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发帖数: 351 | 1 Email on ngAgo sent to the ISTT mailing list：
Dear colleagues,
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: http://www.ncbi.nlm.nih.gov/pubmed/24531762).
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.
On paper.
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 results.
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
https:[email protected]/* *//my-experience-with-natronobacterium-
gregoryi-argonaute-ngago-3ed8909b410c#.bo9y6mf9u
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 develops...
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! |
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