韓春雨的實驗被其他實驗室重複出來了嗎？

12-30

求問韓春雨的實驗有沒有另外的實驗室確切的重複出來了？如果有，請問是哪個實驗室？
在此提問的目的只是想進行試驗細節的討論，希望能有重複得好的實驗室給無法重複的實驗室指點一下，並無任何潑髒水的意思。希望大家多多討論實驗細節和更新最新的實驗進展。畢竟這一研究是令人振奮的發現，大家都希望第一時間能夠利用好這一工具。
討論實驗請注意文明用語。

更新~~~~~~
NgAgo對於基因編輯領域確實是一個大新聞，一出來，領域內的實驗室應該很多都第一時間開始在重複，再次重申開此貼的目的只是為了討論實驗細節，找出無法重複的具體實驗細節，以便大家都能夠重複出文章的Fig.4結果（切割內源基因），請勿做有罪推定。其實所有的質疑，只要有一家實驗室能夠重複出來，都會煙消雲散。
今天得到韓老師的公布實驗細節需要注意的事項，正是開貼最希望看到的，希望此貼成為大家交流試驗技術細節的平台。
歡迎討論實驗細節，請勿人身攻擊和有罪推定。

本人沒有重複出來，，，胚胎幹細胞-mcherry穩定株敲除，，，，多組不同量的組合測試均表示無效---流式發現沒有一丁點敲除的跡象-相對於陽性的穩定株，，，是引物合成公司給我保證99%以上磷酸化了的，聽說做出來是用激酶磷酸化的，難道化學上兩者還不一樣了？我會再測一遍以見真章，我相信我的實驗室養細胞和轉染的能力，，，

風口浪尖時啊！我本人不確定也不清楚那段話是不是韓春雨本人所回復。單論回復的內容看，我個人有很多疑問。第一：什麼是Ago蛋白對污染的細胞很敏感？為什麼，難道其他蛋白就不敏感了，Ago在污染的細胞里就不能切割了？第二：做Figure3實驗的時候，單轉oligo看到GFP knock down 之後，就是假陽性，說明是細胞污染的可能性。那麼是什麼污染呢？真菌污染可以很明顯的看見，如果是支原體污染也可以檢測。而且照他這個理論，這個方法都可以開發出來用來檢測支原體污染了。第三：如果是細胞污染，不可能單轉oligo就能看到GFP 表達下降，這是什麼機制啊！單轉NgAgo怎麼就看不到GFP down 呢。第四：為什麼要一再強調讓其他人重複Figure3，正如某位知友所說，大家更關心Figure4，這篇文章的重點就是Figure4，如果沒有切割內源基因的作用，這篇NBT也不會有這麼大的關注度。我們對bac來源的NgAgo蛋白的表達做了個時間點檢測，可以很明確的說，bac來源的NgAgo，就算沒密碼子優化，絲毫不影響表達，轉染後4h後就可以看到表達，16h依然在增強。所以Figure4重複的難度在哪裡？？

補：這個領域的人都很憤怒啊，大家花了這麼多錢，這麼多精力，在嘗試NgAgo的工作。真正的科研精神，應當是善意競爭。同行之間應該既交流合作又相互競爭。一個系統只有做的人多，大家都爭相優化，才能夠有影響力。就跟ips 假如只有yamanaka一個實驗室做，他怎麼可能有如今的地位。我不知道

