英国《自然》报道关于韩春雨实验结果无法重复的争议

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Replications, ridicule and a recluse: the controversy over NgAgo gene-editing intensifies
08 August 2016
SHIJIAZHUANG, CHINA


%E9%9F%A9%E6%98%A5%E9%9B%A81_CMweb.jpg

Biologist Han Chunyu receives dozens of harassing calls and texts each day, but he is convinced that his gene-editing paper is sound.

A controversy is escalating over whether a gene-editing technique proposed as an alternative to the popular CRISPR–Cas9 system actually works.

Three months ago, Han Chunyu, a biologist at Hebei University of Science and Technology in Shijiazhuang, reported that the enzyme NgAgo can be used to edit mammalian genes. Now, an increasing number of scientists are complaining that they cannot replicate Han’s results — although one has told Nature that he can.

Han says he receives dozens of harassing calls and texts each day, mocking him and telling him that his career is over — but he is convinced that the technique is sound. He also told Nature that he had submitted a detailed protocol to the online genetic-information repository Addgene on 8 August, at Addgene’s request, and hopes that this will help efforts to reproduce his work. Nature Biotechnology, which published the research, is investigating the matter.

The stakes are high. Over the past few years, the CRISPR–Cas9 system has transformed biology. But it has also made scientists hungry for other methods to expand the gene-editing toolkit: NgAgo is one of several that have emerged. “A lot of us are really cheerleading and hoping that it works,” says geneticist George Church of Harvard Medical School in Boston, Massachusetts.

CRISPR–Cas9 uses small genetic sequences to guide an enzyme to cut DNA in a particular location. Inspired, Han looked through the literature for other guidable protein ‘scissors’, and came across a family of proteins called Argonaute, or Ago, that fitted the bill. Others had flagged the proteins as potential gene editors.

eyevine3.14137556_900px.jpg

Former biochemist Fang Shimin alleged on his website that the NgAgo paper was irreproducible.

In the paper, Han’s team reports using a wide variety of genetic sequences to guide one of these proteins, NgAgo, to edit eight different genes in human cells and to insert genes at specific points on chromosomes (F. Gao et al. Nature Biotechnol.34, 768–773; 2016).

Crucially, NgAgo very specifically cut only the target genes, says Han, unlike CRISPR–Cas9, which sometimes edits the wrong genes. And whereas CRISPR–Cas9 requires a certain genetic sequence to be near the cutting site to initiate its activity, NgAgo does not, which could broaden its potential applications, adds Han.

The initial reaction to the work in China was laudatory, including a visit to the lab by China Central Television. It was overwhelming, says Han, who is a reclusive figure. His hobbies include collecting teas and playing an ancient stringed instrument called the guqin. He doesn’t like to travel and has never left China: a trip to visit a collaborator in Hangzhou in March was the first time the 42 year old had boarded a plane. Before his paper came out, “I was completely unknown”, says Han, who spoke to Nature at his laboratory and a nearby restaurant.

Doubts about the research first surfaced at the beginning of July, when Fang Shimin, a former biochemist who has become famous for exposing fraudulent scientists, wrote on his website New Threads (xys.org) that he had heard reports of failed reproduction efforts, and alleged that Han’s paper was irreproducible. Criticism grew on various Chinese sites.

Gaetan-Burgio-in-lab-2015_CM_900px.jpg

Geneticist Gaetan Burgio published failed attempts to replicate the NgAgo experiments on his blog.

On 29 July, the controversy went international when Gaetan Burgio, a geneticist at the Australian National University in Canberra, posted thorough details of his failed attempts to replicate the experiment on his blog. Normally, his posts get a few dozen hits, but this one spiked to more than 5,000.

On the same day, geneticist Lluís Montoliu, at the Spanish National Centre for Biotechnology in Madrid, e-mailed his colleagues at the International Society for Transgenic Technologies to recommend “abandoning any project involving the use of NgAgo” to “avoid wasting time, money, animals and people”. The e-mail was leaked and posted on Fang’s website.

Since then, an online survey by Pooran Dewari, a molecular biologist at the MRC Centre for Regenerative Medicine in Edinburgh, UK, has found only 9 researchers who say that NgAgo works— and 97 who say that it doesn’t.

Two researchers who initially reported success with NgAgo in an online chat group now say that they were mistaken. Debojyoti Chakraborty, a molecular biologist at the CSIR-Institute of Genomics and Integrative Biology in New Delhi, says that he repeated a specific section of Han’s paper that described using NgAgo to knock out a gene for a fluorescent protein that had been introduced into a cell. The glow was reduced, so Chakraborty assumed that NgAgo had disabled the gene. But after sequencing the DNA, he found no evidence of gene editing. He now says that the reduction in fluorescence must have had some other cause.

