can efficiently guide sequence‐specific cleavage of DNA targets in vitro and in vivo (Gao and Zhao, 2014). RGR can be transcribed from any promoters and thus allows for cell‐and tissue‐specific genome editing if appropriate promoters are chosen.