Questions about NgAgo

Gao et al. published data in Nature Biotechnology (Nat Biotechnol. 2016 May 2) showing that DNA-guided genome editing using the Natronobacterium gregoryi Argonaute (NgAgo) protein targeted 47 mammalian genomic loci with a 100% success rate and an efficiency of 21.3%-41.3% at various targets. This report led us to test NgAgo's utility in various cells and organisms such as mouse and zebrafish for gene editing. In most cases, a codon-optimized NgAgo for vertebrate animals was first synthesized and tested with appropriate guide oligos targeting specific genes using techniques similar to what has been utilized for the CRISPR/ Cas9 system. After failing to confirm any NgAgo induced genomic DNA editing in any experiments, some of us switched to use an NgAgo expression vector (CMV-NLS-NgAgo-SK) used and provided by Han, the senior author of this paper, available from Addgene (#78253) since June or directly from his lab. Again, no success editing endogenous genomic DNA was achieved. As controls, the ability of this construct to induce indels was tested, targeting the same genes in cultured human 293T cells as those reported in Fig. 4 of Gao et al. Several researchers in different laboratories independently performed the experiments but no indels were observed at targeted loci, as assayed by T7E1 digestion, PAGE and/or sequencing. Representative data that directly repeat Fig. 4 of Gao et al from eight laboratories are shown in Fig. 1 and protocols used are detailed in supplementary information. We also include additional results from testing NgAgo in various systems by laboratories of signees of this letter in supplementary information. None of these studies proves that NgAgo has any genome editing activities.SCI(E)中国科技核心期刊(ISTIC)中国科学引文数据库(CSCD)LETTERLCheng2@jhmi.edu; shuolin@g.ucla.edu; wswei@pku.edu.cn; wanghaoyi@ioz.ac.cn12913-915