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c********6
发帖数: 693 | 1 杨辉于2012-2013年在白头研究所的Rudolf Jaenisch实验室总共做了10个月的博士后,
在这期间, 杨辉以第一作者或共同第一作者的身份总共发表两篇Cell文章。这两篇文章
都是第一次成功并且完美的将CRISPR质粒注射进入合子中, 一步到位的构建突变小鼠。
Yang H, Wang H, Shivalila CS, Cheng AW, Shi L, Jaenisch R. One-step
generation of mice carrying reporter and conditional alleles by CRISPR/Cas-
mediated genome engineering. Cell. 154: 1370-9. PMID 23992847 DOI: 10.1016/j
.cell.2013.08.022
Wang H, Yang H, Shivalila CS, Dawlaty MM, Cheng AW, Zhang F, Jaenisch R. One
-step generation of mice carrying mutations in multiple genes by CRISPR/Cas-
mediated genome engineering. Cell. 153: 910-8. PMID 23643243 DOI: 10.1016/j.
cell.2013.04.025
然而，该技术发表５年之后全世界有将近１７家实验室公开谴责该方法涉嫌造假。
https://www.the-scientist.com/news-opinion/study-challenges-crispr-method-
for-making-conditional-knockout-mice--64875
Researchers from 17 labs report low efficacy rates for the popular technique.
Sukanya Charuchandra
Sep 28, 2018
Aconsortium of 17 laboratories worldwide has presented results contradicting
a highly cited study that described a technique to create conditional
knockout mice using CRISPR. The preprint, published on bioRxiv on September
1, shows a much lower efficiency rate for the technique compared to the
original report.
The results of the new study indicate the limitations of the original study,
whose success appears to be relegated to deleting a specific gene within a
hybrid mouse strain. The lead author of the first report, cited nearly 1,000
times by Google Scholar’s count, stands by the strength of his method.
Before the original study, published in 2013 by geneticist Rudolf Jaenisch
at the Whitehead Institute of Biomedical Research and colleagues, embryonic
stem cells were used to prepare conditional knockout mice—animals with a
gene engineered to be turned off on command that are missing a gene—a
process that could take years and had only a 1 percent efficacy rate. The
CRISPR technique was presented as one-stop-shop to obtaining conditional
knockout mice with a 16 percent success rate. By injecting zygotes with the
CRISPR machinery, Jaenisch’s team successfully sandwiched the to-be-deleted
gene between two LoxP sites (a step called floxing) that allow the gene to
be conditionally regulated.
“The original Jaenisch paper was a landmark,” says geneticist John
Schimenti, director of Cornell University’s Stem Cell and Transgenic Core
Facility, who was not involved in either study. “It certainly demonstrated
that you can get a high efficiency and high efficiency mutagenesis at
specific loci.”
Conditional knockout mice are extremely important in biomedical research as
they let scientists delete essential genes in specific tissues in the
organism and at particular times during development. While the Jaenisch
method was promising, research groups that have tried to generate mice using
the technique have not been as successful.
“Everybody who’s tried to make floxed alleles by the method they
originally proposed is generally met with failure,” says Schimenti, who has
observed the “same exact issues” in Cornell’s transgenic facility as
those noted in the preprint.
The Jaenisch method often results in off-target mutations, deletions, or a
failure to insert both LoxP sites in the correct orientation, notes
Schimenti. “This is generally recognized in the community, it’s been very
difficult to recapitulate anything close to the numbers that Jaenisch’s
group reported,” he says.
Talks between a close-knit research community at conferences and elsewhere
about the challenges other labs were having with the technique led Gaetan
Burgio, who runs the transgenesis facility at the Australian National
University, and his colleagues to try and determine what was going wrong.
To begin with, three groups replicated the original experiment targeting the
same gene in a different strain of mice and had zero success. Next, 17 labs
, including the original three, independently repeated the experiment on a
total of 56 genes and two intergenic regions in the mouse genome across five
different strains of mice. The combined dataset from all the labs included
17,887 microinjected or electroporated mouse zygotes and a resultant 1,718
live mice, of which only 15 possessed both of the inserted LoxP sites needed
for conditional control. Across all the mice that were tested, off-target
deletions or mutations were observed in lieu of the correct insertion of the
LoxP sites.
