No evidence of genome editing activity from Natronobacterium gregoryi Argonaute (NgAgo) in human cells.
No evidence of genome editing activity from Natronobacterium gregoryi Argonaute (NgAgo) in human cells.
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DOI：
10.1371/journal.pone.0177444
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发表时间：
2017
期刊：
影响因子：
3.7
通讯作者：
Lu B
中科院分区：
文献类型：
--
作者：
Javidi-Parsijani P;Niu G;Davis M;Lu P;Atala A;Lu B
来自嗜热菌Thermus thermophilus的argonaute蛋白在高温下显示出DNA引导的DNA干扰活性，使其在哺乳动物细胞中的应用复杂化。最近的一项工作报告说，来自格氏嗜盐杆菌（NgAgo）的argonaute蛋白在哺乳动物细胞中具有DNA指导的基因组编辑活性。我们并排比较了NgAgo和金黄色葡萄球菌Cas9（SaCas9）在人HEK293T细胞中的基因组编辑活性。EGFP报告基因测定和DNA测序一致地揭示了SaCas9的高基因组编辑活性。然而，这些测定没有证明NgAgo的基因组编辑活性。我们证实，所述条件允许NgAgo表达质粒DNA和DNA引导物的同时转染，以及NgAgo在HEK293 T细胞中的异源表达。我们的数据表明NgAgo不是一个强大的基因组编辑工具，尽管它在其他条件下可能具有这种活性。
The argonaute protein from the thermophilic bacterium Thermus thermophilus shows DNA-guided DNA interfering activity at high temperatures, complicating its application in mammalian cells. A recent work reported that the argonaute protein from Natronobacterium gregoryi (NgAgo) had DNA-guided genome editing activity in mammalian cells. We compared the genome editing activities of NgAgo and Staphylococcus aureus Cas9 (SaCas9) in human HEK293T cells side by side. EGFP reporter assays and DNA sequencing consistently revealed high genome editing activity from SaCas9. However, these assays did not demonstrate genome editing activity by NgAgo. We confirmed that the conditions allowed simultaneous transfection of the NgAgo expressing plasmid DNA and DNA guides, as well as heterologous expression of NgAgo in the HEK293T cells. Our data show that NgAgo is not a robust genome editing tool, although it may have such activity under other conditions.