Hiroyuki MorisakaKazuto YoshimiYuya OkuzakiPeter GeeYayoi KunihiroEkasit SonphoHuaigeng XuNoriko SasakawaYuki NaitoShinichiro NakadaTakashi YamamotoShigetoshi SanoAkitsu HottaJunji TakedaTomoji MashimoCenter for iPS Cell Research and ApplicationInstitute of Medical Science The University of TokyoHiroshima UniversityOsaka UniversityNational Institute of Genetics MishimaKochi UniversityMahidol UniversityDatabase Center for Life Science (DBCLS)2020-01-272020-01-272019-12-01Nature Communications. Vol.10, No.1 (2019)204117232-s2.0-85076283383https://repository.li.mahidol.ac.th/handle/20.500.14594/50021© 2019, The Author(s). 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.Mahidol UniversityBiochemistry, Genetics and Molecular BiologyChemistryArticleSCOPUS10.1038/s41467-019-13226-x