PMID- 31811138 OWN - NLM STAT- MEDLINE DCOM- 20200302 LR - 20231020 IS - 2041-1723 (Electronic) IS - 2041-1723 (Linking) VI - 10 IP - 1 DP - 2019 Dec 6 TI - CRISPR-Cas3 induces broad and unidirectional genome editing in human cells. PG - 5302 LID - 10.1038/s41467-019-13226-x [doi] LID - 5302 AB - 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. FAU - Morisaka, Hiroyuki AU - Morisaka H AUID- ORCID: 0000-0002-1158-8116 AD - Department of Genome Biology, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. AD - Department of Dermatology, Kochi Medical School, Kochi University, Kochi, 783-8505, Japan. FAU - Yoshimi, Kazuto AU - Yoshimi K AD - Genome Editing Research and Development Center, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. AD - Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. AD - Division of Animal Genetics, Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan. FAU - Okuzaki, Yuya AU - Okuzaki Y AUID- ORCID: 0000-0002-7212-4366 AD - Center for iPS Cell Research and Application (CiRA), Department of Clinical Application, Kyoto University, Kyoto, 606-8507, Japan. FAU - Gee, Peter AU - Gee P AUID- ORCID: 0000-0002-2481-5103 AD - Center for iPS Cell Research and Application (CiRA), Department of Clinical Application, Kyoto University, Kyoto, 606-8507, Japan. FAU - Kunihiro, Yayoi AU - Kunihiro Y AUID- ORCID: 0000-0001-7153-1667 AD - Genome Editing Research and Development Center, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. FAU - Sonpho, Ekasit AU - Sonpho E AD - Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. AD - Department of Biology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand. FAU - Xu, Huaigeng AU - Xu H AUID- ORCID: 0000-0002-4415-1287 AD - Center for iPS Cell Research and Application (CiRA), Department of Clinical Application, Kyoto University, Kyoto, 606-8507, Japan. FAU - Sasakawa, Noriko AU - Sasakawa N AD - Center for iPS Cell Research and Application (CiRA), Department of Clinical Application, Kyoto University, Kyoto, 606-8507, Japan. FAU - Naito, Yuki AU - Naito Y AD - Database Center for Life Science, Mishima, 411-8540, Japan. AD - National Institute of Genetics, Mishima, 411-8540, Japan. FAU - Nakada, Shinichiro AU - Nakada S AD - Institute for Advanced Co-Creation Studies, Osaka University, Osaka, 565-0871, Japan. FAU - Yamamoto, Takashi AU - Yamamoto T AD - Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan. FAU - Sano, Shigetoshi AU - Sano S AD - Department of Dermatology, Kochi Medical School, Kochi University, Kochi, 783-8505, Japan. FAU - Hotta, Akitsu AU - Hotta A AUID- ORCID: 0000-0002-2619-7441 AD - Center for iPS Cell Research and Application (CiRA), Department of Clinical Application, Kyoto University, Kyoto, 606-8507, Japan. akitsu.hotta@cira.kyoto-u.ac.jp. FAU - Takeda, Junji AU - Takeda J AD - Department of Genome Biology, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. jjjtakeda@biken.osaka-u.ac.jp. AD - Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan. jjjtakeda@biken.osaka-u.ac.jp. FAU - Mashimo, Tomoji AU - Mashimo T AD - Genome Editing Research and Development Center, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. mashimo@ims.u-tokyo.ac.jp. AD - Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan. mashimo@ims.u-tokyo.ac.jp. AD - Division of Animal Genetics, Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan. mashimo@ims.u-tokyo.ac.jp. LA - eng PT - Journal Article PT - Research Support, Non-U.S. Gov't DEP - 20191206 PL - England TA - Nat Commun JT - Nature communications JID - 101528555 RN - 0 (CRISPR-Associated Proteins) RN - EC 3.6.4.- (DNA Helicases) SB - IM MH - CRISPR-Associated Proteins/classification/*genetics/metabolism MH - CRISPR-Cas Systems/*genetics MH - Clustered Regularly Interspaced Short Palindromic Repeats MH - DNA Cleavage MH - DNA Helicases/metabolism MH - Exons MH - Gene Editing/*methods MH - Gene Expression Regulation/genetics MH - Gene Knockout Techniques/methods MH - HEK293 Cells MH - Humans MH - Induced Pluripotent Stem Cells MH - Muscular Dystrophy, Duchenne/genetics MH - Sequence Deletion PMC - PMC6897959 COIS- The authors declare no competing interests. EDAT- 2019/12/08 06:00 MHDA- 2020/03/03 06:00 PMCR- 2019/12/06 CRDT- 2019/12/08 06:00 PHST- 2019/09/19 00:00 [received] PHST- 2019/10/29 00:00 [accepted] PHST- 2019/12/08 06:00 [entrez] PHST- 2019/12/08 06:00 [pubmed] PHST- 2020/03/03 06:00 [medline] PHST- 2019/12/06 00:00 [pmc-release] AID - 10.1038/s41467-019-13226-x [pii] AID - 13226 [pii] AID - 10.1038/s41467-019-13226-x [doi] PST - epublish SO - Nat Commun. 2019 Dec 6;10(1):5302. doi: 10.1038/s41467-019-13226-x.