Does the new gene editing method NgAgo work or not? If not, what happened? The answers to both questions seem to depend on who you ask and what you read.
As much as CRISPR has been the revolutionary in the genetic modification technology arena over past methods, could CRISPR itself in the next few years become obsolete having been replaced by other new technologies such as the upstart NgAgo? I doubt it.
The odds for NgAgo making a run in this field may have gone down lately, at least based on a comment left by Sheng Qiang on my original post on NgAgo:
“A war of word broke out on the reproducibility of Han’s work these days, especially on the Mitbbs website. The doubters, represented by Zhouzi Fang, said that no labs have repeated Han’s work, especially the Figure 4 results. The supporters claimed that 20 labs in China already repeated Han’s work, yet no data have been shown to support the claim. The doubters suspect that this is another STAP cell incident for China. To be fair, we should probably give more time for labs around the world to repeat Han’s work, which was trumpeted in the Chinese media to be a Nobel prize worthy scientific breakthrough. Let’s just hope that this will not go down the same path as the STAP cells.”
The Zhouzi Fang mentioned seems to most likely be Fang Shimin (方是民), pictured above, who has a Wikipedia page here that mentions his role as a popular science writer who campaigns against pseudoscience and fraud. It also discusses a number of controversies in which he has been involved. I wonder if he might be like Japan’s juuichijigen who played a key role in uncovering STAP. I don’t know.
I’m hoping to learn more about this NgAgo situation so that we all can better judge what the status of NgAgo research might be. The notion that this could be another STAP-like situation would be very unfortunate, but it seems there’s not enough information now to judge and that’s a serious thing to assert. I agree with the commenter that more time is needed before we can be sure what’s what here.
So what is out there on discussions over NgAgo as to whether it works or not?
I did find this page on an “NgAgo” search onMitbbs (which when Google crudely translates it) seems to fit with what the commenter says about a war of words, but I have no idea if that page is reliable.
I also found this Chinese-language science news site reporting on the NgAgo controversy.
This Google group page on NgAgo also has some researchers reporting it doesn’t work for them, but others said it did work.
Overall, I’d say the jury is out, but it’s clear there are strong opinions both ways on NgAgo.
First official conclusion from Gaetan Burgio lab.
https://medium.com/@GaetanBurgio
Swarts DC, Jore MM, Westra ER, Zhu Y, Janssen JH, Snijders AP, Wang Y, Patel DJ, Berenguer J, Brouns SJ, van der Oost J. DNA-guided DNA interference by a prokaryotic Argonaute. Nature. 2014, 507(7491): 258-61
https://patentscope.wipo.int/search/en/detail.jsf;jsessionid=434BE029C1F2BA7D5CF35646B9335B64.wapp2nA?docId=WO2014189628&recNum=1&tab=PCTDocuments&maxRec=&office=&prevFilter=&sortOption=&queryString=
What’s your point, randomwalk?
The genome engineering field already knows about van der Oost’s work. That particular Argo only works for in vitro applications because of the relatively high (to mammalian physiological conditions) temperature required for it to work.
If I recall right, Swarts was supposedly working on modifying it in his own newish(1-2 years old?) lab.
@ a non stem cell guy
Your problems sound more like an issue with the strain of bacteria you’re transforming in rather direct issues with the plasmid itself.
It is standard practice to resequence all plasmids you obtain from an outside source.
I would suggest that you go with ZFNs, TALENs, or CRISPR/Cas9 unless your experiments absolutely depend on NgAgo (in which case I would strongly urge you reconsider). Nothing is more disheartening than watching other biologists waste time on non-useful experiments. Only so many “this doesn’t work as advertised” posts are useful to the community, and I’m pretty sure we’ve already hit critical mass for that.
Well did you guys check your plasmids ?
We are currently trying it and I can tell you that just the cloning of it is a pain if you try to drive the expression of ngAgo with a pol II promoter.
In a way the bacteria detects it as a foreign construct and it gets degrades, so far we got many truncated ng Ago clones and none of them could be used for genome editing.
So it could explain why nobody can do anything if the plasmids contain small deletions.
We tell you if it work 😉
Your problem looks quite strange, actually funny. Bacteria never knows what is pol II promoter, and it only works in mammalian cells. Sometime, you get truncated plasmid (for example, lentivirus or LTR containing) because the special sequence or structure. Under such condition, you can try other stains, like XL1-Blue or Stbl. If you clone some fragment into pcDNA3.1 or pEGFP-N1, and bacteria don’t like it. That will be really funny.
Also, given some of Chunyu Han’s recommendations on how to get it to “work (several of which fly in the face of 30-40 years of molecular biology observations), my money’s on this is sorta, kinda, another STAP-like situation.
Regardless, the efficiencies presented in Han’s paper aren’t good enough for me to justify ever switching.
So far on the Genome Engineering Google Group, no one has been able to replicate or generalize the reported indel-generating ability of NgAgo.
Gaetan Burgio claims that he has indels on Twitter, but the evidence was PCR products on a gel with no sequencing reported (yet). Other commenters have pointed out that the ssDNA guide persists for a long time in the cell and could act as an additional primer in the PCR (!!!).
My thoughts are maybe it’s an alternative to dead-Cas9 for activation and repression purposes, but it will probably be limited to extreme edge-use cases. It seems a little to finicky to use otherwise.
If you could not understand that Chinese-language science news site report
here is an English version might help, http://bloge.us/han-rain-ngago-gene-editing-technology-repeatability-cited-questioned/