Compared to the original study’s 16 percent efficiency rate of obtaining
conditional knockout alleles in mice, Burgio and others had a success rate
of merely 0.87 percent. “The success rate of the method . . . is equivalent
to the classical methods with embryonic stem cells,” says Burgio.
The replication team aimed to figure out the possible factors responsible
for successful conditional knockout mice and found that the simultaneous
insertion of two LoxP sites was critical for the success of the technique.
Jaenisch considers the difference between the mouse strains used in the two
studies to be a sticking point and an underlying reason for the high
variation between the two studies. “I have to discount these data as being
serious,” says Jaenisch, who questions the quality of the recent study.
Schimenti agrees that the genetic background should have been taken into
consideration when replicating the original study. “I think it’s kind of a
flaw in the bioRxiv study—if they were testing the Jaenisch results, they
should have used the exact same types of animals.” However, both Burgio and
Schimenti raise the point that the strain of mice used in the Jaenisch
paper is uncommon compared to the mice used in the new study.
Schimenti also suggests the possibility that the original 16 percent success
rate may have been representative of the single locus in the specific
strain of mice used in that study. “I think it’s clear that this is a very
problematic technique,” says Schimenti. “There needs to be a workaround.
” | m**********r
发帖数: 26 | 2 So many labs and transgenic facilities followed that Cell paper and wasted a
lot of money, effort, and time trying to make conditional mice. What I am
disappointed with this is that in the end, he is going to be all fine. But
if he keeps doing this kind of crappy work, which I suspect he will as
indicated by this new Cell paper, he could get caught eventually and suffer
the consequences. | g********0
发帖数: 6201 | | L***C
发帖数: 1 | | F*Q
发帖数: 3259 | 5 一朝为贼，终生为贼。杜撰、造假或剽窃的人，只要没有付出过代价，他们肯定会不断
地尝试。
---剽窃过本人的院士加HHMI investigator就是例子。
【在 L***C 的大作中提到】
: 每一个传奇人物都有不堪回首的青葱岁月？
| m**********r
发帖数: 26 | 6 这个基本上是这样的，但在中国基本上没有代价。我知道的一个老印，刚知道他时就听
很多人说他造假，我认识的他的一个博后做了个结果，和预期想反，他说你肯定把样上
反了，还不让她重复。她一看这样马上逃离了那个实验室。老印最牛时5个ro1, 甚至当
了系主任，后来被抓到，很多文章撤稿，被停职，只好回印度了。
【在 F*Q 的大作中提到】
: 一朝为贼，终生为贼。杜撰、造假或剽窃的人，只要没有付出过代价，他们肯定会不断
: 地尝试。
: ---剽窃过本人的院士加HHMI investigator就是例子。
| P****R
发帖数: 22479 | 7 烙印胆子不小。
【在 m**********r 的大作中提到】
: 这个基本上是这样的，但在中国基本上没有代价。我知道的一个老印，刚知道他时就听
: 很多人说他造假，我认识的他的一个博后做了个结果，和预期想反，他说你肯定把样上
: 反了，还不让她重复。她一看这样马上逃离了那个实验室。老印最牛时5个ro1, 甚至当
: 了系主任，后来被抓到，很多文章撤稿，被停职，只好回印度了。
| x*********7
发帖数: 427 | | m**********r
发帖数: 26 | | c********6
发帖数: 693 | 10 现在看来当年杨辉跟着李劲松在上海细胞所读博士的时候, 发表的那篇关于在单倍体细
胞里做基因编辑的方法也有很大嫌疑是造假了(当年他们实验室可是大肆宣扬号称在7个
月之内解决了世界性的难题), 直到现在大家都是还是公认这个单倍体细胞的编辑仅仅
停留在猜想阶段,并没有被攻克。想当年他可是凭借那篇文章拿尽了奖学金。 | | | S***i
发帖数: 144 | 11 那篇好像挺火的
没人follow么
【在 c********6 的大作中提到】
: 现在看来当年杨辉跟着李劲松在上海细胞所读博士的时候, 发表的那篇关于在单倍体细
: 胞里做基因编辑的方法也有很大嫌疑是造假了(当年他们实验室可是大肆宣扬号称在7个
: 月之内解决了世界性的难题), 直到现在大家都是还是公认这个单倍体细胞的编辑仅仅
: 停留在猜想阶段,并没有被攻克。想当年他可是凭借那篇文章拿尽了奖学金。
| c********6
发帖数: 693 | 12 肯定是有人去试图重复的, 如果是只有另外1-2家(不会超过)号称也做出来了的话, 不
能够相信的. 就比如当年韩春雨发表NBT之后, 在那么大的争议下, 上海神经所 (跟杨
辉一个所)的仇子龙不是号称自己重复出来了嘛. 要不是大家都拈起棍子打这条狗的话,
他也不会后来发表申明说他们并没有重复出来. 所以后续即使有报道也不排除是沽名
钓誉之辈的人云亦云编造的data.
