Arbor Biotechnologies, Cambridge, MA 02139, USA.
National Center for Biotechnology Information, National Library of Medicine, National Institutes of Health, Bethesda, MD 20894, USA.
Science. 2019 Jan 4;363(6422):88-91. doi: 10.1126/science.aav7271. Epub 2018 Dec 6.
Type V CRISPR-Cas systems are distinguished by a single RNA-guided RuvC domain-containing effector, Cas12. Although effectors of subtypes V-A (Cas12a) and V-B (Cas12b) have been studied in detail, the distinct domain architectures and diverged RuvC sequences of uncharacterized Cas12 proteins suggest unexplored functional diversity. Here, we identify and characterize Cas12c, -g, -h, and -i. Cas12c, -h, and -i demonstrate RNA-guided double-stranded DNA (dsDNA) interference activity. Cas12i exhibits markedly different efficiencies of CRISPR RNA spacer complementary and noncomplementary strand cleavage resulting in predominant dsDNA nicking. Cas12g is an RNA-guided ribonuclease (RNase) with collateral RNase and single-strand DNase activities. Our study reveals the functional diversity emerging along different routes of type V CRISPR-Cas evolution and expands the CRISPR toolbox.
V 型 CRISPR-Cas 系统的特点是单个 RNA 指导的 RuvC 结构域包含效应物 Cas12。尽管亚型 V-A（Cas12a）和 V-B（Cas12b）的效应物已被详细研究，但尚未表征的 Cas12 蛋白的独特结构域架构和分化的 RuvC 序列表明存在尚未探索的功能多样性。在这里，我们鉴定和表征了 Cas12c、-g、-h 和 -i。Cas12c、-h 和 -i 表现出 RNA 指导的双链 DNA（dsDNA）干扰活性。Cas12i 表现出明显不同的 CRISPR RNA 间隔互补和非互补链切割效率，导致主要的 dsDNA 切口。Cas12g 是一种 RNA 指导的核糖核酸酶（RNase），具有附带的 RNase 和单链 DNA 酶活性。我们的研究揭示了 V 型 CRISPR-Cas 进化不同途径中出现的功能多样性，并扩展了 CRISPR 工具包。