Jan Winter, a PhD student in genomics at the German Cancer Research Center in Heidelberg, says that he had a similar experience. “I will retry the experiment in the upcoming weeks, but so far I think it won’t work,” he says.

Lluis_2015_entire_900px.jpg

Geneticist Lluís Montoliu recommends abandoning any project involving the use of NgAgo for gene-editing.

Han says that he has only got the system to work on cells cultured in his laboratory, and it failed in cells that he purchased. He later found the purchased cells to be contaminated with bacteria called mycoplasma, and says that others might be having the same problem. He adds that some graduate students might be working too fast and not being careful with reagents. Winter disagrees: “I do not think it is a problem of the scientists doing something wrong.”

One researcher in China, who works independently from Han’s research group and who doesn’t want his name to be entangled in the public controversy, told Nature that he had tested NgAgo in a few kinds of cell and found that it was able to induce genetic mutations at the desired sites — a finding that he verified by sequencing. He adds that the process was less efficient than CRISPR–Cas9, and requires tweaking to improve the efficiency. “But, in short, it worked,” he says.

Two more Chinese scientists, who also asked not to be named, say they have initial results showing that NgAgo works but still need to confirm with sequencing.

“It might, might work,” says Burgio, “but if so, it’s so challenging that it’s not worth pursuing. It won’t surpass CRISPR, not by a long shot.”

The failure of NgAgo “would be disappointing, but then there is work for us left to do to see whether other Argonaute systems can get it to work somehow,” says microbiologist John Van der Oost of Wageningen University in the Netherlands, a co-author of the 2014 analysis of Argonaute proteins that laid the groundwork for their use in gene editing (D. C. Swaarts et al. Nature 507, 258–261; 2014).

This week, Nature Biotechnology sent a statement to Nature’s news team, saying that “several researchers” have contacted the journal to report that they cannot reproduce the results, and that “the journal is following established process to investigate the issues”. A spokesperson declined to comment on the nature or duration of the investigation. (Nature Biotechnology is published byNature’s publisher, Springer Nature; Nature’s news and comment team is editorially independent of the publisher’s research editorial teams.)

Hebei University says that it will ask Han to repeat the experiment so that it can be verified by an independent party within a month, according to Chinese state media.

Nature
536,
136–137
(11 August 2016)

 

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小保方晴子,汉化版
 
【重磅消息】韩老师公布基因编辑的protocol了


A general protocol of NgAgo/gDNA-mediated genome editing



1. Cell culture

293T cells are maintained in high-glucose DMEM (Gibco) supplemented with 10% FBS (Gibco) and penicillin/streptomycin at 37°C with 5% CO2 incubation. When cells reach their ≈60% confluence, perform transfection. Before transfection, cells are changed to medium containing 2% FBS(heat inactivated). Since 293T cells are not firmly attached, be gentle and do not disturb cells when changing medium.


2. Transfection

2-1 NLS-NgAgo expressing plasmid is extracted with Wizard® Plus SV Minipreps DNA Purification System (Promega), and is adjusted to 100 ng/μl in water (pH 8.0, alkalization by NaOH).

2-2 5’ phosphorylated ssDNA guides are dissolved to 100 ng/μl in water (PH 8.0) For each well of a 24-well plate, 200-250 ng NLS-NgAgo expression plasmid and 100-300 ng guides are diluted in 50 μl Opti-MEM (Gibco); 1.25 μl Lipofectamine® 2000 is diluted in 50 μl Opti-MEM. Incubate the DNA mix and lipofectamine mix for 5 min.

2-3 Combine the DNA mix and lipofectamine mix with gentle pipetting and incubate for 20 min. The DNA/lipo mixture is then added into each well of cells.


*Since NgAgo follows “one-guide faithful” rule, i.e. guides can only be loaded when NgAgo protein is in the process of expression, to improve the efficiency of gDNA loading to NgAgo, sometimes multiple transfection of gDNA helps (e.g. depending on the different kinetics of NgAgo expression in different cells, a second transfection of gDNA can be performed 8, 12 or 24 hours after the primary transfection)

*As stated below, cells will be harvested 48-60 hours after transfection. 90% confluence of the cells on harvesting is ideal. Cell overplating significantly weakens the efficacy of genome editing. Taking HDR as an example, it occurs only during S and G2 phases.


3. Genomic DNA extraction

3-1 48-60 hours after transfection, cells are harvested by trypsin digestion. Four wells of cells are combined into a 1.5 ml EP tube.