至少到目前为止, 任何一家正常的实验室包括大型的公司, 当他们需要构建老鼠系的时
候, 绝对不会说用单倍体细胞来做基因编辑.
【在 S***i 的大作中提到】
: 那篇好像挺火的
: 没人follow么
| c********6
发帖数: 693 | | F*Q
发帖数: 3259 | 14 在造假、剽窃盛行的群体里，他如鱼得水而已。
: 辉哥胆子很大的!
【在 c********6 的大作中提到】
: 辉哥胆子很大的!
| f****y
发帖数: 104 | 15 这叫弯道超车
【在 F*Q 的大作中提到】
: 在造假、剽窃盛行的群体里，他如鱼得水而已。
:
:
: 辉哥胆子很大的!
:
| f****y
发帖数: 104 | 16 这个杨辉就是一个小偷，这篇cell也是偷来的。
【在 x*********7 的大作中提到】
: 最近这哥们又发了一篇cell
| Y******U
发帖数: 1 | 17 我把这篇文章发给杨辉了，这是他的回应：
这是之前几天回复质疑的信，抱歉，附件和原邮件不能给你，response还在审稿中。
对我每篇学术文章的质疑，欢迎发信给杂志社或者我来讨论，我只会回复学术的部分，
谢谢！
关于我在MIT的文章，Genome Biology 文章刚在线的时候，我已经和editor取得联系，
她欢迎我们写response回复，因为涉及到一些实验，最近刚刚和文章共同第一作者
Haoyi Wang与通讯作者Rudolf Jaenisch准备好，文章正在投稿中。以下是回复，详见
附件：
Gurumurthy et al. [1]recently reported that a method developed by Yang et al
. to generate floxed allele (designated as “two donor method” by
Gurumurthy et al.) [2] had poor reproducibility. They claimed that three
centers could not reproduce our results on generating conditional alleles of
the Mecp2 locus and that the “two-donor method” had very low successful
rate on other loci.
Here, we provide our responses to these claims:
1. Our results on Mecp2 locus published by Yang et al have been
reproduced by independent experiments in the Jaenisch (8-10% correct alleles
), Yang (8% correct alleles) and Hatada’s groups (2-6% correct alleles) [3]
, respectively.
2. The conditions used by Gurumurthy et al. [1] do not correspond to the
conditions used in our paper. The concentrations of CRISPR reagents used in
the Gurumurthy et al.’s study [1]on the Mecp2 locus (10 ng/μl for Cas9
mRNA, 10 ng/μl for sgRNA and 10 ng/μl for oligos) were much lower (10 fold
lower RNA and 20 fold lower oligo donor concentration) than those used in
the Yang et al.’s experiments (Cas9 100 ng/μl, sgRNA 50 ng/μl and 100 ng/
μl for each oligo) [2] and Yang et al.’s previous [4] and following
publications [5-8]. It is well known that the concentrations of CRISPR
reagents are well correlated with the genome editing efficiency.
3. We utilized piezo-driven zygote injection method in our original paper
, which allows for injecting CRISPR components at much higher concentration.
The difference between this method and pronuclear injection method used by
Gurumurthy et al. might also contribute to the difference of successful
rates.
