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Development of CRISPR-Cas3 system for human genome editing

Kazuto Yoshimi, Yoko Yamauchi, Hiromi Taniguchi, Tomoaki Fujii and Tomoji Mashimo

Although single-component Class 2 CRISPR sys-tems, such as type II Cas9 or type V Cas12a (Cpf1), are widely used for genome editing in eukaryotic cells, the application of multi-component Class 1 CRISPR has yet to be developed. We demonstrate that type I-E CRISPR, which is composed of Escherichia coli Cas-cade, Cas3, and programmable pre-crRNA, mediates distinct DNA cleavage activity in human cells. Nota-bly, Cas3, which possesses helicase and nuclease ac-tivity, predominantly triggered several thousand base pair deletions upstream of the 5’-ARG proto-spacer adjacent motif (PAM), without prominent off-target activity. This Cas3-mediated directional and broad DNA degradation can be used to introduce functional gene knockouts and knock-ins. As an ex-ample of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne mus-cular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broad-en our understanding of the Class 1 CRISPR system, which may serve as a novel and unique genome edit-ing tool in eukaryotic cells distinct from the Class 2 CRISPR system.

Rapid and accurate detection of novel coronavi-rus SARS-CoV-2 using CRISPR-Cas3

Kazuto Yoshimi, Kohei Takeshita1, Seiya Yamay-oshi2, Satomi Shibumura3, Yuko Yamauchi, Masaki Yamamoto1, Hiroshi Yotsuyanagi4,Yoshihiro Kawa-oka2,5, and Tomoji Mashimo : 1Life Science Research Infrastructure Group,Advanced Photon Technology Division, RIKEN SPring-8 Center, Hyogo 679-5148 Japan, 2Division of Virology, Department of Micro-biology and Immunology, Institute of Medical Sci-ence, University of Tokyo, Minato-ku, 108-8639, To-kyo, Japan, 3C4U Corporation, Osaka 565-0871, Japan, 4Division of Infectious Diseases and Applied Immunology, Institute of Medical Science, Universi-ty of Tokyo, Minato-ku, 108-8639,Tokyo, Japan, 5Department of Pathobiological Sciences, School of Veterinary Medicine, University of Wisconsin-Mad-ison, Madison 53711, Wisconsin, USA

Novel coronavirus SARS-CoV-2 outbreaks have rapidly spread to multiple countries, highlighting the urgent necessity for fast, sensitive, and specific diag-nostic tools for virus surveillance. Here, the previous-ly unknown collateral single-stranded DNA cleavage we observed with type I CRISPR-Cas3 highlights its potential for development as a Cas3-mediated rapid (within 40 min), low-cost, instrument-free detection

Center for Experimental Medicine and Systems Biology

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

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

教　授　博士（人間・環境学）　真　下　知　士講　師　博士（医科学）　　　　吉　見　一　人

Genome engineering technologies, such as Zinc finger nucleases (ZFNs), TAL effector nucleases (TALENs), and clustered regularly interspaced short palindromic repeats (CRISPR)-associated (Cas) nucleases (CRISPR/Cas), have widely used in life science and medical science. We are developing novel genome editing tools to overcome technical and patent limitation of CRISPR-Cas9 system. We are also developing the efficient genome editing strategies with these tools in rodents. These technologies facilitate easy and flexible gene editing in living organisms.

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method for SARS-CoV-2. This Cas3-based assay is comparable with Cas12- and real-time reverse-tran-scriptase PCR-based assays in its speed and sensitivi-ty, but offers greater specificity for single-base-pair discrimination while negating the need for highly trained operators. These findings support the use of CRISPR diagnostics for point-of-care testing in pa-tients with suspected SARS-CoV-2 infections.