如果這篇文章只是做出來Fig3c的話，根本沒有人會去關注他，關鍵data是Fig4，他能

夠引起所有人注意的一個點在於，設計的幾十個基因位點無一例外，全部都能夠驚人的

高效切割內源DNA，所謂重複出來的同志們，關鍵是要重複Fig4切割內源基因，並且有

測序的結果。韓也在電話里說了（第三軍醫大的一小伙給韓打了電話，並錄音），

review重複出來了他的Fig3c，我並不懷疑Fig3c的結果，並且也不在意Fig3c的結果，

我們關注的還是內源基因！電話錄音里韓一個勁叫人做Fig3c，但是明明Fig4才是關鍵

，如果用Cas9的話，一個沒有基礎的實習生，只需要兩個星期的訓練，便能夠做出一個

漂亮的T7E1和測序的data。轉染和T7E1都不是需要多麼高超技巧的技術，令人納悶的是

，為何如此多熟練操作基因編輯的實驗室，都無法重複出內源基因的knock-out。

補：

1.單純轉染dna產生GFP的表達抑制，有一部分可能是類似antisense效應（從文章看，fig3.c有一半的oligo是antisense dna）。但明確的，所有的核酸酶工作與否，都應該通過測序來檢測，是不是假陽性，沒必要糾結這個不完備的實驗結果，直接測序就好。2.所謂高超的技巧，是一個成熟實驗室的基本規則。污染控制，DNA 230/260/280檢測，PCR，跑PAGE，銀染，都屬於用了幾十年的老技術，門檻根本不高。不過控制轉染時的細胞密度和用血清控制細胞生長屬於非常規操作，如韓老師能提供詳細的protocol就好了。3.目前在重複實驗的實驗室，至少一半已經做了密碼子優化。其實好多人是用了密碼子優化的質粒做不出，又反過來去addgene來嘗試韓老師提供的未優化版本。4.不太能理解為什麼不能用agarose和EB。銀染並非解析度高，而是敏感度高。銀染技術本身非常不適合做定量，因為反應太靈敏，很容易「過曝」。5.認同推薦用KI來檢測系統。

順便說一句，真理越辯越明，質疑不等於反對。那些不討論科學問題本身，一切從動機出發，亂扣帽子的別煩我，沒時間磨嘰。

匿了

目前大家還在吵，bbs上有人說能重複但是沒有提交證據。微信上也有個圈內微信群說有人做出來了。不過按著文章是沒人做出來的。

可能性如下

1 作者隱瞞了關鍵步驟。

我覺得這種可能性最大，ssdna是比較特殊的。

nature系列壓文章一貫臭名昭著的，如果隨隨便便能被重複出來根本輪不到他發。就算他發出來，第二篇也輪不到他。國際上大組的動作太快了。

老外其實也玩隱瞞這一套的，他們寫grant也很注意不要泄密。

2 造假

不排除，不過看他極其爽快的把質粒提交給addgene又不像。

我猜還是關鍵細節被隱瞞了，比方說是否需要形成dna-rna雜合鏈，是否需要高磷酸化率，是否需要二磷酸化之類(胡謅的)

現在的情況是關係近的組，能夠做出來的都不會出來講，等文章吧。

你看，當時就說仇子龍藥丸。已被打成水貨騙子，乞丐級科學家。

畢竟這個東西實在太爆炸了。

更新仇子龍的回復

新論文：無效！No evidence of genome editing activity from Natronobacterium gregoryi Argonaute (NgAgo) in human cells

Parisa Javidi-Parsijani,
Guoguang Niu,
Meghan Davis,
Pin Lu,
Anthony Atala,
Baisong Lu
Published: May 11, 2017

不怕別人重複不出來，只要能在第三方的監督下由自己實驗室重複出來也是可以自證清白的。目前來看，能重複出來的實驗室一方面肯定悶聲繼續往前推進了，另一方面，即使真的重複出來了，目前在這樣的風口浪尖，恐怕也沒幾個人會站出來明確表示自己做出來了，以避免惹禍上身。從實驗的角度來看，重複不出來有很多原因，但首先自然不是去質疑對方造假，而是懷疑自己水平不行。據我所知，是有人號稱自己重複出來了的，但這中間是否有「皇帝的新裝」這種可能也是不得而知。

目前方舟子已經盯上韓春雨，雖說方的名聲不好，但個人認為，這種質疑是沒有問題的。科學的來講，對任何理論和結果我們都可以去質疑。但科學研究本身是一種道德自律性很強的行業，審稿人一般都會選擇相信作者提供的數據，就是假定你不會騙我。但在文章發表出來了，就應當直面所有人的質疑。如果韓老師有百分百的自信，自然是不會害怕來自任何方面的質疑，即使是觸犯到了大牛的利益，但是只要自己能重複出來，那也不由得別人不相信了。

現在的問題是，韓老師打算如何應對這些質疑，還是說不打算應對。個人對他的結果保留懷疑的權利，實際上我們可以對任何已發表的工作隨時保持懷疑。但還是希望他能重複出來。

做實驗不穿白大褂不帶手套不戴護目鏡移液槍平放你還指望他的實驗能重複出來？

韓教授弟子高峰做實驗(下圖)和他老師一個毛病

再來幾張特寫

這才叫擺拍，韓教授終於學會怎麼擺拍了

轉Burgio的最新評論：

My experience with Natronobacterium gregoryi Argonaute (NgAgo)

