Gaetan Burgio New Data & Theory on NgAgo versus CRISPR

By Gaetan Burgio

CRISPR/Cas9 genome editing has dramatically changed our way to perform biological experiments. While highly efficient and easy to use, one limitation with CRISPR/Cas9 mediated genome editing technology is the occurrence of off-target effects and the restriction of the PAM recognition sequence. Many modifications from the original system have been proposed to improve its efficiency, specificity and to avoid off-target effects. Recently a new system based on the bacteria Natronobacterium gregory Argonaute (NgAgo) was proposed as a serious alternative to CRISPR/Cas9. NgAgo is based on a DNA recognition pattern and unlike all the systems based on CRISPR doesn’t require a PAM recognition sequence. The target specificity is mediated from a phosphorylated oligonucleotide on the 5′ end. As it doesn’t require any cloning or in-vitro transcription, it was sought to be a serious alternative to the CRISPR-Cas9 system.

Recently an astonishing paper published in Nature Biotechnology from Chunyu Han’s group in China proposed NgAgo as a simple system to edit cell lines. As many, I was particularly interested to establish the protocol in my laboratory. The recent availability of the plasmid from Addgene encouraged us to establish this protocol and this is what I tried to do in the last two months or so. Below is a summary of my experience with NgAgo.

Reproducing Han’s paper results:

Firstly I decided to not repeat Han’s experiment stricto sensu as my group works primarily on mouse zygotes. The sequences targeted in this paper were all specific to the human genome. Instead I’ve chosen a gene that I’ve been working on for a very long time (Beta-spectrin) and used it to make my first CRISPR/Cas9 edited mouse line over 2 and 1/2 years ago. Usually to establish a new technique, I use a set of highly efficient sgRNA targeting this gene. These sgRNA are working extremely well and are extremely helpful to improve the technology in my hands.

We had a first attempts on Beta-spectrin gene by co-injecting the NLS-NgAgo-GK plasmid at 5 ng/µl with various concentrations of 5′ phosphorylated oligo (2.5, 25 and 50 ng/µl) purchased from IDT into the mouse zygote. After co-injection of the mix into the pronucleus, we cultured the zygotes for 4 days to blastocyst stage and extracted the DNA for PCR and Sanger sequencing.

Many extra bands on the gel electrophoresis:

The first results from our PCR are below (Figure 1) and were very exiting for us. It showed many extra-bands on the gel. I thought these were products of the edited genome as I see often with CRISPR/Cas9. At that time I was at the TAGC conference in Orlando, USA. I showed the results to my colleagues and after few discussions with them I decided to release this gel picture below (Figure 1) from my twitter account.

Burgio Figure 1

Figure 1: PCR on mouse Blastocysts after NgAgo Pronuclear injection in zygotes
We then performed the T7 endonuclease assay on these PCR products (Figure 2) and surprisingly we couldn’t see a clear difference with the original PCR, which was very strange.

We then performed the T7 endonuclease assay on these PCR products (Figure 2) and surprisingly we couldn’t see a clear difference with the original PCR, which was very strange.

Figure 2: T7E treatment on the PCR product of the NgAgo injected Zygotes Interestingly at higher concentration of 5' phosphorylated oligo and the same primer set, these extra bands almost disappeared (see Figure 3). We saw this with others genes too (Tet1 and Tet2).

Figure 2: T7E treatment on the PCR product of the NgAgo injected Zygotes
Interestingly at higher concentration of 5′ phosphorylated oligo and the same primer set, these extra bands almost disappeared (see Figure 3). We saw this with others genes too (Tet1 and Tet2).

Interestingly at higher concentration of 5′ phosphorylated oligo and the same primer set, these extra bands almost disappeared (see Figure 3). We saw this with others genes too (Tet1 and Tet2).

Burgio Figure 3

Figure 3: PCR and electrophoresis gel on mouse Blastocysts after NgAgo Pronuclear injection in zygotes with high concentration of 5′ Phosphorylated oligo

Meanwhile I discovered from many discussions on my Twitter account, at the TAGC meeting, emails I have received and from this interesting Google group discussion thread that many have tried to replicate Han’s results using his experimental setup, in human cell lines, mouse or zebrafish with NgAgo DNA, mRNA or protein. They all failed to edit the genome.

First Sanger sequencing results:

We then performed a first round of Sanger sequencing and the chromatograms were an absolute mess (Figure 4) to a point that we couldn’t properly identified any sequences (Except from the wild type allele) as many alleles were amplified. However, by matching the guide to the sequences, I had the suspicion that 2 samples were edited (from Figure 1, samples 3 and 8)

Burgio Figure 4

Figure 4: Typical Sanger sequencing run we had from NgAgo, 5′ phosphorylated oligo injection into zygotes, culture to Blastocyst, DNA extraction, PCR amplification and sequencing.

