◇◇新语丝(www.xys.org)(xys8.dxiong.com)(xys.ebookdiy.com)(fangzhouzi.me)◇◇ 国际转基因技术协会原主席Montoliu博士建议不要再试图重复韩春雨实验结果   澳大利亚国立大学Gaetan Burgion今天撰写长文介绍他试图重复河北科大韩 春雨创立的NgAgo基因编辑技术的经过,得出结论:韩春雨的实验无法重复,而 且与韩在论文所说矛盾;《自然·生物技术》应要求韩公布原始数据;该技术显 然没有前途。   国际转基因技术协会创建者和原主席Lluis Montoliu博士根据这一结果以 及其本人实验室的实验结果,向国际转基因技术协会会员发出一封公开信,建议 停止继续试图验证韩春雨的实验,不要再浪费时间、金钱、人员和项目。   CRISPR谷歌群针对这项技术和韩春雨的文章的调查表明,140个调查回复中, 只有一个(中国神经所仇子龙?)回答该技术起作用,73个回答无效,63个回答 在验证。 From: istt_list-bounces@transtechsociety.org on behalf of Lluis Montoliu To: ISTT List Subject: [ISTT_list] Great disappointment with Ago: Long life to CRISPR ! Date: Friday, July 29, 2016 9:49:22 AM Dear colleagues, the publication by Gao et al in May in Nature Biotechnology (http://www.ncbi.nlm.nih.gov/pubmed/27136078) triggered an enormous expectation. This Chinese team led by Chunyu Huan reported that the Argonaute (Ago) protein from a rare haloarchaea, Natronobacterium gregoryi, (NgAgo) would efficiently work for gene editing purposes in human cells. Ago had been described as an DNA-guided endonucleases two years before, through a Dutch-Spanish microbiologist collaborating team (Swarts et al. 2014, Nature: http://www.ncbi.nlm.nih.gov/pubmed/24531762). On paper, the new (fourth) Gene Editing system looked great. An endonuclease, using ssDNA guides (5' phosphorylated though) and not RNA guides, without a PAM, requiring 24 nucleotides (and not 20nt), hence with higher specificity, and apparently with fewer off-target issues, since modifications in just one position of the DNA guide resulted in >90% decrease of the protein activity. On paper. I must confess we read the Huan paper in my lab with some disappointment, after two years battling, unsuccessfully, with Ago from Thermus, through a collaboration with my friend and colleague J. Berenguer, from the neighbouring reserch centre CBMSO, and one of the co-authors of the Nature 2014 paper. We had been scooped. We repeteadly failed to find any gene editing activity using Ago from Thermus thermophilus (TtAgo) in mammalian cells, through a variety of conditions and we didn't understand why, though we always suspected that these proteins would not be too comfortable at "too cold" temperatures as physiological +37C. After reading the Gao paper we concluded we simply missed the right bug and congratulated them for being smarter and lucky and for finding this archaea. Perhaps the trick was in using NgAgo instead of TtAgo. Shortly after NgAgo was released from Addgene, beginning of June, many labs, including mine, jumped onto it to try experiencing the anticipated great expectations and joy associated with this new tool of prokaryotic origin. But soon it was clear that something wasn't quite right. Rumours began spreading during June and July at congresses, through social networks, list emails and discussions groups that NgAgo didn't appear to work as reported. Actually, didn't work at all. Some colleagues that I absolutely trust at scientitic and technological levels started to indicate that they could not reproduce Huan's paper results. At the recent TAGC meeting (where IMGS was contributing to, merging in along with other Genetics Societies) Gaetan Burgio, from ANU, Camberra, Australia, presented some very preliminary data with a gel with some intermediate bands that would suggest NgAgo would be working and editing at the expected places. But, shortly thereafter, Gaetan engaged his lab in an OpenScience project, tried to characterize all these bands and.... found nothing. So, again, another evidence confirming NgAgo is not working as a gene-editing tool. Gaeatan just released today his experience using NgAgo, openly sharing his failures and providing details and some explanations for them. My experience with Natronobacterium gregoryi Argonaute (NgAgo) Gaetan Burgio Group leader at JCSMR, ANU https://medium.com/@GaetanBurgio/my-experience-with-natronobacterium-g regoryi-argonaute-ngago-3ed8909b410c#.bo9y6mf9u At first, KUDOS to Gaetan. Many thanks to him for sharing their efforts trying to confirm some gene-editing activity associated with NgAgo. There is apparently none. In his view, NgAgo might be working as a ligase at physiological conditions. Similar to our negative results using TtAgo it would appear that NgAgo requires some higher temperatures to work as initially reported. This of course seeds some doubts on the Gao et al. publication and Gaetan, among other, is requesting to Nature Biotechnology to request the Huan's lab to reveal and share their raw data. We will see this part of the history how it develops... But, now, the most important message to convey is: NgAgo does not work for gene editing in mammalian cells. Be aware and do not waste your time, your money, your peoople and projects. If anyone has any positive hint suggesting Ago is indeed working as a genomic editor, please share the results, for the sake of Open Science, as Gaetan beautifully and most generously did. Many thanks to Gaetan! Unfortunately, this is a great disappointment. But, it also highlights the uniqueness and the robustness of the CRISPR-Cas systems. Long life to CRISPR! Lluis phone: +49621- 1703 6210/6204 ___________________________ Dr. Lluis Montoliu Investigador Cientifico - Research Scientist CSIC Centro Nacional de Biotecnologia (CNB-CSIC) Campus de Cantoblanco C/ Darwin, 3 28049 Madrid (Spain) Tel. +34-91-5854844 / Fax +34-91-5854506 e-mail: montoliu@cnb.csic.es WEB: http://www.cnb.csic.es/~montoliu/ U756 CIBERER: http://www.ciberer.es Spanish EMMA node: http://www.infrafrontier.eu ISTT: http://www.transtechsociety.org At present, I would recommend everyone abandoning any project involving the use of NgAgo. And avoid wasting time, money, animals and people. Results are clear in many labs. The results we have in Madrid are that TtAgo does not work in mammalian cells. The results that Gaetan has in Australia are that NgAgo does not work in mammalian cells. I know of many other colleagues who also tried and failed, but have not reported these failures publicly. This is why I posted this message. I think Ago might have some potential but we don't seem to have found yet the adequate version of it (or the right bug). As a side history, Francis Mojica struggled to find Grants to support his pioneer experiments with CRISPR and got a couple of projects turned down. That is why, instead of using Streptococcus pyogenes (Doudna and Charpentier) or thermophilus (Siksnys), buga more difficult and expensive to grow, he chose to work in Escherichia coli CRISPR systems, and faced many difficulties, only to find out, many years later that E.coli CRISPR-Cas system was of type I (not type II, as Cas9) and, furthermore, was mostly inactive. Please, let's focus in CRISPR-Cas9, Cpf1, C2c2,... et al... and leave alone Ago while microbiologists don't find out the right one. Lluis (XYS20160729) ◇◇新语丝(www.xys.org)(xys8.dxiong.com)(xys.ebookdiy.com)(fangzhouzi.me)◇◇