CRISPR/Cas9 genome editing has dramatically changed our way to perform experiments. While highly efficient and easy to use, one limitation is the occurence of off-target effects and the restriction of PAM recognition sequences. Many modifications of the original system have been propose to improve the efficiency, specificity of the system and to avoid off-target effects. Recently a new system based on the bacteria Natronobacterium gregory Argonaute (NgAgo) was proposed as a serious alternative to CRISPR/Cas9. NgAgo is based on a DNA recognition pattern and unlike all the systems based on CRISPR locus doesn』t require a PAM recognition sequence. The target specificity is offered from a phosphorylated oligonucleotide. As it doesn』t require any cloning or in-vitro transcription, it was sought to be a serious alternative to the CRISPR-Cas9 system.

Recently an astonishing paper published in Nature Biotechnology from Chunyu Han』s group in China proposed NgAgo as a simple system to edit cell lines. As many, I was particularly interested to establish the protocol in my laboratory. The recent availability of the plasmid from Addgene encouraged us to establish the protocol and this is what I tried to do in the last month or so. Below is a summary of my experience with NgAgo.

Reproducing Han』s paper results:

Firstly I decided to do not repeat Han』s experiment per se in his paper as my group works primarily on mouse zygotes. The sequences targeted in this paper were all specific to the human genome. Instead I』ve chosen a gene that I』ve been working on for a very long time (Beta-spectrin) and made my first CRISPR/Cas9 edited mouse line over 2 and 1/2 years ago. Usually to establish a new technique, I use a set of sgRNA targeting this gene. These sgRNA are working extremely well and I have been trialling different improvements of the techniques using those guides.

We had a first attempts on Beta-spectrin gene by co-injecting the NLS-NgAgo-GK plasmid at 5 ng/μl with various concentrations of phosphorylated oligo (2.5, 25 and 50 ng/μl) purchased from IDT into the mouse zygote. After co-injection of the mix into the pronucleus, we cultured the zygotes for 4 days to blastocyst stage and extracted the DNA for PCR and Sanger sequencing.

Many extra bands on the gel electrophoresis:

The first results from our PCR are below (Figure 1) and were very exiting. It showed many extra-bands on the gel. I thought were product of the edited genome as I see often with CRISPR/Cas9. At that time I was at the TAGC conference. I showed the results to my colleagues and after few discussions with them decided to release this gel picture below (Figure 1) from my twitter account.

Figure 1: PCR on mouse Blastocysts after NgAgo Pronuclear injection in zygotes

We then performed the T7 endonuclease assay on these PCR products (Figure 2) and we couldn』t see a clear difference with the original PCR, which was very strange.

Figure 2: T7E treatment on the PCR product of the NgAgo injected Zygotes

Interestingly at higher concentration of 5" phosphorylated oligo, these extra bands almost disappeared using the same primer pair (see Figure 3). We saw this with other genes too (Tet1 and Tet2).

Figure 3: PCR and electrophoresis gel on mouse Blastocysts after NgAgo Pronuclear injection in zygotes with high concentration of 5" Phosphorylated oligo

Meanwhile I discovered from many discussions on my Twitter account, at the TAGC meeting, emails I have recieved and from this insteresting Google group discussion that many have tried to replicate Han』s results using his experimental setup, in human cell lines, mouse or zebrafish with NgAgo DNA, mRNA or protein. They all failed to edit the genome.

First Sanger sequencing results:

We then performed a first round of Sanger sequencing and the chromatgrams were an absolute mess (Figure 4) to a point that we couldn』t identified the sequences as many alleles were amplified. However, by matching the guide to the sequences, I had the suspicion of editing on 2 samples (from the Figure 1 sample 3 and 8)

Figure 4: Typical Sanger sequencing run we had from NgAgo, 5" phosphorylated oligo injection into zygotes, culture to Blastocyst, DNA extraction, PCR amplification and sequencing.

