An update on my NgAgo experiments

After a very interesting last 3 to 4 weeks, here is a short update on my NgAgo experiments. Firstly I would like to thank all the people for their support and their interest in my previous little piece (https://medium.com/@GaetanBurgio/my-experience-with-natronobacterium-gregoryi-argonaute-ngago-3ed8909b410c#.ofd8tq6nq). Following all the questions I received regarding my data, I wish to reply to couple of comments from various people.

Firstly, as stated before, my work is not an exact replicate of Han’s work. This work is done in mouse zygotes, not human cell lines. I have adapted the protocol to a system (the mouse zygote) I know extremely well. I have no intend doing any work with NgAgo in human cell line.

Secondly, I won’t comment here on the dispute. Many things have been said and well summarised here (http://www.nature.com/news/replications-ridicule-and-a-recluse-the-controversy-over-ngago-gene-editing-intensifies-1.20387). I would like to clarify that I am only interested on the technique. Many true or untrue things were said and I have strictly no intent to comment on this. The others aspects of this story outside of the troubleshooting of the technique have little to no interests to me.

Lack of primers specificity:

This is a very interesting question and was raised quite rapidly after I posted the results. The specificity of the primers were checked multiple times and indeed these are specific without any doubt. No extra-bands were observed.

The results were showed only for one gene are these are not reproducible for other genes:

As explained in my previous piece, I showed the results only for one gene (Ank-1). We could reproduce the same pattern (multiple bands on the gel from the PCR amplification, figure 1 on my previous piece) for 3 other genes (Tet1, Tet2 and Tet3). One other consistent observation was significantly more bands were observed for low concentration of 5'P oligonucleotides (2.5 ng/µl) micro-injected. We are still trying to understand why.

The Ank-1 gene was in fact edited?

I found this interesting post (http://blog.sciencenet.cn/blog-533418-994090.html) claiming a frameshift mutation was observed in the chromatogram I posted (see below from this blog post) and I might miss it.

While I acknowledge the chromatograms are confusing, messy and looks like edited, it turns out that the genome was not edited for 3 reasons. Firstly, the surveyor assay did not reveal any presence of a DNA cleavage. Secondly and importantly, the end of the chromatogram of this sequence showed no frameshift at all (Figure 1). We discovered that these multiple alleles seen in the Figure 4 of my previous post were in fact semi-random amplification from other genomic regions. Finally we performed 2 or 3 additional micro-injection sessions and genotyped the pups that were born. Strictly none of the pups we have genotyped from these 4 genes we have microinjected were edited whereas the editing frequency with CRISPR/Cas9 was in average 90–100% for Ank-1, Tet-1, Tet-2 and Tet-3.

Figure 1: end of chromatogram of Ank1 gene micro-injected with NgAgo

The 5'P oligonucleotide functioned as a primer:

This is certainly a very interesting point. One hypothesis that came up the most is these extra-bands on the Figure 5 from my previous post resulted of the 5'P oligo functioned as a primer. this enabled a chain reaction and semi-random amplifications of the genome. To investigate this further we performed 3 additional experiments. Firstly we purified the DNA and as expected these extra-bands almost disappeared. Secondly we added to the control DNA, the Forward and Reverse primers as well as the 5'P oligonucleotide at various concentrations. Part of the result of this experiment is shown in Figure 2 (Lanes B6 and B6-P). The concentration of 5'P oligo was 2.5 ng/µl. We observed only 2 bands on the gel corresponding to the amplicon length from the Forward primer to the 5'P oligo and from the Forward to the Reverse primer both at the expected size. From this experiment the 5'P oligo functions as a second reverse primer. This was confirmed from all the genes we have tested. We then added the 5'P oligo to the blastocyst DNA, incubated at 50ºC and performed the PCR. The results are in figure 2 (6, 6-P, 11 and 11-P). We could see some extra-bands (lane 6-P and 11-P) compared to without the 5'P oligo (lane 6 and 11). Once again, no editing was evidenced from a surveyor assay and Sanger sequencing.

Figure 2: Electrophoresis gel on Tet-1 and Tet-2 genes of PCR from micro-injected zygotes in a presence or not of NgAgo that have matured to Blastocysts. B6 represent C57BL/6 DNA control. B6-P represents C57BL/6 DNA in presence of 5'P oligo. 6 and 11 are DNA from blastocystes microinjected with NgAgo (5 ng/µl of plasmid concentration) and cultured for 4 days. 6-P and 11-P are DNA from blastocystes microinjected with NgAgo and cultured for 4 days. 5'P oligo was incubated with the samples and the PCR was performed.

How NgAgo works?

In my previous piece, I hypothesised that NgAgo could function not as an endonuclease, but as a ligase. Since, I have investigated this hypothesis. I have analysed some additional results on this gene and on other genes. We have identified that it required at 4 to 12 bp overhang from both 3' ends of the Forward and Reverse primers to amplify those extra-PCR products. The rest of the primer sequence (8 to 16 nt) fused with these 4 to 12 overhang nucleotides to allow the amplification of these genomic regions to occur. This was observed after incubation at 56–58 ºC. I still don’t know if this is related to NgAgo (I have some indirect evidence it is) or whether it is something else. We are still investigating this and this will take probably a fair bit of time.

The NgAgo protocol on Addgene:

I am still really puzzled how NgAgo works and why so many of us have failed to replicate those results. Recently the transfection protocol in cell line was updated on Addgene (https://www.addgene.org/78253/). However out of the 3 pages of protocols, 2 were copied and pasted from the supplementary data of the Nature biotechnology paper. The third page supplied additional information on the protocol but are basic tissue culture and molecular biology techniques. The bottom line is what was provided didn’t add much to what we already know. Once again, if the authors of this paper wish to restore any form of credibility over their technique, they must share their raw data and full and detailed protocols for us to understand why we are unable to replicate this. The distillation of few tips through the media outlets is irritating at least. It has started with the mycoplasma infection, via the multiple transfections of the 5'P oligo towards the addition of Mg or an increase of the pH in the culture media. I would suggest to the authors to write a very detailed protocol and to take part of a group discussion on line to help us to troubleshoot this.

On open science:

To conclude I would reiterate this chase to high impact publication is a total absurdity. Many papers make absurd claims that are not even remotely true (see http://science.sciencemag.org/content/332/6034/1163) or are impossible to reproduce. I think this is having a significant negative impact on the advancement of science. This should really change. We should embrace preprints, post publication peer review process and open science. This would be beneficial for everyone in the scientific community and to the public too.