[3S02a] 05. Genome Editing Technology and its Applications - Part 2

3:10 PM - 3:25 PM JST(6:10 AM - 6:25 AM UTC)

[3GET-11] CRISPR-Cas type I-D (TiD-X) enables efficient, precise and versatile induction of diverse mutation patterns in human cells

○Naoki Wada¹, Emi Murakami¹, Kazuya Marui¹, Yuriko Osakabe², Keishi Osakabe¹ (1. Graduate School of Technology, Industrial and Social Sciences, Tokushima University (Japan), 2. School of Life Science and Technology, Science Tokyo (Japan))

Keywords:

Genome editing,CRISPR-Cas type I-D,TiD-X

The development of genome editing technologies has led to remarkable advances across a wide range of fields, including microbial engineering, crop improvement, and gene therapy. In recent years, technical innovation has been driven by both the expanding applications of the CRISPR-Cas9 system and the discovery and utilization of novel CRISPR-Cas systems. We previously identified a novel CRISPR-Cas type I-D system from cyanobacteria and developed it into a genome editing tool, termed “TiD”. TiD has been successfully applied to genome editing in both plants and mammalian cells (Osakabe et al., 2020, Osakabe, Wada et al., 2021). Furthermore, we identified a highly active TiD system “TiD-X” from another cyanobacterial species (Wada et al., in preparation). TiD-X consists of six Cas proteins and crRNA that recognizes longer target sequence (35-36 bp) compared to Cas9 (20 bp). This extended target recognition enables genome editing with higher specificity than other existing technologies. Notably, we found that TiD-X induces bi-directional long-range deletions at target sites, a unique feature that distinguishes it from other technologies. In this study, we developed an efficient gene knockout system using TiD-X. To enrich for TiD-introduced cells, we employed the visible marker system based on Tripartite Fluorescence Complementation (TriFC). Using the TiD-X in combination with the TriFC system, we successfully generated B2M gene knockout cells with efficiencies of up to 75% in human diploid HCT116 cells, without the need for antibiotic selection. The induced long-range deletions ranged from 2 kb to over 18 kb. These results demonstrate that the combination of TiD-X and the TriFC system provides a highly efficient gene knockout platform in human cells. Taking advantage of its unique bi-directional long deletion activity, we also developed a strategy to efficiently induce designed deletions. Using this approach, we successfully generated deletions of various sizes and types as intended. In addition, we also achieved base substitution at target sequence. These findings indicate that TiD-X can be used to induce a wide spectrum of genetic modifications, ranging from small mutations to long-range deletions (e.g. 10 kb). We are currently exploring the application of this technology to exon skipping for gene therapy. Overall, TiD-X is expected to open new avenues for genetic modification in the fields such as agriculture, industrial biotechnology, and biomedicine.