Second Sanger sequencing results:

We then performed again the PCRs and cut every single extra band from the electrophoresis gel and send those to Sanger sequencing to determine whether these were sequences from the plasmid, from the edited beta-spectrin gene or primer dimers. Couple of discussions I had on twitter or elsewhere mentioned that the 5" Phosphorylated oligo could act as a primer and amplify the genome, which is possible. The results are in Figure 5 and show convincingly that these extra bands were amplification of random sequences.

Figure 5: Electrophoresis gel of mouse zygotes micro-injected with NgAgo

I must made 2 important comments there: 1) The primers are specific to the sequence of interest. we have performed tones of PCRs using these primers and we never seen had these extra bands. 2) This is specific to low 5" phosphorylated oligo concentration and are almost inexistent with 25 ng/μl of 5" phosphorylated oligo.

Clustal alignement:

Initially I thought these sequences were random but I wasn』t quite sure. To test this hypothesis, I aligned all these sequences together using Clustal to see wheter I could identify a common pattern. The results are presented in Figure 6 using the results from Sanger sequencing (forward primers). The results are similar for the Reverse primers and I won』t show it there.

Figure 6: Typical Clustal alignement of all the sequences cut from the elecrophoresis gel. The Ank-1 is the reference sequence.

There is clearly a common pattern which doesn』t match at all the 5" phosphorylated oligo. However it matches with the sequences from the Forward and Reverse primers but quite imperfectly and I will come back to this. The hypothesis that came in my mind is my primers are unspecific. Although it doesn』t explain 1) Why at 25 ng/μl of 5" Phosphorylated oligo I don』t see this pattern, 2) I should have for a long time noticed this given I have genotyped and Sanger sequenced over 100 CRISPR/Cas9 edited mice using these primers and 3) the initial PCRs (Figure 1,2 and 3) showed no extra-bands for the B6 (C57BL/6) DNA control or the water.

To investigate this further, I hypothesised that a foreign DNA sequence (plasmid or other nucleotides from the mouse genome) integrated to these amplified sequences. To test this, I Blast the sequences to the mouse genome and the primer pairs for each sequence that were cut from the gel. One expample is presented in Figure 7. I found the same pattern for the Forward and Reverse primers.

Figure 7: chromatogram of one typical sequence (here Reverse primer)

The figure 7 shows two features. Firstly the first 6 to 9 nucleotides from the Forward and the Reverse primers match perfectly with the endogenous sequence. Secondly the remaining 13 to 16 nucleotides from the primer pairs were added to the endogenous sequence. This explains the amplification of these extra bands on the gel (Figure 1). This primer pair was not phosphorylated and no ligase was added to the PCR and sequencing reactions.

NgAgo: A ligase enzyme?

From these results, my hypothesis is as following: The NgAgo plasmid was injected the zygotes and NgAgo was transcribed and translated into a protein, possibly at zygote stage. The enzyme certainly persisted to blastocyst stage at 37oC and remained intact after DNA isolation from the blastocysts. The PCR reaction certainly activated the NgAgo enzyme, which functionned as 『a ligase』 under the classical PCR conditions and added the 10 to 15 nucleotides to the endogenous sequences that were matched with the first 6 to 10 nucleotides of the primer pairs. Interestingly this 『ligase』 activity from NgAgo seems to be inhibited at high concentration of 5" Phosphorylated oligo. My hypothesis is this might have degraded the NgAgo enzyme.

My Hypothesis on how NgAgo function:

After these series of experiments, these are my thoughts on NgAgo. Firstly, as many, I have found strictly NO EVIDENCE for genome editing with NgAgo after multiple attempts. Secondly I found instead a 『Ligase』 like activity of NgAgo under normal PCR conditions, which has strictly nothing to do with the endonuclease activity claimed in Han』s paper. It seems to me that the NgAgo enzyme needs to be heated over 50oC to function, which is in direct contradiction to the Han』s paper.

My take on all these failed experiments trying to reproduce Han』s paper is basically the incubation temperature of the cells is too low for the enzyme to function or the enzyme/5" phosphorylated oligo complex is raidly degraded within the cells explaining possibly why nobody has been able to reproduce Han』s experiment. NgAgo may or may not have an endonuclease activity creating a double strand break but under so specific conditions that are almost impossible to reproduce and too restrictive for a broad use of this system if this is real. Additionally I do have some serious doubt on NgAgo over its endonuclease activity. Nature Biotechnology should ask Han to release all his raw data + experimental condition to the public. This is a duty of care from the journal. Finally I do believe strongly that whatever happens with NgAgo. CRISPR/Cas9 system will be there for a very long time and NgAgo will be rapidly abandoned after such failed attempts. There is clearly no bright future for NgAgo.

