[P005] Poster

12:10 PM - 1:00 PM JST(3:10 AM - 4:00 AM UTC)

[P03-315] Development of Target-G, a novel genome editing tool that enables local gene evolution

○Ken-ichiro Taoka¹, Zenpei Shimatani¹, Hitoshi Mitsunobu¹, Rie Terada¹, Keiji Nishida¹ (1. Engineering Biology Research Center, Kobe University (Japan))

Keywords:

genome editing

Genome editing is a technology that introduces desired mutations into genome DNA, and it has become increasingly important in life sciences, medicine, and agriculture in recent years. Among genome editing tools, CRISPR-Cas9 has played a pivotal role due to its simplicity and high editing efficiency. In CRISPR-Cas9, Cas9 protein specifically binds to a genomic sequence defined by gRNA, and the nuclease activity of Cas9 causes a double-strand break in the DNA. The damaged DNA is sometimes repaired incompletely, resulting in mutations at specific locations within the genome. However, the introduced mutations are unpredictable, and many of them generally lead to disruption of gene function. To overcome the problems, a technology called base editing has been developed. Base editing enables precise genome editing at the single-nucleotide level by using a base editor in which an engineered deaminase is fused to nCas9 with nickase activity. However, the restriction of the editing window sometimes makes practical use difficult. These two editing tools are used when the region to be edited is predetermined. On the other hand, an evolutionary engineering approach is also becoming important that introduces random mutations into a wide region of a gene to create genetic diversity and then searches for novel mutants suitable for the purpose. Here, we report a novel genome editing tool that can introduce random mutations in a wide DNA region, named Target-G. In Target-G, a DNA glycosidase is modified and designed to introduce mutations near the region defined by gRNA by fusing with dCas9, a nuclease dead Cas9. We investigated how Target-G works using rice, a model plant for monocots. In rice callus transformed with the Target-G vector, mutations were introduced in a region of approximately 100 bp containing the gRNA area, and some of these mutations were stably inherited by plants of the T1 and T2 generation. This technology is expected to be a unique molecular breeding tool that enables artificial evolution of genes and the generation of diversity in the genome.