3-2 For genomic DNA extraction, 500 μl of cell lysate buffer (50 mM Tris,100 mM EDTA,0.5% SDS,pH 8) and 10 μl proteinase K (10 mg/ml)are added into each tube and mixed gently and sufficiently. Bathe the tubes at 55℃ for 2 hours.

3-3 200 μl Tris-Phenol and 200 ul trichloromethane are added into each tube and mixed gently and sufficiently. After incubation for 5 min, samples are spun at 12,000 rpm for 15 min to separate aqueous phase from Phenol phase.

3-4 Carefully collect the aqueous phase into a clean EP tube.

3-5 Repeat the Steps 3-3 and 3-4 once and pool the aqueous phase.

3-6 Add 500 μl trichloromethane into the collected aqueous phase, mix gently and sufficiently, stay for 5 min, and then spin the sample at 12,000 rpm for 15 min to separate aqueous phase from Phenol phase. Carefully remove the aqueous phase into a clean EP tube.

3-7 Repeat the Steps 3-6 once.

3-8 Add 900 μl EtOH to the collected aqueous phase and incubate at -20°C for 30 min.

3-9 Centrifuge the sample at 12,000 rpm for 10 min, and then the DNA sediment is washed with 500 μl 75% EtOH thrice.

3-10 Air-dry the DNA sediment, add 50 μl 0.5 x TE and then adjust the genome DNA to 100 ng/μl for later use.



Note:

1. NgAgo/gDNA system is extremely sensitive to contamination of intracellular bacteria (including mycoplasma) which are widespread and leave no visible signs of presence. Carefully excluding the presence of intracellular bacteria before performing experiments.

2. Because Agos need magnesium, avoid EDTA when detaching and seeding cells into the plates for transfection. Alternatively, supplementing Mg2+ to 5 mM (may need optimization to your cell type).

3. Avoid using Lipofectamine® 3000 since the supplement P3000 interferes with ssDNA transfection. Lipofectamine® 2000 is a good choice. Other transfection methods such as electroporation are yet to be tested.

4. Ideally, 5' phosporylation of ssDNA guide by using T4 PNK (Biolab):

T4 PNK 2 μl

T4 ligase buffer (containing ATP): 12 μl

1OD ssDNA (about 33 μg) in H2O: 100 μl

(alternatively) additional ATP (25 mM) 2 μl

Add H2O to a final volume of 120 μl

Incubate at 37℃ overnight

After 5' phosphorylation, the resultant ssDNA needs no purification, dilute by water (PH8) to 300 μl with a final concentration of 10 nM or 100 ng/μl)

0


免责声明:部分文章和信息来源于互联网,不代表本订阅号赞同其观点和对其真实性负责。如转载内容涉及版权等问题,请立即与小编诸葛神通联系,我们将迅速采取适当的措施。本订阅号原创内容,转载需授权,并注明作者和出处。
 
【重磅消息】韩老师公布基因编辑的protocol了

A general protocol of NgAgo/gDNA-mediated genome editing


公布实验的protocol根本就是应该的而且是必须的,否则怎么让其他人重复?根据这个protocol重复不出其实验结果才是重磅消息,说明其实验protocol无效或做假。
 
希望这个protocol是真的,那韩就是牛人了。
 
【重磅消息】韩老师公布基因编辑的protocol了


A general protocol of NgAgo/gDNA-mediated genome editing



1. Cell culture

293T cells are maintained in high-glucose DMEM (Gibco) supplemented with 10% FBS (Gibco) and penicillin/streptomycin at 37°C with 5% CO2 incubation. When cells reach their ≈60% confluence, perform transfection. Before transfection, cells are changed to medium containing 2% FBS(heat inactivated). Since 293T cells are not firmly attached, be gentle and do not disturb cells when changing medium.


2. Transfection

2-1 NLS-NgAgo expressing plasmid is extracted with Wizard® Plus SV Minipreps DNA Purification System (Promega), and is adjusted to 100 ng/μl in water (pH 8.0, alkalization by NaOH).

2-2 5’ phosphorylated ssDNA guides are dissolved to 100 ng/μl in water (PH 8.0) For each well of a 24-well plate, 200-250 ng NLS-NgAgo expression plasmid and 100-300 ng guides are diluted in 50 μl Opti-MEM (Gibco); 1.25 μl Lipofectamine® 2000 is diluted in 50 μl Opti-MEM. Incubate the DNA mix and lipofectamine mix for 5 min.

2-3 Combine the DNA mix and lipofectamine mix with gentle pipetting and incubate for 20 min. The DNA/lipo mixture is then added into each well of cells.