My view on Open Science:

Finally I would like to conclude my post by acknowledging all the people in my laboratory, on Twitter and elsewhere that have contributed to this story. It was my first open science experience and I found the discussion with my pairs highly stimulating. I think rather than to chase high impact publications and be secretive, we should be more open and share our results to avoid everyone wasting their time on results that are irreproducible and pointless. In my opinion this is the way Science should work.

update on 2016/08/29

「最美教師」韓公布新的protocol已經月余，仍然沒人能重複出其實驗結果，其造假可以肯定了。網上實名為他辯護的，剩下了一個叫岳東曉的物理博士要指導生物學家們怎麼做分子生物學實驗，因為過於熱情和荒唐，被外國科學家懷疑是韓春雨的馬甲。---by 方舟子

最大的可能性，韓誇大了效率。

用Google 查到國外重複這個實驗的研究者目前有兩個實驗室取得進展，等待後續結果，相關鏈接如下：

https://groups.google.com/forum/#!topic/crispr/V1FALNbspCo

因為被噴，所以摘錄了重複出韓春雨NgAgo實驗的印度實驗室的發言：

Dr. Debojyoti Chakraborty

Room 129, CSIR-Institute of Genomics and Integrative Biology

Mathura Road,

New Delhi - 110020

+919433790132

Jun 29

Hi guys,

We
have used this system and it works. We have followed the same protocol
as mentioned in the paper and were able to confirm knockout both on FACS
as well as western. We purified the oligos on gel after T4 PNK and saw
the phenotype after 48 and 72 h.

cheers

debo

just a small correction: we did not purify the oligos. We used 700 ng ngAgo and 500ng sgDNA for Lipo3000 mediated transfection.

Here"s the protocol we followed:

We first phosphorylated the
gDNA using the PNK kit from Ambion for 1 hr followed by heat
inactivation at 95 degrees for 5 min. We then went on to transfect
100,000 cells in 12 well plates as follows:

Mix 1:

Lipo reagent- 3ul

Optimem- 43ul

EGFP N1- 1 ug

NgAgo- 900 ng

gDNA- 500 ng

Mix 2:

Lipo 3000- 2ul

Optimem- 48ul

Mix 3:

Media- 250ul

Optimem- 150ul

Mix1 and Mix2 was incubated for 10min each and then both were mixed together and incubated at RT for 30min

This was now added to cells. To each well, 50 ul of Mix 3 was added on top. No further media was added to the cells.

After 12h, the mix was removed and fresh media was added.

個人觀點：（在目前結果尚無定論前，仍然不好妄自評判）

1.首先，無論究竟是否像某些社會人士所找的借口闡述涉及到核心技術或國家機密，韓老師在面對同行質疑時所表現出的公關手段及處理程序，與一個成熟的卓越的科研工作者的形象是不匹配的，並且相比海外教育，其受到傳統中國教育的謹慎言行的知識分子氣息看不出來，網路中的言行反而有點戾氣，一些答覆可以說有點不講道理的詭辯。我見過很多學者，甚至是院士，就算在有的問題上存在裝睡的情況，但那大多是面對外行人，比如政府領導，面對同行，都是坦坦蕩蕩，就算玩陰的也是直來直去。

2.網上流傳出的官方的韓老師實驗宣傳照，很遺憾，我是真沒有見過在實驗室里是這樣做實驗的科研人員，可能是擺拍需要，連實驗服都不穿，手套也不帶，甚至在宣傳照中出現了較不嚴謹的實驗操作，連出現在鏡頭中的論文第一作者韓的弟子高峰也是如此，韓在這篇文章中排序為第四，為通訊作者。如果是宣傳需要，我想如果為了宣傳連自己的職業精神都不尊重，不如去拍時尚大片。

3.其實這事最後的走向，是否涉及造假，在我看來，當前圈子內爭議如此巨大之時，政府的相關部門能否摒棄政績觀與傳統和稀泥的手段，直面質疑，啟動全面的調查，無論對於科研形象也好，還是還韓老師以清白也好，都是極其必要的。畢竟李總理點了贊，處理和調查的程序上必須嚴謹，不然對科研的影響是致命的。

