韩春雨验证工作困难，已有64.5%实验室选择放弃

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    Nathan Blow于当地时间8月22号在《Biotechniques》发表《To Edit or Not: The NgAgo Story》（继续试验还是停止，关于韩春雨NgAgo的故事）。调查表明: 世界上超过64%的实验室都停止了韩春雨试验的验证工作，大家在等待别小组的重复情况，也在等待Nature Biotechnology或者河北科技大学的调查结果。

    现在唯一能沉得住气的，就是我们的科技大神韩春雨——任凭你怎么质疑，依旧不声不响。

    下面将《Biotechniques》的英文原文和小纳君用自己蹩脚的英语给出的译文放在下面，请君阅览！由于并非相关专业，不当之处，请多多包涵！

    With an ever-growing array of applications being reported in the scientific literature, CRISPR/Cas9 remains the hot molecular biology technology of the moment. Yet there continues to be significant interest in refining and expanding current genome-editing tools for more efficient use. On the heels of the expansion of CRISPR, researchers introduced an argonaute-based genome editing approach that seemed to overcome some of CRISPR’s downsides.

    With thoughts of a new tool to add to the genome-engineering toolbox, scientists set off on a whirlwind of experiments—which led to more questions as data analysis began.

    越来越多的应用在科学文献中被报告，在当前CRISPR / Cas9仍然是分子生物学技术的热点。然而，仍有致力于改善和拓展目前的基因编辑工具以高效使用的兴趣。紧随CRISPR的扩张，研究人员发现了一个argonaute-based基因组编辑方法，似乎可以克服CRISPR的一些缺点。

    随着一种新的工具加入到基因工程工具箱，科学家引发了实验的旋风，这导致了像数据分析开始那样更多的问题。

A New Way to Edit Genes  韩春雨创造新的基因编辑工具

    In May 2016, Hebei University researchers Chunyu Han and his colleagues reported in the journal Nature Biotechnology that Natronobacterium gregoryi Argonaute (NgAgo) could function as a DNA-guided endonuclease capable of genome editing in human cells. The results were powerful since, unlike Cas9, NgAgo did not seem to require a protospacer-adjacent motif (PAM), making it easier to use for targeted genome editing. In addition, Han’s data showed that NgAgo resulted in low guide-target mismatch and high efficiency when editing difficult templates, such as GC-rich genomic targets. These features made the new system particularly attractive to researchers, and many were eager to test the method for themselves. In fact, according to a blog on the Addgene website (a plasmid repository) posted by MRC Centre for Regenerative Medicine postdoctoral fellow Pooran Dewari, more than 400 requests for the NgAgo plasmids have been made since publication.

    2016年5月，河北大学研究人员韩春雨和他的同事在《自然生物技术》报道，Natronobacterium gregoryi Argonaute (NgAgo)可以作为DNA-guided核酸内切酶在人类细胞基因组中进行编辑。不同于Cas9，NgAgo的结果是强大的，因为其似乎并不需要protospacer-adjacent基序(PAM)，使其更容易对目标基因组编辑。此外，韩的数据显示，在编辑困难模板如富含GC的基因组时，NgAgo导致低错配率和高效性。这些特性使新系统对研究人员特别有吸引力，许多研究者急于测试该方法。事实上，根据一篇Addgene网站（一个质粒库）上MRC再生医学中心博士后Pooran Dewari发布的博客，自从该技术报道以来，已经发出超过400个NgAgo质粒的请求。

    The NgAgo system is not without its own shortcomings though—in vitro assembly of the NgAgo/single-stranded DNA (ssDNA) complex for targeting requires incubation at 55°C, a high temperature indeed for enzymes. Plus, the system involves co-transfection of the NgAgo plasmid with the targeting ssDNA guide, another cumbersome step. Still, with the possibility of little off-target editing, researchers could see potential for the tool.

    NgAgo系统并非没有自己的缺点，虽然在体外组装NgAgo /单链DNA(ssDNA)复合体以进行基因编辑，需要在55 ℃进行孵育，酶所需要的温度。另外，该系统需要NgAgo质粒与靶向ssDNA的引导链共转，也是一个繁琐的步骤。不过，伴随着编辑脱靶的可能性，研究人员可以看到该工具的潜能。

    The scientific community weren’t the only ones who had taken note of the publication; NgAgo gene editing quickly made its way into the general media. One Chinese news outlet quoted Han as saying that in the future, “With this technique, middle-aged men with bald heads can probably gain their hair though genetic repair.” The South China Morning Post detailed Han’s work in a story on underfunded researchers making significant contributions to science. And on the Nature Biotechnology website, Han’s NgAgo original article was getting more downloads and views than any other. Expectations and interest in NgAgo were sky-high out of the gate.

