2020 Update: early NgAgo reports have been mostly discredited and there is doubt on its function as a gene-editing method.
What could be better than CRISPR for gene editing?
A new genetic modification technology called NgAgo has some researchers really excited. How does it compare to CRISPR?
I’ll admit it that as a scientist who works on genetics and genomics, I am really enjoying the power and simplicity of CRISPR-Cas9 type technology for genome editing. We are working with it extensively in my lab. One of the remarkable things about CRISPR is how fast the technology has evolved in just the last 2 years.
Despite all that warp speed for CRISPR, some are asking: could the new method zoom past CRISPR?
While NgAgo is indeed a nifty new genome editing technology based on DNA guides instead of RNA guides, it’s not going to immediately race ahead of CRISPR…not yet any way. Still it’s got people buzzing.
A recent Nature Biotechnology paper from Chunyu Han’s lab, DNA-guided genome editing using the Natronobacterium gregoryi Argonaute, is a must-read for genome editing folks. Their team sums up the new method’s potential pluses this way (emphasis mine):
“The useful features of NgAgo for genome editing include the following. First, it has a low tolerance to guide–target mismatch. A single nucleotide mismatch at each position of the gDNA impaired the cleavage efficiency of NgAgo, and mismatches at three positions completely blocked cleavage in our experiments. Second, 5′ phosphorylated short ssDNAs are rare in mammalian cells, which minimizes the possibility of cellular oligonucleotides misguiding NgAgo. Third, NgAgo follows a ‘one-guide-faithful’ rule, that is, a guide can only be loaded when NgAgo protein is in the process of expression, and, once loaded, NgAgo cannot swap its gDNA with other free ssDNA at 37 °C. All of these features could minimize off-target effects. Finally, it is easy to design and synthesize ssDNAs and to adjust their concentration, which is difficult with the Cas9-sgRNA system, if the sgRNA is expressed from a plasmid and the normal dosage of an ssDNA guide is only ~1/10 of that of a sgRNA expression plasmid.
This might be a more orderly way and perhaps even simpler way to go about genome editing than CRISPR, but the jury is still out on that until there are more papers and data. The NgAgo edit efficiency at this preliminary stage of technology development seems very strong (see Figure 5 from the paper above).
NgAgo is a nifty new kid on the block for genome editing so let’s see what more we learn about it in coming months via the almost certain flood of additional papers that tell us more about it.
Hat tip to my colleague Dave Segal on the NgAgo paper.
@Sheng Qian,
Could you please provide links to the webpage(s) that relate to your comment?
Thanks,
Paul
Genomeweb has a report on this, although it’s a paid page and I can’t open it.
Link to the page:
https://www.genomeweb.com/gene-silencinggene-editing/new-genome-editing-tool-elicits-interest-concern-about-reproducibility?utm_content=37423724&utm_medium=social&utm_source=facebook
If you register (free) I think you should be able to read it. I can and I never paid anything.Paul
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.
@Jim Bacon
From what limited information is at that link, it’s one of three things:
1) Crossovers (most common in meiosis); not really exciting
2) AAV with homology arms, like the paper from Mark Kay’s lab a couple years ago. Interesting, but I can’t tell if any of these Homology Medicines people are associated with them or just using the same idea (Side note: Steve Dowdy’s lab has shown that combining this approach with CRISPR/Cas9 results in extremely high efficiency of correct targets with the caveat of selection being required).
3) Smoke and mirrors, people, smoke and mirrors. All aboard the hype train! Choo-choo!
Homology Medicines is also hinting that it has developed a gene editing technology that trumps CRISPR:
http://www.fiercebiotech.com/can-5am-upstart-homology-leapfrog-gene-editing-pioneers
“”In-Vivo gene editing, with high fidelity … has arrived,” Tweeted Arch’s Robert Nelsen this morning. “Not using Crispr or talens.” “
NgAgo replys on 5′-P ssDNA which only cannot be generated via a plamisd, thus cannot delivered via plasmid or virus. It only can use the synthesized 5′-P DNA and delivered via transfection which could be a main bottemneck for this DNA based editing. And it’s editing result may be consistent since the transfection delivery is uncontrollable.
@Bud, that’s a great point. Entirely or nearly entirely egineered editing systems may yield the best of the best features of different natural systems.
@Chris, Yeah, for those of us who have invested time and resources into CRISPR it doesn’t make sense to switch right now. For certain applications that won’t work with CRISPR that well because of PAM issues or whatever though it might be worth trying NgAgo. Simple enough. Thanks for the comment. Paul
@Anonymous, you make some good points. I do think DNA guides are going to be easier though and as Chris mentioned and I should have in my article, the lack of a PAM requirement may prove very important in certain cases. The bottom line is that if NgAgo is better, people will switch to it. If it isn’t, they probably won’t. Let’s see what the data in the next year or so tell us.
Paul
Now that we have a few examples of naturally occurring gene altering nucleases, can completely lab created nucleases be far behind?
Hey Paul,
I thought this was a hugely exciting paper, but mainly in the sense that it highlights how young this field of gene editing it.
With regards to CRISPR vs. Ngago, I think there’s some benefits to the latter (no PAM requirements, smaller (complete list from me here: https://www.horizondiscovery.com/gene-editing/ngago) but if you’re already set up and running CRISPR-Cas9, there aren’t going to be huge advantages to leaping across at this stage.
That said, for labs or companies that have not yet gone CRISPR it presents an exciting alternative, and (dare I say it) may be a less convoluted route when it comes to IP.
We will see, but I suspect Ngago won’t be the last new nuclease for genome editing we see in 2016
Chris
Do you think it could be possible to edit genes in adults by this technique, too?
The quick answer is to invoke Betteridge’s law of headlines: no.
To go by the point-by-point breakdown:
1) Low tolerance to mismatch is also a feature of Cas9. We’ve learned that by looking at sequenced iPSC genomes from the Church lab.
2) The rationale for misguiding is actually the most ridiculous thing I’ve heard for genome engineering problems. This is a non-issue. (Also, phosphorylated ssDNA may be viewed as DNA damage or an infection, both of which would be undesirable for genome manipulation).
3) I can’t recall if anyone has actually looked at the sgRNA swapout rate for Cas9, so this might be an advantage, maybe, kinda, if one is worried about extremely unlikely things happening?
4) It is easy to design and synthesize sgRNAs as well, as long as you do it yourself and are careful during the synthesis step.
Regarding Figure 5, that editing efficiency actually seems fairly low to me for HEK293T cells (you know, the cell type where everything always works well). They also reference a supplementary figure (S8) when describing Figure 5, where their claim is Southern blotting reveals off-target insertion with Cas9. If one looks at the blot, the “off-target” insertion seems to be something else entirely and not related to editing.
Stability and consistency might by the keyword, now we had two studies on human embryo showing CRISPR-Cas9 does not necessarily reach the target genes: NgAgo is betting on this aspect to gain popularity. Now, only the future will tell which of the two technique swill prevail, but let’s expect it will create a healthy competition environment.
Or maybe project with most funding will prevail at the expense of better tech. Has happened before.