4.當然，如果韓老師的成果最後走向了大家最不願看到的一面，首先這件事會讓中國科研學術界集體蒙羞，大大降低基礎科研在社會中所營造的積極形象，但個人覺得解決當前問題的關鍵不是韓是否造假，而是主管部門乃至校方怎麼處理這次事件，以及隨後的督查程序。

5.我覺得之後對於國內的影響才是長久的，高等學府作為科研主力軍，頂級名校的資源聚集優勢將會更為明顯，普通二本乃至中西部普通學校的青年研究者的科研環境將愈發惡劣，國家寧願將基礎科研的有限資源集中在有著高大上實驗室，完備團隊及成熟自查程序的北青復交，也不會再傾向於多年教育人士們奔走疾呼的教育公平化的普通學校及二本之中，科研結構的二元化將更為典型和集中。作為一個普通二本的青年教師，本還想做點實事，現實感憂心。如果我是主管部門，我也寧願把發CNSP的重大任務全交給一流名校，普通本科就回歸到教育本質上來。什麼河北科技大學，好好做好本科教育和研究生教育就好了。

6.中國當前的社會民眾大多本就處於一個蒙昧的階段，說實話，那就是該懂的不懂，不該懂的也不懂，這也應該歸結於長久以來的唯利是圖和宣傳口徑隱晦化的社會風氣離不開。但韓春雨老師的出現，其實給了社會上一個很不好的點和現象，也給了民眾一個宣洩的出口。我許多同事甚至以這個例子說，就算十多年不發論文不做科研，最後也能積澱出成果，中國人最大的特點就是給自己的混找一個借口，當然如果我五十多歲了我可能也會開始混了，但我沒有臉面說得那麼振振有詞。

科研說到底也是一份工作，而這份工作的普遍存在一個門檻較高的情況，而民眾借這個特例，把基礎科研看得很庸俗化、功利化。我說實話，如果一個五十多歲的老師，不關心科研，十年磨一劍，我還可以理解，但一個當打之年的青年教師，十多年一篇文章不發，我是有點覺得在正確的時間沒有做正確的事的看法。

這個事情發展到最後，長此以往，基礎科研在社會形象中必將一落千丈，在一個有限科研經費下的普通高校的諾獎級成果，國家與民眾或許再也不信。

貼一張圖，供懂得人參考

來源：jessecai@mitbbs

反對有人根據PCR電泳圖出現的水平邊界認為其拼貼造假的說法，我從http://www.nature.com/nbt/journal/v34/n7/extref/nbt.3547-S1.pdf下載了補充圖表並在Photoshop中加大對比度分析。

Supplementary Figure 9 j, for Fig 4 a: T7E1 (DYRK1A).

Supplementary Figure 9 t, for Supfig 5: T7E1 (HBA2,GATA4,GRIN2B,HRES1,APOE).

可以看出電泳圖的所有地方，包括拼貼毫無意義的地方，都出現了水平邊界，並且其高度均為12px。這顯然並非拼貼造成的現象，而應該是將電泳圖合併在Supplementary Figure 9中因為圖片有損壓縮造成的現象。

9月2日最新結果進展——無法重複新增一例：

實驗結束：未能利用NgAgo敲除目的基因

發信人: volontaire (volontaire), 信區: Biology

標題: 實驗結束：未能利用NgAgo敲除目的基因

發信站: BBS 未名空間站 (Fri Sep 2 18:21:27 2016, 美東)

已完成用韓春雨在自然生物技術所報道的NgAGO做基因敲除的實驗，按照韓春雨提供的

Genbank號找到了序列，做了密碼子優化，在蛋白N端加了V5標籤，C端加了SV40核定位

信號，全基因合成了這個蛋白的基因序列克隆到了lenti病毒載體，用包裝的病毒轉導

了A549細胞，可以檢測到蛋白表達，克隆了轉導細胞拿到了穩定表達NgAgo的細胞系，

按照韓春雨文章中列出的方法設計了一對gDNA 靶向基因RNASEL的起始密碼子區域，10

倍於韓春雨所示DNA的量轉染細胞，48小時後克隆轉染細胞，一共篩選了36個克隆，未

見到任何RNASEL敲除的表型，大家看著辦吧，我做不出來

某些韓的支持者，請不要把問題往民族主義上引！

重複不出來那就應該出來解釋，科學的一大特點就是可重複性。

某些韓的支持者就跟某些小鮮肉的粉絲似的，張口閉口你知道##有多刻苦嗎，##發著250度的高燒還在訓練，你們黑他還是人嗎？請記住，科研成果不談情懷，談情懷請出門左拐找羅胖子。

