Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses.
Programmable RNA-guided endonucleases are widespread in eukaryotes and their viruses.
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可编程RNA引导的核酸内切酶广泛存在于真核生物及其病毒中。
DOI：
10.1101/2023.06.13.544871
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发表时间：
2023
期刊：
影响因子：
--
通讯作者：
Gootenberg,JonathanS
中科院分区：
文献类型：
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作者：
Jiang,Kaiyi;Lim,Justin;Sgrizzi,Samantha;Trinh,Michael;Kayabolen,Alisan;Yutin,Natalya;Koonin,EugeneV;Abudayyeh,OmarO;Gootenberg,JonathanS
可编程RNA引导的DNA核酸酶在原核生物中发挥许多作用，但它们在原核生物外的传播程度尚不清楚。Fanzors是原核生物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，但其在真核生物中的活性和功能尚不清楚。在这里，我们的特点范佐RNA可编程的DNA内切酶，使用生化和细胞的证据。我们发现了经常与各种真核转座酶相关的各种Fanzor。Fanzors进化的重建揭示了真核生物中含有RuvC的TnpB同系物的多重辐射。Fanzor基因捕获了内含子，蛋白质获得了核定位信号，表明在真核细胞中广泛的、长期的适应性功能。Fanzor核酸酶含有RuvC结构域的重排催化位点，类似于TnpB的不同子集，并且缺乏附带切割活性。我们证明了Fanzors可以用于人类细胞中的基因组编辑，突出了这些广泛存在的真核RNA引导核酸酶在生物技术应用中的潜力。
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.