4. Multiple peer-reviewed publications [3, 9-12] have successfully used
our method to create conditional knockout (CKO) mice (9 out of 11 loci
succeeded, 2.5% to 18% efficiency). We note that the efficiency of
generating CKO mice by CRISPR/Cas9 is highly dependent on the professional
skills and well-built platforms. Thus, it is inappropriate to calculate the
editing efficiency based on data from different labs and “core facilities”
with varying capability and using different methodologies.
5. With any genome editing method or strategy being used, the
efficiencies at different genomic loci are often highly variable. In the
2013 proof of concept paper, we showed the feasibility of generating floxed
allele at Mecp2locus using CRISPR. As a X-linked gene, Mecp2has a higher
chance of having two independent loxP insertion events residing on the same
X chromosome, since half of the embryos are males. To assume the efficiency
we demonstrated at Mecp2locus will be directly translated to the successful
rate at other genomic loci seems premature.
We agree with the Gurumurthy et al’s comment that the “one-donor method”
offers higher success rate for generating floxed alleles in general, while
the efficiency of “one-donor method” is also variable depending on the
genomic loci and donor plasmid design. Before the publication of Gurumurthy
et al., we also noted this, and developed a “one-donor method”, termed “
Tild-CRISPR” method [8], and demonstrated the feasibility and high
efficiency in generating CKO mice.
With the fast improvement of genome editing technologies, we and many others
constantly optimize our protocols. We welcome all discussions about the
choice of optimal strategy for particular applications, however, we think
the reproducibility of any published work can only be validated by using the
exact same experimental methods and technical parameters.
PS: 和Genome Biology的editor 私下交流，这篇文章原本在Nature Methods审稿，但
是有一个reviewer就是用我们这种方法日常做CKO小鼠，所以文章拒了。转投Genome
Biology的时候，也有一个reviewer用我们这种方法日常做CKO小鼠，所以他们的文章关
注点改为比较我们的CKO方法和最新发展的"one-donor method"，才发表出来。我一直
不明白，许多人已经重复出我们的实验结果，也有许多人用该方法做CKO小鼠，这篇文
章也能说造假吗？数据不可信吗？当然更多人做不出来，我认为很大原因是因为本身的
实验体系不好，这篇文章的所有实验室（绝大大数是core facility）都没有先严格重
复我们的实验结果，再在此基础上设计新的实验。我认为这不是受过好的科研训练的人
应有的做事方式，我也没有收到他们的任何来信询问我该实验的实验细节和tricks。
关于我“其他文章也有很多问题”，我将我之前发表的文章都放到附件中，欢迎质疑者
发信给我和大家，一一指出，我会做一一答复。
我也将我自博士期间以来发表的一些重要文章做一个简明阐述：
博士期间：Publication list 30: 孤雄单倍体的建立，这个已有一些实验室同样建立
