Department of Molecular and Cell Biology, University of California, Berkeley, CA, USA.
California Institute for Quantitative Biosciences, University of California, Berkeley, CA, USA.
Nature. 2019 Feb;566(7743):218-223. doi: 10.1038/s41586-019-0908-x. Epub 2019 Feb 4.
The RNA-guided CRISPR-associated (Cas) proteins Cas9 and Cas12a provide adaptive immunity against invading nucleic acids, and function as powerful tools for genome editing in a wide range of organisms. Here we reveal the underlying mechanisms of a third, fundamentally distinct RNA-guided genome-editing platform named CRISPR-CasX, which uses unique structures for programmable double-stranded DNA binding and cleavage. Biochemical and in vivo data demonstrate that CasX is active for Escherichia coli and human genome modification. Eight cryo-electron microscopy structures of CasX in different states of assembly with its guide RNA and double-stranded DNA substrates reveal an extensive RNA scaffold and a domain required for DNA unwinding. These data demonstrate how CasX activity arose through convergent evolution to establish an enzyme family that is functionally separate from both Cas9 and Cas12a.
RNA 指导的 CRISPR 相关（Cas）蛋白 Cas9 和 Cas12a 为抵御入侵的核酸提供了适应性免疫，并且作为基因组编辑的强大工具在广泛的生物中发挥作用。在这里，我们揭示了一种名为 CRISPR-CasX 的第三种、完全不同的 RNA 指导的基因组编辑平台的基本机制，该平台使用独特的结构进行可编程双链 DNA 结合和切割。生化和体内数据表明，CasX 可用于大肠杆菌和人类基因组修饰。CasX 与其指导 RNA 和双链 DNA 底物在不同组装状态下的 8 个冷冻电镜结构揭示了一个广泛的 RNA 支架和一个用于 DNA 解旋的结构域。这些数据表明 CasX 如何通过趋同进化产生活性，从而建立一个与 Cas9 和 Cas12a 在功能上完全不同的酶家族。
