《自然》：同行评议的论文升级了NgAgo基因编辑争议

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《自然》：同行评议的论文升级了NgAgo基因编辑争议

记者：David Cyranoski （翻译：方舟子）2016年11月23日

6个月前，中国研究人员报告说一种叫NgAgo的酶能够用于编辑哺乳动物基因，而它可能比流行的CRISPR–Cas9基因编辑技术更精确、更通用。但是其他科学家几乎马上就在互联网论坛抱怨说他们没能重复出该实验结果。

现在，一篇有20个作者的论文在《蛋白质与细胞》上发表，列出多次试图重复原始实验但是失败的结果，而另一篇发表在《细胞研究》上的论文表明，把NgAgo注射入斑马鱼胚胎中，它可能只是在遏制而不是编辑基因。

位于石家庄的河北科技大学生物学者韩春雨是第一个报告NgAgo基因编辑实验的，他说他坚持原来的主张。

《蛋白质与细胞》论文的20个作者描述了他们如何试图用NgAgo编辑各种基因组而未能成功。其中8个实验室用尽可能与韩春雨实验相同的方式，使用韩春雨提供的材料，针对相同的基因，也将该技术用于人类细胞，再次做了尝试。他们全都失败了。

该论文呼吁韩团队“澄清围绕NgAgo的不确定性，提供能重复当初非常重要的结果的所有必需细节”。但是其中一个作者、北京大学分子生物学者魏文胜已经对NgAgo有了结论。“它根本就不工作，句号，”他说。

北京出版的《蛋白质与细胞》曾在2015年发表关于在人类胚胎使用基因编辑技术的第一篇报道，它的编辑张晓雪说，该杂志尽量快地发表该NgAgo论文，是因为围绕着该研究的辩论还在持续。“在中国，它不仅仅是一个科学问题。它也是一个道德和政治问题，”她说。

在《细胞研究》论文中，研究人员报告他们试图用NgAgo编辑一个斑马鱼胚胎的基因，该基因被认为与眼睛发育有关。有些胚胎发育出一个非常小的眼睛和一个基本正常的眼睛，还有的胚胎的眼睛融合在一起，长在头顶“就像一个独眼龙”，这是人们预料如果NgAgo把那个基因敲除掉后会出现的结果。但是研究人员在对斑马鱼基因组进行测序之后，惊讶地发现该基因还是完整的。

该论文的首要作者、江苏省南通大学一名分子生物学者刘东提出了一种解释：NgAgo分子与基因组结合，但是没有切除目标基因，而只是降低了其表达。因为NgAgo蛋白很容易在实验室制备，刘东说这种功能能够让它作为暂时遏制斑马鱼基因功能的更便宜、更容易得到的另一种方法。但是如果他是对的，那么NgAgo并不会产生能够传递给下一代的永久变化，因此不会被认为是一种基因编辑器。

刘东不据此对围绕最初NgAgo实验的争议提供什么见解，他注意到该实验是用人类细胞做的。他说，他的论文只是提供了NgAgo一种新的可能用处，用来作为遏制而不是编辑基因的便宜工具。

但是批评者说，刘东的论文提供了进一步的证据表明最初NgAgo论文的说法站不住脚。“这是发表在同行评议期刊上的另一篇报道证实了NgAgo不能作为基因编辑器发挥作用，”马德里的西班牙国家生物技术中心的遗传学者Lluis Montoliu说，他以前已批评过韩春雨的论文，“有必要强调这一点。”

堪培拉的澳大利亚国立大学的遗传学者Gaetan Burgio是最早在网上报告未能重复韩春雨实验的研究人员之一，他说他不相信NgAgo能够在韩春雨或刘东实验使用的温度下具有功能。两个实验使用的温度都比该蛋白质的来源——一种细菌——生活的环境温度低得多。

Burgio认为斑马鱼的异常结果可能是由于与NgAgo活性无关的毒性导致的。刘东说他的数据让他确信NgAgo能够可靠地遏制基因，但是承认他需要更多的证据决定性地证明这一点。他说他现在正在汇总这些证据。