，比如周琪老师组，同时李劲松老师组也有许多后续的工作。但我相信更多实验室建立
不了这个体系，技术要求太高，所以很难像CRISPR那样广泛使用。不过李劲松老师建立
的孤雄单倍体平台会促进该技术的进一步应用。
博后期间：Publication list 27, 28: 首次报道利用CRISPR技术可以制作各种基因修
饰小鼠，两篇文章都有数千次引用，也有无数个实验室重复和应用。当然技术还在不断
优化和进步，我建立实验室后也发表过相应的文章，见Publication list 14, 20
独立PI期间：Publication list 18: 首次报道利用CRISPR可以敲除整体染色体。同期
其他实验室也有报道，得到验证。
Publication list 7, 11：首次报道单碱基编辑技术的脱靶安全性问题，这两个工作都
是back to back发表，相互很好的印证了彼此的结论。同时我们通过生物学改造获得高
保真单碱基编辑工具的文章也已经在Nature Methods接收，同期Nature Biotechnology
的David Liu的文章的结果也跟我们有很好的印证。值得一提的是，我们在Science文章
中建立的高敏感检测脱靶的GOTI技术，技术要求很高，只有少数实验室具备这个实验条
件，但这些并不影响我们实验数据和结论的可靠性。
Publication list 1, 15：利用各种基因编辑技术做胶质细胞向神经元转分化研究，并
且在最近的Cell文章中利用该方法治疗帕金森及RGC loss的小鼠模型。这篇文章两种疾
病的治疗效果皆是该领域最好的，势必引起很大关注和争议，但我们相信在不久之后，
许多实验室都能在自己各自的系统中得到验证。
最后说说我对最近一些事情的整体看法：
我个人对于外界的评论和质疑其实并不关注或看重，只要不是杂志编辑部或者同行邮件
的询问质疑，都尽量置之不理。我非常珍惜现在中国良好的科研环境，所以格外珍惜每
一点时间。只需一心在科研上面，不停有好的工作展示给同行，就足够了。也很钦佩像
蒲老师、王晓东老师、施一公老师那样，呵护着我们这群青年PI，不用花费过多时间申
请经费，有很好的core facility，在文章思路或写作上给予我们一定的指导。所有这
些让我们有更多时间聚焦于科学本身。
此外，“最近一堆年轻人认为杨辉在MIT的文章涉嫌造假”。我相信绝大多数不是这个
领域的专家，我认为正确的方式是写信给Cell的editor，质疑这篇文章的真实性、可靠
性，而不是靠公众的舆论来挑取自己想要接收的信息。他们似乎不在乎质疑我们工作的
文章的数据可靠性，也更接受10个月（其实不到半年时间）发两篇Cell只有造假才有的
可能。然后近一年又把CNS都发了一遍，造假就实锤了。我觉得这不是一个好的风气，
似乎许多人接受不了同代人比自己出色太多。
张锋一直是我追敢的目标，从PhD开始就一直很出色，很快的fellow经历，刚独立就发
表了最重要的工作，后续更是一系列的重要文章，也成立了自己的公司，将CRISPR技术
第一次应用于人治疗失明。
而我，PhD和Postdoc相对都很顺，但是回国独立头三年没有发表一篇文章，直到第5年
才陆续有重要工作出现。也开始向临床迈进，希望两年内能够国内上临床，最终造福国
内的患者。虽然时间有点落后张锋了，但我仍然坚信有赶上和超过他的一天。
所以希望““最近一堆年轻人”能找到自己追赶的目标，再为之努力，而不是传一些不
在自己学术水平范围内能够judgement的事情。学术圈毕竟不是娱乐圈。
杨辉 | f****y
发帖数: 104 | | P****R
发帖数: 22479 | 19 付向东怎么对付他？
【在 f****y 的大作中提到】
: 偷付向东，还有以前偷其他人的东西，怎么摔锅？
| c********6
发帖数: 693 | 20 不仅是小偷, 而且还胆大包天.
【在 f****y 的大作中提到】
: 偷付向东，还有以前偷其他人的东西，怎么摔锅？
| | | c********6
发帖数: 693 | 21 付向东能拿他怎么办?还不是只能打掉牙往肚子里咽. 只能给其他人提个醒, 将来不要
去上海那边做报告, 小心被人偷idea.
【在 P****R 的大作中提到】
: 付向东怎么对付他？
| c********6
发帖数: 693 | 22 欺世盗名罢了!
【在 L***C 的大作中提到】
: 每一个传奇人物都有不堪回首的青葱岁月？
| c********6
发帖数: 693 | 23 杨辉比韩春雨还要不要脸, 在实验室公然宣扬胆子小的学生就毕不了业. 什么玩意儿! | c********6
发帖数: 693 | 24 杨辉越来越不要脸了,居然把通过造假骗钱成立的皮包公司必做生物界的"华为".好大口
气! | c********6
发帖数: 693 | | c********6
发帖数: 693 | 26 种种迹象表明杨辉的品行确实有很大的问题!
半年发2篇Cell这种事情反反复复在杨辉身上发生, 只有两种可能, 第一就是造假(专挑
那种超高难度的造,反正别人也重复不出来),第二就是剽窃(偷人家的idea,知道人家的
课题已经做到哪一步了,然后赶在别人文章出来之前,通过编造数据的方式快速的把人家
的课题发表出去). | c********6
发帖数: 693 | | s******r
发帖数: 1245 | 28 杨辉要真这么做的不算不意外，投干细胞的大paper作者经常会要求exclude审稿人找
Jaenisch和他的关联人 | d*****h
发帖数: 61 | 29 这种造假不用坐牢吗？类似吃兴奋剂拿了冠军，让第二第三名一生遗憾。何况这是生命
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