    并不是只有科学界在关注NgAgo，其也迅速进入了公众视野。一位中国新闻机构援引韩，“在未来，用这种技术修复基因，脱发的中年男性可以获得他们的头发。”《南华早报》用一个故事详细报道了韩的工作，其在经费不足情况下对科学做出了重大贡献。在自然生物技术网站，韩的NgAgo原创文章获得了最多的下载和浏览。对NgAgo有着极高的期望和兴趣。

Wrinkles Appear 争端源于谷歌对外报道其没能获得预期结果

    The internet has become a haven for researchers of all disciplines looking to find suggestions and tips to improve their experiments. Open sharing of ideas and strategies has become increasingly commonplace online, so it is no surprise that there is an active genome-engineering Google Group focused on methods and tools for genome modification.

    互联网已经成为所有学科的研究人员去寻找建议和提高他们实验技巧的天堂。在线公开分享想法和策略已经越来越普遍，所以毫不奇怪的是，有一个活跃的基因工程组织，谷歌集团，也专注于基因组修饰的方法和工具。

    The first entry in the Google genome-engineering group associated with NgAgo appeared June 23, 2016. It was from a researcher who tried to transfect with an NgAgo plasmid acquired from Addgene and a 5´P oligonucleotide targeting GFP for modification. This should have been a simple experiment where the editing results in a decrease in GFP fluorescence. The problem was that there was no change in fluorescence after transfection, according to the post. The researcher wondered if anyone else had experienced similar issues with the NgAgo system. A day later, another poster reported they too had tried targeting genes with the same NgAgo plasmid, and also failed to see the expected editing result.

    与NgAgo有关的报道第一次进入谷歌基因工程集团是在2016年6月23日。一位研究人员试图转染一个载有Addgene和5´P寡核苷酸的NgAgo质粒对GFP进行修饰。这应该是一个简单的实验，基因编辑导致GFP荧光的减少。问题是与编辑前相比，在转染后没有荧光的变化。研究人员想知道其他研究者是否经历过NgAgo系统类似的问题。一天后，另一个报道表示，他们用相同的NgAgo质粒去靶定基因，基因编辑也没有看到预期的编辑结果。

    It’s not unusual for researchers to have hiccups with new techniques in their first outing—there might be specific steps in a protocol that need to be exactly adhered to for achieving the intended result. But following those posts on June 23 and 24, more scientists posted their data. In time, trouble arose as there seemed to be two camps: some believed that the system might be working, while others agreed with the first two posts after they also did not see the expected results. Confusion wove its way through various group posts.

    在第一次涉足新技术时，研究者会有打嗝是正常的，可能需要特殊的步骤才能准确地得到预期结果。但是在6月23日和24日的报道中，更多的科学家发布了他们的数据。此时麻烦出现了，似乎是分成了两个阵营：一些人认为该系统可能会工作，在未看到预期结果后其他人同意前两个报道。通过各种群的帖子，混乱编织着。

    It’s worth taking a step back here and noting that life science research is in the middle of a reproducibility crisis. Several recent high profile reports that eventually had to be retracted (regarding STAP cells for example) have led many to question new findings to a greater extent than in the past. As of early July 2016, vetting of the NgAgo technique became increasingly intense as more researchers posted their results and the details of how they did their experiments, and requested assistance in getting the technique to work.

    值得退一步指出的是，生命科学研究处在再现性危机中。最近的一些高调报道，最终不得不收回（例如STAP细胞），这导致了比过去更大程度上许多问题的新发现。截至2016年7月初，随着越来越多的研究人员发布了他们的研究结果和他们如何做实验的细节，以及对实验技术指导的请求，对NgAgo技术的审查变得越来越激烈。

    In an effort to gain some insight on the NgAgo controversy and where the technique stood, Dewari sent out a survey to users to assess their NgAgo research success. By early August, the results were in. When asked if indels (insertions and deletions) had been seen using the NgAgo system, only 5.1% said yes, while 51.5% said no. Using NgAgo for other genome editing applications yielded similar numbers. Based on the percentages, NgAgo was in trouble, but the question remained whether the problem lay in the technique itself or the researchers trying to implement it in other labs.

