[Apr. 8] 121st IRCMS Seminar

March 17 2025

We would like to inform you that the 121st IRCMS seminar has been scheduled as below.
* This IRCMS seminar is open to everyone.

Date      : April 8, 2025 (Tuesday)

Time      : 16:00-17:00

ZOOM　: Meeting ID: 964 6814 3938
            　 Passcode: Sem08Apr

Speaker : Dr. Masaki Kawamata (Kyushu University)

Title        :  Safe and Precise Genome Editing by Fine-tuning CRISPR-Cas9 Activity

Abstract 

CRSIPR-Cas9 system has opened up the avenue to versatile genome editing. However, together with known off-target effects, the current CRISPR-Cas9 platform has raised several concerns, including severe cytotoxicity, large genomic alteration, and dysregulation of allele-selective editing. It has been recently suggested that the cause of these problems is due to the excessive Cas9 activity. Therefore, strategies to regulate its activity have been proposed, but few practical methods are available.

We here developed a novel technology for fine-tuning of Cas9 activity through a simple design modification of sgRNA ("safeguard sgRNA"). Addition of cytosine stretches to the 5'-end of conventional sgRNAs enabled length-dependent tuning Cas9 activity by multiple mechanisms. Overall, short cytosine extension reduced p53-dependent cytotoxicity and enhanced homology-directed repair, while maintaining bi-allelic editing capacity. Long extension lowered on-target activity but improved specificity, thereby facilitating mono-allelic and precise editing. The integration of a fluorescence-based allele-specific indel monitor system (AIMS), computational simulation, and systematic validation established distinct optimal windows of Cas9 activity for diverse applications, including precise one-step generation and correction of disease-associated single-nucleotide substitutions. Together, the "safeguard sgRNA" platform represents a promising strategy to maximize safety and efficiency of diverse editing through optimizing Cas9 activity, which will contribute to advances in research and medical applications.

2-3 major papers:

1. Kawamata M*, Suzuki HI*, Kimura R, Suzuki A*. Optimization of Cas9 activity through the addition of cytosine extensions to single-guide RNAs. Nature Biomedical Engineering, 2023 (*Corresponding)

2. Katsuda T, Kawamata M, Hagiwara K, Takahashi RU, Yamamoto Y, Camargo FD, Ochiya T. Conversion of Terminally Committed Hepatocytes to Culturable Bipotent Progenitor Cells with Regenerative Capacity. Cell Stem Cell, 5;20(1):41-55. 2017

3. Kawamata M, Ochiya T. Generation of genetically modified rats from embryonic stem cells. PNAS, 10;107(32):14223-8, 2010

Flyer: (Click to enlarge)