CRISPR-Cas3 induces broad and unidirectional genome editing in human cells

Hiroyuki Morisaka, Kazuto Yoshimi, Yuya Okuzaki, Peter Gee, Yayoi Kunihiro, Ekasit Sonpho, Huaigeng Xu, Noriko Sasakawa, Yuki Naito, Shinichiro Nakada, Takashi Yamamoto, Shigetoshi Sano, Akitsu Hotta (), Junji Takeda () and Tomoji Mashimo ()
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Hiroyuki Morisaka: Osaka University
Kazuto Yoshimi: Osaka University
Yuya Okuzaki: Kyoto University
Peter Gee: Kyoto University
Yayoi Kunihiro: Osaka University
Ekasit Sonpho: Graduate School of Medicine, Osaka University
Huaigeng Xu: Kyoto University
Noriko Sasakawa: Kyoto University
Yuki Naito: Database Center for Life Science
Shinichiro Nakada: Osaka University
Takashi Yamamoto: Hiroshima University
Shigetoshi Sano: Kochi University
Akitsu Hotta: Kyoto University
Junji Takeda: Osaka University
Tomoji Mashimo: Osaka University

Nature Communications, 2019, vol. 10, issue 1, 1-13

Abstract: Abstract Although single-component Class 2 CRISPR systems, 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 been less developed. Here we demonstrate that type I-E CRISPR mediates distinct DNA cleavage activity in human cells. Notably, Cas3, which possesses helicase and nuclease activity, predominantly triggered several thousand base pair deletions upstream of the 5′-ARG protospacer 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 example of potential therapeutic applications, we show Cas3-mediated exon-skipping of the Duchenne muscular dystrophy (DMD) gene in patient-induced pluripotent stem cells (iPSCs). These findings broaden our understanding of the Class 1 CRISPR system, which may serve as a unique genome editing tool in eukaryotic cells distinct from the Class 2 CRISPR system.

Date: 2019
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DOI: 10.1038/s41467-019-13226-x

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