发信人: abyssdragon (魔龙出山), 信区: Biology
标 题: 韩春雨事件梳理
发信站: BBS 未名空间站 (Thu Sep 13 19:10:25 2018, 美东)
竟NgAgo未经过系统的密码子优化---照着online method上protocol for genome
editing and T7E1做（小经验，PCR最好不要有引物二聚体，如果引物二聚体多请重设
对于Fig4，若是不习惯做银染，也可以直接做KI：doner 用GFPcoden+polyA signal，
扩（防止GFP-N1污染的假阳性）出来纯化，500-800ng共转（for each well of 24
协会创建者Lluis Montoliu根据Gaetan Brugio和自己实验室的结果，向国际转基因技
From: firstname.lastname@example.org on behalf of Lluis
Montoliu To: ISTT List
Subject: [ISTT_list] Great disappointment with Ago: Long life to
Date: Friday, July 29, 2016 9:49:22 AM
the publication by Gao et al in May in Nature Biotechnology
(http://www.ncbi.nlm.nih.gov/pubmed/27136078) triggered an enormous
expectation. This Chinese team led by Chunyu Huan reported that the
Argonaute (Ago) protein from a rare haloarchaea, Natronobacterium
gregoryi, (NgAgo) would efficiently work for gene editing purposes in
human cells. Ago had been described as an DNA-guided endonucleases two
years before, through a Dutch-Spanish microbiologist collaborating
team (Swarts et al. 2014, Nature:
On paper, the new (fourth) Gene Editing system looked great. An
endonuclease, using ssDNA guides (5' phosphorylated though) and not
RNA guides, without a PAM, requiring 24 nucleotides (and not 20nt),
hence with higher specificity, and apparently with fewer off-target
issues, since modifications in just one position of the DNA guide
resulted in >90% decrease of the protein activity.
I must confess we read the Huan paper in my lab with some
disappointment, after two years battling, unsuccessfully, with Ago
from Thermus, through a collaboration with my friend and colleague J.
Berenguer, from the neighbouring reserch centre CBMSO, and one of the
co-authors of the Nature 2014 paper. We had been scooped. We
repeteadly failed to find any gene editing activity using Ago from
Thermus thermophilus (TtAgo) in mammalian cells, through a variety of
conditions and we didn't understand why, though we always suspected
that these proteins would not be too comfortable at "too cold"
temperatures as physiological +37C. After reading the Gao paper we
concluded we simply missed the right bug and congratulated them for
being smarter and lucky and for finding this archaea. Perhaps the
trick was in using NgAgo instead of TtAgo.
Shortly after NgAgo was released from Addgene, beginning of June, many
labs, including mine, jumped onto it to try experiencing the
anticipated great expectations and joy associated with this new tool
of prokaryotic origin. But soon it was clear that something wasn't
quite right. Rumours began spreading during June and July at congresses,
through social networks, list emails and discussions groups that NgAgo
didn't appear to work as reported. Actually, didn't work at all. Some
colleagues that I absolutely trust at scientitic and technological
levels started to indicate that they could not reproduce Huan's paper
At the recent TAGC meeting (where IMGS was contributing to, merging in
along with other Genetics Societies) Gaetan Burgio, from ANU, Camberra,
Australia, presented some very preliminary data with a gel with some
intermediate bands that would suggest NgAgo would be working and
editing at the expected places. But, shortly thereafter, Gaetan
engaged his lab in an OpenScience project, tried to characterize all
these bands and.... found nothing. So, again, another evidence
confirming NgAgo is not working as a gene-editing tool.
Gaeatan just released today his experience using NgAgo, openly sharing
his failures and providing details and some explanations for them.
My experience with Natronobacterium gregoryi Argonaute (NgAgo) Gaetan
Burgio Group leader at JCSMR, ANU
At first, KUDOS to Gaetan. Many thanks to him for sharing their
efforts trying to confirm some gene-editing activity associated with
NgAgo. There is apparently none. In his view, NgAgo might be working
as a ligase at physiological conditions. Similar to our negative
results using TtAgo it would appear that NgAgo requires some higher
temperatures to work as initially reported. This of course seeds some
doubts on the Gao et al. publication and Gaetan, among other, is
requesting to Nature Biotechnology to request the Huan's lab to reveal
and share their raw data. We will see this part of the history how it
But, now, the most important message to convey is: NgAgo does not work
for gene editing in mammalian cells. Be aware and do not waste your
time, your money, your peoople and projects. If anyone has any
positive hint suggesting Ago is indeed working as a genomic editor,
please share the results, for the sake of Open Science, as Gaetan
beautifully and most generously did. Many thanks to Gaetan!
Unfortunately, this is a great disappointment. But, it also highlights
the uniqueness and the robustness of the CRISPR-Cas systems.
Long life to CRISPR!
Lluis phone: +49621- 1703 6210/6204
Dr. Lluis Montoliu
Investigador Cientifico - Research Scientist
CSIC Centro Nacional de Biotecnologia (CNB-CSIC)
Campus de Cantoblanco
C/ Darwin, 3
28049 Madrid (Spain)
Tel. +34-91-5854844 / Fax +34-91-5854506
U756 CIBERER: http://www.ciberer.es
Spanish EMMA node: http://www.infrafrontier.eu
At present, I would recommend everyone abandoning any project
involving the use of NgAgo. And avoid wasting time, money, animals and
people. Results are clear in many labs. The results we have in Madrid
are that TtAgo does not work in mammalian cells. The results that
Gaetan has in Australia are that NgAgo does not work in mammalian cells.
I know of many other colleagues who also tried and failed, but have
not reported these failures publicly. This is why I posted this message.
I think Ago might have some potential but we don't seem to have found
yet the adequate version of it (or the right bug).
As a side history, Francis Mojica struggled to find Grants to support
his pioneer experiments with CRISPR and got a couple of projects
turned down. That is why, instead of using Streptococcus pyogenes
(Doudna and Charpentier) or thermophilus (Siksnys), buga more
difficult and expensive to grow, he chose to work in Escherichia coli
CRISPR systems, and faced many difficulties, only to find out, many
years later that E.coli CRISPR-Cas system was of type I (not type II,
as Cas9) and, furthermore, was mostly inactive.
Please, let's focus in CRISPR-Cas9, Cpf1, C2c2,... et al... and leave
alone Ago while microbiologists don't find out the right one.
※ 修改:·abyssdragon 於 Sep 13 21:22:35 2018 修改本文·[FROM: 173.]
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