韓老師條件有多苦，實驗條件有多差，人品有多好，這些都不是科學所關注的，關注的只有你的成果的意義以及真實性！

這倆是一個人吧，在這帶節奏

希望韓老師的成果早日重複，不然對國內底層科研界的打擊太大了。

方舟子說的很客觀也很實在，另外韓老師不應該顧忌別人重複出來河北工業大學就成吊車尾了，你是第一個做出來的，世界的同行都知道的，別人做出來的發揚你的工作不是好事嗎？》

建議大家自己動手檢驗真偽，把韓的pdf原文從nature網站下載之後截圖sup fig9j，然後用Image J軟體打開，點image--&>adjust--&>brightness/contrast,適當調整一下，就能發現原圖ps的痕迹非常明顯，一看便知，電泳條帶明顯粘貼上去的，不相信你自己試試吧

——————————————

應一些評論人士的要求，我貼一下我處理的圖片和我的主觀分析。

上面這張圖是從nature網站下載的韓文獻的pdf版本，之後截取的sup 9j圖。

上面這張圖是Image J軟體的圖片處理路徑，下面有具體參數截圖。

上面這張圖是具體處理後的情況，調節的參數也在裡面顯示著。下面我再貼一張標註有疑問地方的圖。

這張圖是在上面一張的基礎上對電泳條帶做了標註，分別是1,2,3個框表示。我們用這張圖進行分析：

疑點1. 條帶1和3的拖尾都是向上的，但是條帶2的拖尾是向下的，做過電泳的人都知道這不科學，不應該出現這種情況。

疑點2, 條帶2框住的部分具有明顯的邊界，大家可以對照上面一張原圖看看，ps時候整個條帶的粘貼就會出現這種情況。

疑點3，條帶2框住部分的背景噪音整體是橫向條紋，與周圍背景噪音明顯不一樣。

疑點4，條帶3框住部分的拖尾在上方某個位置突然整齊截止，我解釋為ps條帶粘貼的情況。那麼有人會問，是不是圖像壓縮造成的呢，這個問題你可以用同樣的方法處理其他圖，我發現只有幾張圖有這種情況，其餘大部分圖像過渡都比較自然，沒有明顯ps粘貼的情況。大家可以自己試試。

非常詭異的是，這張補充圖對應於正文fig4a，而fig4a確沒有這麼明顯的處理痕迹，難道fig4a後期經過了細緻的修飾，消除了ps痕迹？只是猜測。

本人目前人在美國，有2年多的CRISPR/Cas9經驗，N年的細胞培養經驗，所以請不要質疑這方面的技術或是細胞污染。Well, 我用了韓的Addgene上的質粒，用了文中的targeting GFP的磷酸化的ssgDNA（但不是T4PNK處理的，而是直接從Sigma合成的磷酸化引物），然後按照文中的比例轉染了GFP的穩定細胞系，我連GFP的Knockdown都一點也不顯著，就是說我連假陽性也未出現。Sorry,根據目前的情況，我選擇質疑韓的系統。或者說裡面肯定有關鍵的東西沒有公開。

最近我的論文被某國外研究組引用。本來應該高興的，但是我仔細一看，他們根本就是胡亂引用，根本沒有看懂我的論文。在給國際知名期刊（IF&>6）審稿過程中，也看到很多論文，數據處理這一塊就是錯的，根本不是內行，數據的解釋也非常牽強。還有很多事例，不一一列舉。

因此，本人十分確定，很多研究組就是打醬油的，混飯吃的，根本不是真心做科研。

N多研究組，實驗技術不過關，也沒有自己的研究思路，就是跟風，胡亂做出一些結果，隨便找個地方發表而已。跟他們不必浪費時間和納稅人的錢。各基金資助機構應該把這些研究組列在黑名單上。

把科研留給認真的人去做吧。