gRNA-transient expression system for simplified gRNA delivery in CRISPR/Cas9 genome editing

The clustered regularly interspaced short palindromic repeats (CRISPR) and CRISPR-associated protein 9 (CRISPR/Cas9) system is one of the most powerful tools for genome engineering. However, some of the steps are laborious, reducing its usability. In this study, we have developed a simplified method, called the guide RNA-transient expression system (gRNATES), to deliver gRNA in yeast. In gRNA-TES, a DNA fragment containing the promoter and gRNA is prepared by two simple PCR steps and co-transformed with a DNA module into the host strain; all steps including PCR steps and yeast transformation are completed within 5-6 h in a single day, in contrast to conventional plasmid-based gRNA delivery systems, which require at least 3-4 days to construct and verify the gRNA-expressing plasmids. The performance of gRNA-TES was evaluated by the replacement of 150-kb, 200-kb, 300-kb, 400-kb, and 500-kb regions of yeast chromosome 4 with a DNA module. Increased numbers of transformants with a high frequency of expected replacement of even the 500-kb region were obtained with gRNA-TES as compared with transformation without gRNA-TES. In addition, the integrity of the replaced region was verified in 67%-100% of transformants tested by colony PCR. We believe that gRNATES will vastly increase the accessibility of CRISPR/Cas9 technology to biologists and biotechnologists by offering a simple, fast, and cost-effective tool to deliver gRNA in genome engineering. Furthermore, it might be applied to plant and animal systems if appropriate gene promoters are incorporated in the technology. (C) 2019, The Society for Biotechnology, Japan. All rights reserved.