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

Morisaka, Hiroyuki, Yoshimi, Kazuto, Okuzaki, Yuya, Gee, Peter, Kunihiro, Yayoi, Sonpho, Ekasit, Xu, Huaigeng, Sasakawa, Noriko, Naito, Yuki, Nakada, Shinichiro, Yamamoto, Takashi, Sano, Shigetoshi, Hotta, Akitsu, Takeda, Junji, Mashimo, Tomoji·2019·Nature Communications
DOI 10.1038/s41467-019-13226-xPMC PMC6897959
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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.

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Metadata

Authors
Morisaka, H., Yoshimi, K., Okuzaki, Y., Gee, P., Kunihiro, Y., Sonpho, E., Xu, H., Sasakawa, N., Naito, Y., Nakada, S., Yamamoto, T., Sano, S., Hotta, A., Takeda, J., & Mashimo, T.
ORCID
Venue
Nature Communications
Publisher
Springer Science and Business Media LLC
Published
2019-12-06
Volume
10
Issue
1
ISSN
2041-1723
DOI
10.1038/s41467-019-13226-x
PMC
PMC6897959
Cited by
198
References
7
Language
en
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