Graduate School of Technology, Industrial and Social Sciences, Tokushima University, Tokushima, Tokushima 770-8503, Japan.
Department of Computational Systems Biology, Faculty of Biology-Oriented Science and Technology, Kindai University, Kinokawa, Wakayama 649-6493, Japan.
Nucleic Acids Res. 2021 Jun 21;49(11):6347-6363. doi: 10.1093/nar/gkab348.
Adoption of CRISPR-Cas systems, such as CRISPR-Cas9 and CRISPR-Cas12a, has revolutionized genome engineering in recent years; however, application of genome editing with CRISPR type I-the most abundant CRISPR system in bacteria-remains less developed. Type I systems, such as type I-E, and I-F, comprise the CRISPR-associated complex for antiviral defense ('Cascade': Cas5, Cas6, Cas7, Cas8 and the small subunit) and Cas3, which degrades the target DNA; in contrast, for the sub-type CRISPR-Cas type I-D, which lacks a typical Cas3 nuclease in its CRISPR locus, the mechanism of target DNA degradation remains unknown. Here, we found that Cas10d is a functional nuclease in the type I-D system, performing the role played by Cas3 in other CRISPR-Cas type I systems. The type I-D system can be used for targeted mutagenesis of genomic DNA in human cells, directing both bi-directional long-range deletions and short insertions/deletions. Our findings suggest the CRISPR-Cas type I-D system as a unique effector pathway in CRISPR that can be repurposed for genome engineering in eukaryotic cells.
近年来，CRISPR-Cas 系统（如 CRISPR-Cas9 和 CRISPR-Cas12a）的采用彻底改变了基因组工程；然而，CRISPR 类型 I——细菌中最丰富的 CRISPR 系统——的基因组编辑应用仍不太发达。I 型系统，如 I-E 型和 I-F 型，由抗病毒防御相关的 CRISPR 复合物（“Cascade”：Cas5、Cas6、Cas7、Cas8 和小亚基）和 Cas3 组成，Cas3 降解靶 DNA；相比之下，对于缺乏典型 Cas3 核酸酶的 CRISPR-Cas 类型 I-D，靶 DNA 降解的机制仍不清楚。在这里，我们发现 Cas10d 是 I-D 系统中的一种功能性核酸酶，在其他 CRISPR-Cas I 型系统中发挥 Cas3 的作用。I-D 系统可用于人类细胞中基因组 DNA 的靶向诱变，指导双向长距离缺失和短插入/缺失。我们的发现表明，CRISPR-Cas I-D 系统是 CRISPR 中的一种独特效应途径，可用于真核细胞的基因组工程。
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