Second Sanger sequencing results:

We then performed again the PCRs and decided to cut every single extra band from the electrophoresis gel and send those to Sanger sequencing to determine whether these were sequences from the plasmid, from the edited beta-spectrin gene or primer dimers. Couple of discussions I had on twitter or elsewhere mentioned that the 5′ Phosphorylated oligo could act as a primer and amplify the genome, which is possible and I will come back to this later. The results are in Figure 5 and show convincingly that these extra bands were the amplification of random sequences.

Burgio Figure 5

Figure 5: Electrophoresis gel of mouse zygotes micro-injected with NgAgo

I must make 2 important comments: 1) The primers are specific to the sequence of interest. we have performed tons of PCRs using this primer set and we never saw these extra bands. 2) This result is specific to the low 5′ phosphorylated oligo concentration setup and is almost nonexistent with 25 ng/µl of 5′ phosphorylated oligo.

Clustal alignment:

Initially I thought these sequences were random but I wasn’t quite sure. To test this hypothesis, I aligned all these sequences together using Clustal to see whether I could identify a common pattern. The results are presented in Figure 6 using the results from Sanger sequencing (forward primers). The results are similar for the Reverse primers and I won’t show it here.

Burgio Figure 6

Figure 6: Typical Clustal alignment of all the sequences cut from the electrophoresis gel. The Ank-1 is the reference sequence.

There is clearly a common pattern which doesn’t match at all the 5′ phosphorylated oligo. However it matches with the sequences from the Forward and Reverse primers but quite imperfectly and I will come back to this later. The first hypothesis that came into my mind is my primers are not specific enough. Although it didn’t explain 1) Why at 25 ng/µl of 5′ Phosphorylated oligo I don’t see this pattern, 2) I should have for a long time noticed this given I have genotyped and Sanger sequenced over 100 CRISPR/Cas9 edited mice using these primers and 3) the initial PCRs (Figure 1,2 and 3) showed no extra-bands for the B6 (C57BL/6) DNA control or the water.

To investigate this further, I hypothesised that a foreign DNA sequence (plasmid or other nucleotides from the mouse genome) integrated to these amplified sequences. To test this, I Blast searched the sequences to the mouse genome and the primer pairs for each sequence that were cut from the gel. One example is presented in Figure 7. I found the same pattern for the Forward and Reverse primers for all samples that I have tested.

Burgio Figure 7

Figure 7: chromatogram of one typical sequence (here Reverse primer)

Figure 7 shows two features. Firstly the first 6 to 9 nucleotides from the Forward and the Reverse primers match perfectly with the endogenous sequence. Secondly the remaining 13 to 16 nucleotides from the primer pairs were added to the endogenous sequence. This explains the amplification of these extra bands on the gel (Figure 1). This primer pair was not phosphorylated and no ligase was added to the PCR and sequencing reactions.

NgAgo: A ligase enzyme?

From these results, my hypothesis is as following: The NgAgo plasmid was injected into the zygotes and NgAgo was transcribed and translated into a protein, possibly at zygote stage. The enzyme certainly persisted to blastocyst stage at 37ºC and remained intact after DNA isolation from the blastocysts. The PCR reaction certainly activated the NgAgo enzyme, which functioned as ‘a ligase’ under the classical PCR conditions and added the 10 to 15 nucleotides to the endogenous sequences that were matched with the first 6 to 10 nucleotides of the primer pairs. Interestingly this ‘ligase’ activity from NgAgo seems to be inhibited at high concentration of 5′ Phosphorylated oligo. My hypothesis is this might have degraded the NgAgo enzyme.

My Hypothesis on how NgAgo function:

After these series of experiments, these are my thoughts on NgAgo. Firstly, as many elsewhere found, I have found strictly NO EVIDENCE for a genome editing with NgAgo after multiple attempts with various settings and 3 different genes. Secondly I found instead a ‘Ligase’ like activity of NgAgo under normal PCR conditions, which has strictly nothing to do with the endonuclease activity claimed in Han’s paper. It seems to me that the NgAgo enzyme needs to be heated over 50ºC to function, which is in direct contradiction to the Han’s paper.

My take on all these failed experiments trying to reproduce Han’s paper is basically the incubation temperature of the cells is too low for the enzyme to function or the enzyme/5′ phosphorylated oligo complex is rapidly degraded within the cells explaining possibly why nobody has been able to reproduce Han’s experiment. NgAgo may or may not have an endonuclease activity creating a double strand break but under so specific conditions that they are almost impossible to reproduce and too restrictive for a broad use of this system if this is real. Additionally I do have some serious doubt on NgAgo over its endonuclease activity. Nature Biotechnology should ask Han to release all his raw data + experimental condition to the public. This is a duty of care from the journal. Finally I do believe strongly that whatever happens with NgAgo. the CRISPR/Cas9 system will be there for a very long time and NgAgo will be rapidly abandoned after such failed attempts from everyone in the genome editing field. There is clearly no bright future for NgAgo.

My view on Open Science:

Finally I would like to conclude my post by acknowledging all the people in my laboratory, on Twitter and elsewhere that have contributed to this story. It was my first open science experience and I found the discussion with my peers highly stimulating. I think rather than to chase high impact publications and be secretive, we should be more open and share our results to avoid everyone wasting their time on results that are irreproducible and pointless. In my opinion this is the way Science should work.

