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

Easmin, Farhana, Hassan, Naim, Sasano, Yu, Ekino, Keisuke, Taguchi, Hisataka, Harashima, Satoshi
Journal of bioscience and bioengineering 2019 v.128 no.3 pp. 373-378
CRISPR-associated proteins, DNA fragmentation, chromosomes, cost effectiveness, gene editing, genes, plasmids, polymerase chain reaction, yeasts
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 (gRNA-TES), 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 gRNA-TES 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.