    企图在NgAgo质疑中获得关注，Dewari给用户的一项调查问卷以评估NgAgo研究成功。8月初，结果返回。当被问及使用NgAgo系统所得到的indels(插入和删除)时，只有5.1%的研究者认为可以，而51.5%的人说不。使用NgAgo应用于其他基因组的编辑得到了类似的数字。基于百分比，NgAgo遇到了麻烦，但问题依然是在于技术本身还是在其他实验室的研究人员试图实现它。

Consensus is a Complicated Road 结论直接扑朔迷离

    Han has been quiet for the most part. According to reports, the researcher rarely travels outside of China. And while he has provided an additional protocol and detailed conditions for using NgAgo, as well as noting that researchers in other labs have been able to replicate the technique, the criticism has not been silenced. In fact, the additional protocol actually raised more concerns amongst critics with its specific time requirements and conditions.

    韩长期以来籍籍无名，据报道，其很少出国旅行。尽管韩已经提供了一份NgAgo的详细操作规则，同时亦有研究人员可以重复出该技术，但是批评远未沉寂。事实上，附加的操作规程吸引了更多的关注，需要时间和条件去验证该实验。

    Perhaps the best summation of the curvy road for NgAgo up to this point came from Gaetan Burgio, a group leader at Australian National University. On July 29, 2016, Burgio wrote a comprehensive blog entry on his experiences with trying to get NgAgo to work for gene editing.

    可能最早关注NgAgo的是澳大利亚国立大学的课题组长Gaetan Burgio，在2016年7月29日，Burgio写了一篇博文介绍他的实验以试图让NgAgo编辑基因。

    Initially, Burgio said his PCR results indicated that NgAgo had edited the beta-spectrin gene he targeted using an NgAgo plasmid in a way that was similar to CRISPR/Cas9, vindication for NgAgo and Chunyu. Burgio posted this result on Twitter and mentioned it to colleagues at a conference he attended at the time. But shortly after Burgio’s first PCR results came in and the initial excitement died down, things started to change.

    最初，Burgio表示PCR结果证明NgAgo已对一种利用类似于CRISPR/Cas9的方法靶定的beta-spectrin基因进行了编辑，说明NgAgo是可行的。Burgio在Twitter上公布了这一结果,并在一次会议上与同事提到了它。但在Burgio第一个PCR结果公布后的不久，没有了最初的兴奋，情况开始变化。

    In a follow-up experiment, Burgio began to see small holes to the NgAgo gene editing validation data. Other enzyme tests he tried indicated that NgAgo had not edited the beta-spectrin gene as that first PCR gel suggested. Sequencing the PCR products again showed what Burgio suspected could be edited sequences. However, after isolating individual PCR bands and sequencing them, Burgio finally concluded that those sequences were in fact random and that no NgAgo editing ever occurred in his experiments.

    在后续试验中，Burgio开始观察到NgAgo基因编辑验证数据的小孔洞。他做了其他的酶学试验，显示NgAgo并没有像第一次PCR胶的结果那样对beta-spectrin基因进行编辑。对Burgio怀疑已进行基因编辑的PCR产物进行测序，然而分离单个PCR片段和测序后，结果表明这些序列都是随机的，NgAgo并没有对其进行编辑。

    Burgio’s results and conclusions have only bolstered the contention for many that the NgAgo system simply does not work. Others argue that there are specific cell lines and conditions that are needed for the gene-editing activity of NgAgo to be effective, but critics counter that even if true, this simply means the technique is not robust and therefore a not a viable substitute for CRISPR/Cas9.

    Burgio的实验结果和结论显示NgAgo系统不能简单地工作，其他研究者认为特殊细胞系和条件是NgAgo基因编辑活性所必需的。但批评者反驳说，即使如此，该技术并不坚稳，不能够替代CRISPR / Cas9。

    Nature Biotechnology is currently investigating the NgAgo paper after being contacted by several researchers who were unable to replicate the findings. And Han’s university has also launched an inquiry.

    在数位研究者表示不能重复该项发现后，《自然·生物技术》目前正在调查这篇NgAgo的文章。此外，韩所在的河北科技大学亦表示对此展开调查。

    While the future of NgAgo is still up in the air as scientists work to determine whether or not the enzyme is in fact capable of gene editing, Dewari’s survey indicates most (64.5%) are now holding off on experiments, waiting to see what happens. Time will tell if NgAgo ends up in the genome-engineering toolbox.

    尽管NgAgo技术的未来悬而未决，尚待科学家们进一步证实其是否具有确切的基因编辑酶活性。Dewari的调查显示已有64.5%的研究人员都停止了这方面的实验，等待看这是怎么一回事。时间会告诉我们NgAgo是否会成为基因工程工具中的一员。

    ReferenceGao et al. 2016. DNA-guided genome editing using Natronobacterium gregoryi Argonaute. Nat. Biotechnol. 34, 768-773.

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