韩春雨告诉《自然》他已发现了其他研究人员没有注意到的一个问题，这个问题能够解释为什么其他人难以重复他的结果。他说他目前正在做确认实验，这样他就能发表数据和实验步骤让批评者满意。“我现在不能说，因为无论我说什么中国媒体都会跳，”他告诉《自然》，“我需要一点点时间。”

发表其论文的期刊《自然·生物技术》发言人说，很多批评韩春雨NgAgo论文的人和团体已经与该刊联系，它根据发表研究结果的准则已经或正在仔细对此进行评判。“目前我们对该论文的调查还在继续，”该发言人说。《自然·生物技术》隶属《自然》的出版社Springer-Nature，与《自然》新闻团队的编辑是相互独立的。

有少数几名科学家此前告诉《自然》他们能重复出韩春雨的发现，但还未发表这些结果，现在其中一个说他用NgAgo做与其研究有关的实验，希望不久能够发表。但是另一个此前注意到用NgAgo有初步阳性结果的人现在说，“数据令人困惑”，“我们不能得出结论”。这两人都不愿透露姓名，因为害怕被牵涉到争议当中。

这一大失败让人质疑韩春雨所在大学——河北科技大学——在八月份宣布要建的基因编辑中心项目，该项目由当地政府投资2.24亿元人民币。“没有韩春雨的《自然·生物技术》论文和之后的炒作，该校不可能获得如此巨额的资金来建基因编辑中心，”方舟子说，他以前是生物化学学者，因为揭露造假科学家而闻名，是最早公开批评韩春雨论文的人之一。

他说，除了韩春雨关于NgAgo的主张，该大学并无从事基因编辑研究的专业资质。因此如果韩春雨的工作站不住脚，“该中心就失去了其合理性。”

魏文胜也批评建立该基因编辑中心的决定，他也把它归因于韩春雨论文导致的激动。“考虑到这是一个热门领域，在中国建一个基因编辑中心并不是坏主意，”他说，“但是把这样的中心建在河北的唯一原因就是因为韩春雨的论文。”

河北科技大学拒绝与《自然》新闻团队讨论该中心。

NgAgo gene-editing controversy escalates in peer-reviewed papersOne paper describes surprising results in zebrafish embryos, another lists failed replication efforts.

David Cyranoski23 November 2016

Six months ago, Chinese researchers reported that an enzyme called NgAgo could be used to edit mammalian genes — and that it might be more accurate and more versatile than the popular CRISPR–Cas9 gene-editing technique1. But almost immediately, other scientists complained on Internet forums that they could not replicate the experiment.

Now, a paper with 20 authors, published in Protein & Cell2, lists multiple attempts that failed to replicate the original experiment — while another, published in Cell Research3, suggests that NgAgo may only block, but not edit, genes when it is injected into zebrafish (Danio rerio) embryos.

Han Chunyu, a biologist at Hebei University of Science and Technology in Shijiazhuang who first reported the NgAgo experiment, says he is standing by the original claims.

The 20 authors of the Protein & Cell paper2 describe how they attempted without success to use NgAgo to edit a variety of genomes. Eight of the labs then tried again, adhering as closely as possible to Han’s experiment, using genetic materials provided by Han, targeting the same genes and also applying the technique to human cells. They all failed.

The paper urges Han’s team to “clarify the uncertainty surrounding NgAgo and provide all the necessary details for replicating the initial, very important results”. But one of the authors, Wensheng Wei, a molecular biologist at Peking University has already made his mind up about NgAgo. “It simply doesn’t work, period,” he says.

Zhang Xiaoxue managing editor at Protein & Cell in Beijing China — which also published the first report of gene-editing in human embryos in 2015 — says that the journal made an effort to publish the NgAgo paper quickly because of the ongoing debate over the work. “In China, it’s not just a scientific issue. It’s also an ethical and political issue,” she says.

In the Cell Research paper 3, researchers reports an attempt to use NgAgo to edit a gene thought to be related to eye development in zebrafish embryos. Some of the embryos developed either just one very small eye and one largely normal eye, or eyes that were fused and that formed on the top of the head “like a cyclops”, as one would expect if NgAgo had knocked out the gene. But when the researchers sequenced the genomes of the fish, to their surprise they found that the gene was still intact.

