McGovern Institute for Brain Research at MIT Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Department of Biological Engineering, Massachusetts Institute of Technology, Cambridge, MA 02139, USA.
Sci Adv. 2023 Sep 29;9(39):eadk0171. doi: 10.1126/sciadv.adk0171. Epub 2023 Sep 27.
Programmable RNA-guided DNA nucleases perform numerous roles in prokaryotes, but the extent of their spread outside prokaryotes is unclear. Fanzors, the eukaryotic homolog of prokaryotic TnpB proteins, have been detected in genomes of eukaryotes and large viruses, but their activity and functions in eukaryotes remain unknown. Here, we characterize Fanzors as RNA-programmable DNA endonucleases, using biochemical and cellular evidence. We found diverse Fanzors that frequently associate with various eukaryotic transposases. Reconstruction of Fanzors evolution revealed multiple radiations of RuvC-containing TnpB homologs in eukaryotes. Fanzor genes captured introns and proteins acquired nuclear localization signals, indicating extensive, long-term adaptation to functioning in eukaryotic cells. Fanzor nucleases contain a rearranged catalytic site of the RuvC domain, similar to a distinct subset of TnpBs, and lack collateral cleavage activity. We demonstrate that Fanzors can be harnessed for genome editing in human cells, highlighting the potential of these widespread eukaryotic RNA-guided nucleases for biotechnology applications.
可编程 RNA 引导的 DNA 核酸酶在原核生物中发挥着多种作用，但它们在原核生物以外的传播范围尚不清楚。Fanzors 是原核 TnpB 蛋白的真核同源物，已在真核生物和大型病毒的基因组中检测到，但它们在真核生物中的活性和功能仍不清楚。在这里，我们使用生化和细胞证据将 Fanzors 表征为 RNA 可编程的 DNA 内切酶。我们发现了多种 Fanzors，它们经常与各种真核转座酶相关联。Fanzors 进化的重建揭示了真核生物中含有 RuvC 的 TnpB 同源物的多次辐射。Fanzor 基因捕获了内含子，并且蛋白质获得了核定位信号，表明它们在真核细胞中进行了广泛而长期的适应性进化。Fanzor 核酸酶包含 RuvC 结构域催化位点的重排，类似于 TnpB 的一个独特亚类，并且缺乏旁切活性。我们证明了 Fanzors 可以在人类细胞中用于基因组编辑，突出了这些广泛存在的真核 RNA 引导的核酸酶在生物技术应用中的潜力。
