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发帖数: 693 | 1 9月28日Joe Miano
Most of you have already moved on from this discussion but I wanted to chime
in one last time with our negative data in the mouse. Whether we used
original plasmid or a custom NgAgo that was codon-optimized for mouse with
untranslated sequences, a Kozak Methionine, and polyA tail the results were
uniformly negative. We used a 5' phosphorylated ssDNA (both IDT bought or
in vitro phosphorylated) along with the mRNA to codon-optimized NgAgo and an
HDR template we used successfully to edit a regulatory element (published
in late 2014). Genotyping of more than 50 pups failed to show a single
correctly edited genome (note we did not sequence or test for indels because
of time/cost and because we were only interested to know whether it could
edit as CRISPR did in a 3 component experiment). Thus, in all, we tried
three experiments (one in vitro and two in vivo) and none worked. The
corresponding author of the NgAGo paper communicated with me throughout and
emphasized some dubious claims (e.g., higher magnesium concentration). This
method, touted initially to supplant CRISPR, simply does not work in mouse
genome editing. The author should either come clean with his secret
methodology or a confession for publishing a false story. He is responsible
for many labs spending tens of thousands of dollars in reagents, tech/
postdoc time, and injections of fertilized eggs. He should reimburse those
of us who spent the time and $$ to try and extend his findings with the
millions he has been awarded. He should be held accountable for any
scientific impropriety and apologize to the genome editing community. The
fact that he does not appear at international meetings or even release a
public statement underscores the dubious nature of the work in Nature
Biotechnology. The latter journal should also come clean and at least
publish a letter of concern.
As I know is the case with others before me, this has been a colossal waste
of time, money, and resources.
On Thursday, June 23, 2016 at 9:18:50 PM UTC-4, Davide Seruggia wrote:
Hi,
I tried a couple of transfections with the NgAgo plasmid from Addgene and
the 5'P oligo against GFP from the paper, but I did not see a dramatic
reduction in GFP+ as seen using Cas9 and a GFP sgRNA.
Anybody tried as well and came to the same (dead) end?
Davide
9月28日Ivan Ivandic
Thank you very much for this honest statement, I couldn′t agree more...
- 隐藏引用文字 -
On Wednesday, September 28, 2016 at 6:21:43 AM UTC+2, Joe Miano wrote:
Most of you have already moved on from this discussion but I wanted to chime
in one last time with our negative data in the mouse. Whether we used
original plasmid or a custom NgAgo that was codon-optimized for mouse with
untranslated sequences, a Kozak Methionine, and polyA tail the results were
uniformly negative. We used a 5' phosphorylated ssDNA (both IDT bought or
in vitro phosphorylated) along with the mRNA to codon-optimized NgAgo and an
HDR template we used successfully to edit a regulatory element (published
in late 2014). Genotyping of more than 50 pups failed to show a single
correctly edited genome (note we did not sequence or test for indels because
of time/cost and because we were only interested to know whether it could
edit as CRISPR did in a 3 component experiment). Thus, in all, we tried
three experiments (one in vitro and two in vivo) and none worked. The
corresponding author of the NgAGo paper communicated with me throughout and
emphasized some dubious claims (e.g., higher magnesium concentration). This
method, touted initially to supplant CRISPR, simply does not work in mouse
genome editing. The author should either come clean with his secret
methodology or a confession for publishing a false story. He is responsible
for many labs spending tens of thousands of dollars in reagents, tech/
postdoc time, and injections of fertilized eggs. He should reimburse those
of us who spent the time and $$ to try and extend his findings with the
millions he has been awarded. He should be held accountable for any
scientific impropriety and apologize to the genome editing community. The
fact that he does not appear at international meetings or even release a
public statement underscores the dubious nature of the work in Nature
Biotechnology. The latter journal should also come clean and at least
publish a letter of concern.
As I know is the case with others before me, this has been a colossal waste
of time, money, and resources.
On Thursday, June 23, 2016 at 9:18:50 PM UTC-4, Davide Seruggia wrote:
Hi,
I tried a couple of transfections with the NgAgo plasmid from Addgene and
the 5'P oligo against GFP from the paper, but I did not see a dramatic
reduction in GFP+ as seen using Cas9 and a GFP sgRNA.
Anybody tried as well and came to the same (dead) end?
Davide
9月29日Petr Tomek
Very very true. I thought the Nature Journals would be more credible than
this. Well, that is the way the cookie crumbles for the blockbuster-hungry
journal editors.
Dne středa 28. září 2016 17:21:43 UTC+13 Joe Miano napsal(a): | P****R
发帖数: 22479 | |
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