Combination of NHEJ and HDR  for  efficient  and precise plasmid knock-ins in mice and rats

Kazuto Yoshimi, Yuichiro Oka1,2, Yoshiki Miyasa-ka3, Yuko Kotani3, Misato Yasumura1, Yoshihiro Uno3, Kosuke Hattori3, Arisa Tanigawa3, Manami Oya4, Kazuhiro Nakamura4, Toshihide Yamashita5, Makoto Sato1,2, Natsuki Matsushita6, Kazuto Kobayashi7, and Tomoji Mashimo : 1Department of Anatomy and Neurosciences, Graduate School of Medicine, Osaka University, Osaka 565-0871, Ja-pan, 2Department of Child Development, United Graduate School of Child Development, Osaka Uni-versity, Osaka 565-0871, Japan, 3Institute of Exper-imental Animal Sciences, Graduate School of Medi-cine, Osaka University, Osaka 565-0871, Japan, 4Department of Integrative Physiology, Nagoya Uni-versity Graduate School of Medicine, Nagoya 466-8550, Japan, 5Department of Molecular Neurosci-ence, Graduate School of Medicine, Osaka University, Osaka 565-0871, Japan

6, Division of Laboratory Animal Research, Aichi Medical University School of Medicine, Aichi 480-1195, Japan7, Department of Molecular Genetics, Institute of Biomedical Sciences, Fukushima Medical Universi-ty School of Medicine, Fukushima 960-1295, Japan

CRISPR-Cas9 systems have been widely used for gene targeting in mice and rats. The non-homologous end joining (NHEJ) repair pathway, which is domi-nant in zygotes, efficiently induces insertion or dele-tion (indel) mutations as gene knockouts (KOs) at tar-geted sites, whereas gene knock-ins (KIs) via homology-directed repair (HDR) are difficult to gen-erate. In this study, we used a double-stranded DNA (dsDNA) donor template with Cas9 and two single guide RNAs (sgRNAs), one designed to cut the tar-geted genome sequences and the other to cut both the flanked genomic region and one homology arm of the dsDNA plasmid, resulting in 20%–50% KI efficiency among G0 pups. G0 KI mice carried NHEJ-dependent indel mutations at one targeting site that was de-signed at the intron region, and HDR-dependent pre-cise KIs of the various donor cassettes spanning from 1 to 5 kbp, such as EGFP, mCherry, Cre, and genes of interest, at the other exon site. These findings indicate that this combinational method of NHEJ and HDR mediated by the CRISPR-Cas9 system, named Com-bi-CRISPR, facilitates the efficient and precise KIs of plasmid DNA cassettes in mice and rats.

Publications

1. Photoactivatable Cre knock-in mice for spatiotem-poral control of genetic engineering in vivo. Yoshi-mi K, Yamauchi Y, Tanaka T, Shimada T, Sato M, Mashimo T. Lab Invest. 2020 Sep 5. doi: 10.1038/s41374-020-00482-5.

2. Aspects of Gene Therapy Products Using Current Genome-Editing Technology in Japan. Yamaguchi T, Uchida E, Okada T, Ozawa K, Onodera M, Kume A, Shimada T, Takahashi S, Tani K, Nasu Y, Mashi-mo T, Mizuguchi H, Mitani K, Maki K. Hum Gene Ther. 2020 Oct;31(19-20):1043-1053. doi: 10.1089/hum.2020.156.

3. Combi-CRISPR: combination of NHEJ and HDR

provides efficient and precise plasmid-based knock-ins in mice and rats. Yoshimi K, Oka Y, Mi-yasaka Y, Kotani Y, Yasumura M, Uno Y, Hattori K, Tanigawa A, Sato M, Oya M, Nakamura K, Matsu-shita N, Kobayashi K, Mashimo T. Hum Genet. 2020 Jul 2. doi: 10.1007/s00439-020-02198-4.

4. Rapid and accurate detection of novel coronavirus SARA-CoV-2 using CRISPR-Cas3　Yoshimi K, Takeshita K, Yamayoshi S, Shibumura

S,Yamauchi Y, Yamamoto M, Yotsuyanagi H, Ka-waoka Y and Mashimo T. medRivx:2020-0602 doi: https://doi.org/10.1101/2020.06.02.20119875

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