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Poll: Will NgAgo strongly challenge CRISPR in coming years?






Controversy over CRISPR challenger NgAgo irreproducibility reported

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.

Fang Shimin (方是民) NgAgo

Wikipedia image

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.

Hateful politics infiltrate human genome editing debate in France

By Elliot Hosman

Summary.  A campaign calling for a moratorium on using CRISPR in human embryos was launched by a prominent French organization fighting for narrow understandings of life and family.

A recent campaign calling for a ban on “transgenic” human embryos was launched by one of France’s most prominent organizations fighting for “science”-backed “one-man-one-woman” families, and the exclusion of all other forms.

Stop Baby GMO Campaign

“Stop GMO Baby: Yes to therapeutic progress, no to transgenic embryos” (image via Alliance VITA).

Since March 24, more than 15,500 people in France have signed a petition started by Alliance VITA declaring (translated from French*):

“I ask my country to engage with all urgency to obtain an international moratorium – that is to say an immediate stop – on the genetic modification of human embryos, especially via the technique CRISPR-cas9.”

*all French materials and quotations presented in English in this post have been translated using Google and my college-level French. Suggested revisions to translations are welcome and will be noted. Alliance VITA offers some materials on its website in English.

In that time, volunteers have canvassed cities around France, handing out brochures explaining the breakthrough CRISPR genome editing technology, and tweeting pictures of their advocacy using Flickr and the hashtags: #StopBébéOGM, #ProtectHumanity, and #CRISPR-Cas9.

Alliance VITA’s opposition to using human gene editing for reproduction is widely shared, including by my organization, the Center for Genetics and Society. But a closer look at the Stop GMO Baby campaign in France reveals a troubling and at times explicitly hateful politics infiltrating the human genome editing debate. A polarization of the conversation about heritable human genetic modification along “right to life” and “natural family” fault lines threatens to derail public conversations about responsible regulation of science and medicine that serves the public interest.

Paul also recently flagged Alliance VITA’s Stop GMO Baby campaign, cautioning:

“I’m concerned that these campaigns that specifically target CRISPR could have negative effects on the freedom of us scientists to do responsible CRISPR research in the lab. … at least some of the motivation seems to be related to a “right-to-life” perspective. “

I share this concern, and we’re not alone. In a February article titled Gene editing: The next frontier in America’s abortion wars, the “last scientist in Congress” U.S. Representative Bill Foster (D-IL) told Politico’s Sarah Karlin that he’d been warned by scientists that “‘this issue will get all tied up over the abortion debate,’ interfering with the creation of ‘good policy decisions.’”

The Stop GMO Baby Campaign

Alliance VITA’s campaign materials on CRISPR take as their central point that CRISPR-Cas9 is an ethically neutral and promising technology that could help gene therapy, but that any use in human embryos or gametes is a red line no researcher in the world should cross. In their other words: “GM babies? No!” Here are some examples of their slogans and statements:

  • Campaign slogan: “CRISPR-Cas9: Yes to Therapeutic Progress, No to Transgenic Embryo!” (March 24, 2016) [Brochure PDF]
  • On February 16, 2016, Alliance VITA Research Director Blanche Streb stated on Catholic television: “The technique poses no ethical problems on its own, it’s the application that does.” (YouTube)
  • Alliance VITA General Delegate-CEO Tugdual Derville commenting on Kathy Niakan’s application to the HFEA in January 2016:

“Although this technique might be promising for genetic therapy, Tugdual Derville reminds us that when applied to the human embryo: “the danger is to cause the emergence of custom-made babies, with pre-selected genetic criteria, heritable modifications, with unknown consequences for future generations. The human genome is part of our most precious “heritage of humanity.” Its integrity must absolutely be preserved for future generations.”

In March, Alliance VITA released a study they conducted finding that 76% of French people support gene therapy, but oppose using CRISPR to genetically modify embryos in vitro. Some of their data conform to a number of other recent studies. But the slipperiness of public opinion polls that Pete Shanks describes in a recent survey of public opinion of human heritable genetic modification is on point here, as the framing of questions may lead to an overstatement of the sanctity of the embryo for the people who polled their opposition.

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Shape Shifting NY Times Headline on Gene Drive Becomes More Positive

After the NY Times published an article on June 8th on the exciting, controversial technology called gene drive that can alter the genome of an entire species, strangely the article’s headline changed at least twice.

The article was focused on a National Academy panel studying this technology. The headline gradually evolved to become much more positive in tone. You can see this evolution below in chronological order from top to bottom.

Harmon Gene Drive article headlineThe first headline sounded like there wasn’t much support, while by the time the third title for the same article popped up now the focus was on the panel endorsing gene drive. I’m not sure if the actual text of the piece changed substantively or not, but what gives with the title?

Did the paper get pushback on the original title from someone? Did editors themselves change their minds and decide a more positive title was needed?

Who knows? However, it seems like this kind of headline evolution means something.