*Since NgAgo follows “one-guide faithful” rule, i.e. guides can only be loaded when NgAgo protein is in the process of expression, to improve the efficiency of gDNA loading to NgAgo, sometimes multiple transfection of gDNA helps (e.g. depending on the different kinetics of NgAgo expression in different cells, a second transfection of gDNA can be performed 8, 12 or 24 hours after the primary transfection)

*As stated below, cells will be harvested 48-60 hours after transfection. 90% confluence of the cells on harvesting is ideal. Cell overplating significantly weakens the efficacy of genome editing. Taking HDR as an example, it occurs only during S and G2 phases.


3. Genomic DNA extraction

3-1 48-60 hours after transfection, cells are harvested by trypsin digestion. Four wells of cells are combined into a 1.5 ml EP tube.

3-2 For genomic DNA extraction, 500 μl of cell lysate buffer (50 mM Tris,100 mM EDTA,0.5% SDS,pH 8) and 10 μl proteinase K (10 mg/ml)are added into each tube and mixed gently and sufficiently. Bathe the tubes at 55℃ for 2 hours.

3-3 200 μl Tris-Phenol and 200 ul trichloromethane are added into each tube and mixed gently and sufficiently. After incubation for 5 min, samples are spun at 12,000 rpm for 15 min to separate aqueous phase from Phenol phase.

3-4 Carefully collect the aqueous phase into a clean EP tube.

3-5 Repeat the Steps 3-3 and 3-4 once and pool the aqueous phase.

3-6 Add 500 μl trichloromethane into the collected aqueous phase, mix gently and sufficiently, stay for 5 min, and then spin the sample at 12,000 rpm for 15 min to separate aqueous phase from Phenol phase. Carefully remove the aqueous phase into a clean EP tube.

3-7 Repeat the Steps 3-6 once.

3-8 Add 900 μl EtOH to the collected aqueous phase and incubate at -20°C for 30 min.

3-9 Centrifuge the sample at 12,000 rpm for 10 min, and then the DNA sediment is washed with 500 μl 75% EtOH thrice.

3-10 Air-dry the DNA sediment, add 50 μl 0.5 x TE and then adjust the genome DNA to 100 ng/μl for later use.



Note:

1. NgAgo/gDNA system is extremely sensitive to contamination of intracellular bacteria (including mycoplasma) which are widespread and leave no visible signs of presence. Carefully excluding the presence of intracellular bacteria before performing experiments.

2. Because Agos need magnesium, avoid EDTA when detaching and seeding cells into the plates for transfection. Alternatively, supplementing Mg2+ to 5 mM (may need optimization to your cell type).

3. Avoid using Lipofectamine® 3000 since the supplement P3000 interferes with ssDNA transfection. Lipofectamine® 2000 is a good choice. Other transfection methods such as electroporation are yet to be tested.

4. Ideally, 5' phosporylation of ssDNA guide by using T4 PNK (Biolab):

T4 PNK 2 μl

T4 ligase buffer (containing ATP): 12 μl

1OD ssDNA (about 33 μg) in H2O: 100 μl

(alternatively) additional ATP (25 mM) 2 μl

Add H2O to a final volume of 120 μl

Incubate at 37℃ overnight

After 5' phosphorylation, the resultant ssDNA needs no purification, dilute by water (PH8) to 300 μl with a final concentration of 10 nM or 100 ng/μl)

0


免责声明:部分文章和信息来源于互联网,不代表本订阅号赞同其观点和对其真实性负责。如转载内容涉及版权等问题,请立即与小编诸葛神通联系,我们将迅速采取适当的措施。本订阅号原创内容,转载需授权,并注明作者和出处。
CFC 这里有相关专业的:
@向问天
 
啊欧,自然网上这篇文章的下面有这么一段评价:
DONGXIAO YUE•2016-08-08 08:06 PM
The following is a summary of some very heated debate in China on the Han NgAgo paper. 1. Fang's rash accusation of fraud was a mere result of his misunderstandings of the Han paper and molecular biology in general. Even one of Fang's ardent supporters pointed out to him that he misinterpreted the electrophoresis bands presented in the paper. Fang further mistook distances between target sites for DNA segment size deltas. 2. Another named person, who was Fang's main source, has been discredited in a direct online debate, for failing to consider NgAgo's effect of removing 1 to 20 nts at the target site. 3. As stated in Han's paper, the guide can be loaded only when the NgAgo protein is in the process of expression. Dr. Burgio admitted that he did not follow a starred procedure in Han's published protocol. Thus far, the accusations of fraud have been shown to be unfounded. A single successful replication is sufficient to qualitatively vindicate Han's result. Hopefully, the suspicion and ridicule will trigger more curiosity and research to bring about more definitive answers, instead of misguided abandonment of a potentially potent tool for gene editing.

