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Center for Experimental Medicine and Systems Biology

Division of Genome Engineering
ゲノム編集研究分野

ProfessorTomoji Mashimo, Ph.D.
Senior Assistant Professor Kazuto Yoshimi, Ph.D.

教講授師

博士
（人間・環境学） 真博士（医科学）
吉下見知一士人

Genome engineering technologies such as clustered regularly interspaced shortpalindromic repeats (CRISPR)-associated (Cas) nucleases (CRISPR-Cas) havebeen widely used in life sciences and medicine. We have developed a novel
genome editing tool, CRISPR-Cas3, to overcome the technical and patent
limitations of the CRISPR-Cas9 system. We are analyzing the molecularmechanisms underlying Cas3-mediated genome editing in human cells andimproving this tool for translational research, such as gene therapy and viraldiagnostics. We are also developing some efficient genome editing strategies using
these tools in rodents. These technologies enable easy and flexible gene editing inliving organisms.

Type I-E CRISPR-Cas3 for large-scale genomic
modifications in mice and rats
Kazuto Yoshimi, Akihiro Kuno1, Kosuke Hattori,
Yoko Yamauchi, Kohei Takeshita2, Hiromi Taniguchi, Satoru Takahashi1, Tomoji Mashimo
1, Department of Anatomy and Embryology, Faculty of Medicine, University of Tsukuba
2, Life Science Research Infrastructure Group, Advanced Photon Technology Division, RIKEN
SPring-8 Center
Genome editing technologies are highly effective
tools for genetic engineering in various organisms including experimental animals. Type I-E CRISPR-Cas3
uses an RNA-guided multi Cas-protein complex, Cascade, which detects and degrades foreign nucleic acids via the helicase-nuclease Cas3 protein. However,
it is unclear whether the system can be used for genome editing in fertilized eggs.
We applied the CRISPR-Cas3 system with severalmodification to generate genetically modified animals, and could generated knockout mice and rats inseveral genetic loci with optimizing method for theintroduction into embryos even by using electropora-tion methods. This work with the Type I CRISPR zygote editing system represents a significant leap forward, offering increased flexibility and broader
applications in genetic engineering across multiplespecies.
CRISPR-Cas3-based diagnostics for virus detection and genetic screening
Koji Asano, Rika Hirano, Kohei Takeshita1, YukoYamauchi, Kazuto Yoshimi, Tomoji Mashimo
1, Life Science Research Infrastructure Group,Advanced Photon Technology Division, RIKEN
SPring-8 Center, Hyogo 679-5148 Japan
CRISPR-based diagnostics (CRISPR-dx), including the Cas12-based DETECTR and Cas13-based
SHERLOCK Class 2 CRISPRs, have been used to detect the presence of DNA or RNA from pathogens
such as the 2009 pandemic influenza A (IVA) virus
and the 2019 novel coronavirus SARS-CoV-2. Here,the collateral single-stranded DNA cleavage we observed with class 1 type I CRISPR-Cas3 highlights its
potential for development as a Cas3-mediated, rapid(within 40 minutes), low-cost, instrument-free detec-106
tion method for SARS-CoV-2. This assay, which wehave named Cas3-operated nucleic acid detection(CONAN), not only detects SARS-CoV-2 in clinicalsamples, but also provides specific detection of single

base pair mutations in IVA variants. We are also optimizing protocols for cancer detection by liquid biopsy and genetic screening for inherited diseases suchas trinucleotide repeat disorders.

Publications
1.		Junki Kawakami, Satoshi Hisanaga, Yuki Yoshimoto, Tomoji Mashimo, Takehito Kaneko, NaotoYoshimura, Masaki Shimada, Makoto Tateyama,
Hideto Matsunaga, Yuto Shibata, Shuntaro Tanimura, Kosei Takata, Takahiro Arima, [ ... ], TakeshiMiyamoto		Remnant tissue enhances early postoperative biomechanical strength and infiltration of Scleraxis-positive cells within the grafted tendon in a rat
anterior cruciate ligament reconstruction model		PLOS ONE https://doi.org/10.1371/journal.pone.
02939442.		Natsuki Matsushita, Shigeki Kato, Kayo Nishizawa, Masateru Sugawara, Kosei Takeuchi, Yoshiki
Miyasaka, Tomoji Mashimo, *Kazuto Kobayashi		Protocol for highly selective transgene expression
through the flip-excision switch system by using aunilateral spacer sequence in rodents.		 STAR protocols 4(4) 102667-102667 2023
3.		Hiroki Furuie, *Yuka Kimura, Tatsuhiro Akaishi,
Misa Yamada, Yoshiki Miyasaka, Akiyoshi Saitoh,
Norihiro Shibuya, Akiko Watanabe, Naoki Kusunose, Tomoji Mashimo, Takeo Yoshikawa, Mitsuhiko Yamada, Kazuho Abe, Hideo Kimura		Hydrogen sulfide and polysulfides induce GABA/glutamate/D-serine release, facilitate hippocampal LTP, and regulate behavioral hyperactivity		 Sci Rep 2023 31;13(1):17663.
4.		Yaguang Luo, Shinya Akatsuka, Yashiro Motooka,
Yingyi Kong, Hao Zheng, Tomoji Mashimo, Tatsu-hiko Imaoka, *Shinya Toyokuni		BRCA1 haploinsufficiency impairs iron metabolism to promote chrysotile‐induced mesothelioma
via ferroptosis resistance		 Cancer Sci. 2023, 114(4): 1423–1436.
5.		Masato Kinboshi, Saki Shimizu, Kentaro Tokudome, Tomoji Mashimo, Tadao Serikawa, Hidefumi Ito, Ryosuke Takahashi, Akio Ikeda, *Yukihiro Ohno		Imbalance of glutamatergic and GABAergic neurotransmission in audiogenic seizure-susceptible
Leucine-rich glioma-inactivated 1 (Lgi1)-mutantrats		 Heliyon 9(7) e17984-e17984 2023
6.		Victoria Morley, Karamjit Singh Dolt, Carlos J Alcaide-Corral, Tashfeen Walton, Christophe Lucatelli, Tomoji Mashimo, *Adriana A S Tavares,*Tilo Kunath		In vivo18F-DOPA PET imaging identifies a dopaminergic deficit in a rat model with a G51D α-synuclein mutation		 Frontiers in Neuroscience 2023 24:17:1095761.
7.		Masayuki Mori, Jian Dai, Hiroki Miyahara, YingLi, Xiaojing Kang, Kazuto Yoshimi, Tomoji Mashimo, Keiichi Higuchi, Kiyoshi Matsumoto		Cyba and Nox2 mutant rats show different incidences of eosinophilia in the genetic backgroundand sex-dependent manner		 Exp Anim. 2023 May 17;72(2):233-241.