The lead author, Liu Dong, a molecular biologist Nantong University in Jiangsu province, offers an explanation: the NgAgo molecules clamp onto the genome but instead of cutting the target gene, just reduces its expression. Because the NgAgo protein can be easily prepared in the laboratory, Liu says that this capability could make it a cheaper, more accessible alternative to current methods of temporarily blocking gene function in zebrafish. But if he is right, then NgAgo would not make permanent changes that are passed on to the next generation and would therefore not be considered a gene-editor.

Liu offers little insight into the controversy over the original NgAgo experiments, which he notes were done in human cells, in vitro. Rather, he says, it raises the possibility of a new use for NgAgo, as a cheap tool to block but not edit genes.

But critics say Liu’s paper is further evidence that the claims in the original NgAgo paper1 don’t stand up. “This is another report, now published in a peer-reviewed journal, confirming that NgAgo does not work as gene editor,” says Lluis Montoliu, a geneticist at the Spanish National Centre for Biotechnologyin Madrid, who has previously criticized the Han paper. “This needs to be highlighted.”

Gaetan Burgio, a geneticist at the Australian National University in Canberra and one of the first to post online a failed attempt to replicate’s Han’s experiments, says he doesn’t believe that NgAgo can function at the temperatures used in either Han’s or Liu’s experiment. Both were carried out at much cooler temperatures than the environment in which the protein’s source — a bacterium — lives.

Burgio thinks the zebrafish abnormalities might have formed because of a toxicity unrelated to the activity of NgAgo. Liu says his data convince him that NgAgo can reliably block genes, but acknowledges that he needs more evidence to show this conclusively. He says he is working on assembling that now.

Han told Nature he has discovered a problem that would not have been obvious to others and that could explain why others are having difficulty replicating his results. He says that he is currently running confirmatory experiments so that he can publish data and a protocol that satisfies his critics. “I cannot say right now because the media in China jumps on everything I say,” he told Nature. “I need a little bit of time.”

A spokesperson for the journal that published his paper, Nature Biotechnology, said that a number of individuals and groups critical of Han’s NgAgo paper had contacted the journal, and that it has considered or is considering them carefully, alongside any published critiques of the research. “Our investigations into the paper are continuing at this time,” says the spokesperson. Nature Biotechnology is editorially independent of Nature’s news team and is owned by Nature’s publisher Springer-Nature.

One of the few scientists who previously told Nature he had corroborated Han’s findings — but has not published these results — now says that he is using NgAgo for experiments related to his research, and that he hopes to publish soon. But another who previously noted positive initial results with NgAgo says now that the “data are confusing” and “we cannot make a conclusion”. Neither wanted to be named for fear of being dragged into the controversy.

The debacle has raised questions about a 224-million yuan (US$32-million) gene-editing centre that Han’s university — Hebei University of Science and Technology — announced in August that it would build, to be paid for with local government money. “Without Han’s Nature Biotechnology paper and the hype after that, it’s impossible for the school to get such huge funding to establish the gene editing research centre,” says Fang Shimin, a former biochemist who has become famous for exposing fraudulent scientists and one of the first to publicize criticism of Han’s paper.

He says that aside from Han’s NgAgo claims, the university lacks gene-editing expertise. So if Han’s work doesn’t stand up, “the centre will lose its legitimacy”.

Wei too is critical of the decision to build the gene-editing centre, which he also attributes to the excitement over Han’s paper. “It’s not a bad idea to have one in China considering that it is such a hot area,” he says, “but the only reason such centre is built in Hebei is because of Han’s publication.”

The university has declined the Nature news team’s requests to discuss the centre.

Nature doi:10.1038/nature.2016.21023

References

1.Gao, F., Shen, X. Z., Jiang, F., Wu, Y. & Han, C. Nature Biotech. 34, 768–773 (2016).2.Burgess, S. et al. Protein Cell http://dx.doi/org/10.1007/s13238-016-0343-9 (2016).3.Qi, J. et al. Cell Res. http://dx.doi.org/10.1038/cr.2016.134 (2016).

(XYS20161128)

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