看来韩的发现最终到底有没有价值都跟连滚带爬来摘桃的方教主关系不大咯。:evil:
下一步,教主是不是准备打岳东晓的假呢?:rolleyes: 好像有点不容易呀,岳可是不怕打官司的哦,特别是在美国。:tx:
 
啊欧,自然网上这篇文章的下面有这么一段评价:
DONGXIAO YUE•2016-08-08 08:06 PM
The following is a summary of some very heated debate in China on the Han NgAgo paper. 1. Fang's rash accusation of fraud was a mere result of his misunderstandings of the Han paper and molecular biology in general. Even one of Fang's ardent supporters pointed out to him that he misinterpreted the electrophoresis bands presented in the paper. Fang further mistook distances between target sites for DNA segment size deltas. 2. Another named person, who was Fang's main source, has been discredited in a direct online debate, for failing to consider NgAgo's effect of removing 1 to 20 nts at the target site. 3. As stated in Han's paper, the guide can be loaded only when the NgAgo protein is in the process of expression. Dr. Burgio admitted that he did not follow a starred procedure in Han's published protocol. Thus far, the accusations of fraud have been shown to be unfounded. A single successful replication is sufficient to qualitatively vindicate Han's result. Hopefully, the suspicion and ridicule will trigger more curiosity and research to bring about more definitive answers, instead of misguided abandonment of a potentially potent tool for gene editing.

看来韩的发现最终到底有没有价值都跟连滚带爬来摘桃的方教主关系不大咯。:evil:
下一步,教主是不是准备打岳东晓的假呢?:rolleyes: 好像有点不容易呀,岳可是不怕打官司的哦,特别是在美国。:tx:
岳东晓有什么要打的?
 
岳东晓有什么要打的?
岳东晓说方是民不懂行也没读懂韩的论文,依方是民的脾性,敢对他有任何不敬的都是他的打假对象。估计方是民这会儿正埋头研究看看能不能在岳东晓三十年前的论文里找出一点瑕疵来呢。:monster:
 
方舟子2015年的文章就批过这个岳东晓。

驳斥科学网《方舟子妄批柴静捏造数据的错误》

  科学网首页头条登了一个叫岳东晓的妄人(一个物理博士)谩骂我的文章《方舟子妄批柴静捏造数据的错误》(http://blog.sciencenet.cn/blog-684007-873706.html )。此人多年来一贯发表无知妄言攻击我,我一般不予理会。现在既然被科学网放到了头条,我就赏他个脸。岳东晓指责我读不懂这张图:

tumblr_inline_nl1lhblBcc1t3y4hw.png

  他把该图左纵坐标说成是死亡率每年的增加,然后说每年死亡率都在直线上升。其实是他根本就读不懂这张图,当然更没有看过论文原文。论文原文以及该图的说明说得清清楚楚,该纵坐标是【每天】死亡率(daily mortality)的增加,这个增加是与基数相比的,是以此来表示死亡率的变化。例如,与基数相比,2007年北京循环系统疾病每日死亡率增加2%,而2008年增加1.5%,那么2008年与2007年相比,死亡率实际上是下降的,绝非像岳东晓理解的那样每年死亡率都在直线上升,没过几年北京人都死绝了。

  岳东晓还声称:“吸入雾霾当然不会立刻开始死亡增加,而是有一个积累的延迟。”这也是无知妄言。雾霾导致循环系统疾病是一个积累的过程,而导致循环系统疾病患者的【死亡】却是个即时的过程,即一旦细颗粒物浓度增加,死亡人数也随即上升。这正是那篇论文想要得出的结论。比如,为什么2008年北京死亡率下降了?因为为了开奥运会采取了控制空气污染的措施,立即导致死亡率降下来了。奥运会一开完,这些措施没有了,所以死亡率马上又上升了。

  岳东晓指责我“仔细看图之后竟然连基本的东西都看不懂,反倒轻率指责他人捏造”,谩骂我“方舟子号称博士,一张图都读不懂,而且完全没有脑子”,其实是他本人连基本的东西都看不懂,反倒指责他人看不懂,完全没有脑子的是他。科学网竟把这种不学无术、谩骂攻击他人的文章放到头条,也可见这个打着“科学”旗号的网站其编辑水平低下、作风下作到何等程度。

2015.3.10
 
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