Applications

Temporal and Spatial genome editing
By using ribozyme-gRNA-ribozyme (RGR) cassette design and free of the constrain inherited from the RNA polymerase III promoters, the in vivo expression of guide RNA for CRISPR systems could be controlled using well-understood RNA polymerase II promoters. For example, genes essential for early development stages are difficult to study as null mutants of those genes would not survive to later stages. By using promoters in later development stages to drive guide RNA expression, those genes could be knocked out after the organism passes the early development, allowing us to study the roles of those genes in later development stages. Additionally, we could sequentially knock out several genes by using sequentially active promoters to drive the expressing of guide RNAs targeting those genes. Moreover, CAS9 and guide RNA could be controlled under different and non-constitutive promoters, allowing the genome cleavages to happen only in the cells with both promoters active. Last but not the least, light- or chemically-inducible promoters could be used to control the guide RNA expression, allowing manual control of the genome cleavage events. Many of those applications are difficult or impossible if only the CAS9 expression could be manipulated.

Multiplex gene targeting
One possible and exciting application of ribozyme-gRNA-ribozyme (RGR) cassette design would be targeting multiple genes at the same time, at ease. Similar to the gene clusters organized in the form of operons, multiple RGR cassette could be tandemly joined and expressed using one promoter, and different guide RNAs targeting different sites would be automatically and simultaneously produced upon the transcription of the single primary transcript.

Detecting mutations generated by CRISPR-mediated genome editing
By using ribozyme-flanked guide RNA design (ribozyme-gRNA-ribozyme (RGR) cassette), guide RNA could be easily produced by in vitro transcription. The templates for in vitro transcription could be readily amplified from the RGR constructs using universal primers, and this greatly facilitates large scale genome applications.

It is challenging to efficiently detect the mutations using DNA-sequencing and restriction enzyme/CEL I-based digestions because CRISPR often generates many different mutations in a tissue or in an organism. We use Cas9 / gRNA-mediated digestion in vitro to detect the mutations generated by CRISPR. Any mutations generated by CRISPR will abolish the target site of Cas9/gRNA. Our method can detect a mixture of different mutations. For details, please see our recent paper (DOI: 10.1111/jipb.12152).