CRISPR-Cas3 induces broad and unidirectional genome editing in human cells.,Nature Communications

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CRISPR-Cas3 induces broad and unidirectional genome editing in human cells.
Nature Communications ( IF 14.7 ) Pub Date : 2019-12-06 , DOI: 10.1038/s41467-019-13226-x
Hiroyuki Morisaka _{1,

2} , Kazuto Yoshimi _{3,

4,

5} , Yuya Okuzaki ₆ , Peter Gee ₆ , Yayoi Kunihiro ₃ , Ekasit Sonpho _{4,

7} , Huaigeng Xu ₆ , Noriko Sasakawa ₆ , Yuki Naito _{8,

9} , Shinichiro Nakada ₁₀ , Takashi Yamamoto ₁₁ , Shigetoshi Sano ₂ , Akitsu Hotta ₆ , Junji Takeda _{1,

12} , Tomoji Mashimo _{3,

4,

5}

Affiliation

Department of Genome Biology, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan.
Department of Dermatology, Kochi Medical School, Kochi University, Kochi, 783-8505, Japan.
Genome Editing Research and Development Center, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan.
Institute of Experimental Animal Sciences, Graduate School of Medicine, Osaka University, Osaka, 565-0871, Japan.
Division of Animal Genetics, Laboratory Animal Research Center, Institute of Medical Science, The University of Tokyo, Tokyo, 108-8639, Japan.
Center for iPS Cell Research and Application (CiRA), Department of Clinical Application, Kyoto University, Kyoto, 606-8507, Japan.
Department of Biology, Faculty of Science, Mahidol University, Bangkok, 10400, Thailand.
Database Center for Life Science, Mishima, 411-8540, Japan.
National Institute of Genetics, Mishima, 411-8540, Japan.
Institute for Advanced Co-Creation Studies, Osaka University, Osaka, 565-0871, Japan.
Department of Mathematical and Life Sciences, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, 739-8526, Japan.
Research Institute for Microbial Diseases, Osaka University, Osaka, 565-0871, Japan.

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.

中文翻译：

CRISPR-Cas3在人类细胞中诱导广泛的单向基因组编辑。

尽管单组分2类CRISPR系统（例如II型Cas9或V型Cas12a（Cpf1））已广泛用于真核细胞中的基因组编辑，但多组分1类CRISPR的应用却很少开发。在这里，我们证明IE CRISPR类型在人类细胞中介导了独特的DNA裂解活性。值得注意的是，具有解旋酶和核酸酶活性的Cas3主要触发5'-ARG原间隔子相邻基序（PAM）上游数千个碱基对的缺失，而没有明显的脱靶活性。这种Cas3介导的定向降解和广泛的DNA降解可用于引入功能性基因敲除和敲入。作为潜在治疗应用的一个例子，我们在患者诱导的多能干细胞（iPSCs）中显示了Cas3介导的杜兴氏肌营养不良症（DMD）基因的外显子跳跃。

更新日期：2019-12-06

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