RE: DNA-guided genome editing using the Natronobacterium gregoryi Argonaute

[SKim]

Hello,

I guess most of you are aware of a recent paper published in Nature Biotechnologies.

http://www.nature.com/nbt/journal/vaop/ncurrent/full/nbt.3547.html

It's been a week since the publication and I have been expecting that there would be flaming discussion about this new genome editing tool but this forum is relatively quiet.

We understand that there will be more work to be done for further validation.

However, based on clear advantages over CRISPR/Cas9, I am wondering how many of you are thinking that NgAgo is going to be the one in the next round.

[YIng Dang]

I can't wait to see a follow-up report.

[Shouguang Huang]

Hi, 

I have read the literature and found this technique really attracts our eyes. There are more advantages than CRISPR/Cas9, such as the description in the literature:

1. It has a low tolerance to guide-target mismatch.

2. 5' phosporylated short ssDNA are rare in mammalian cells, which minimizes the possibility of cellular oligonucleotides misguiding NgAgo.

3. NgAgo follows a "one-guide-faithful" rule.

4. it is easy to design and synthesize ssDNA and to adjust their concentration.

In sum, I have to say NgAgo-guide genome editing is a super nice technique. However, I guess most of labs are still focusing on CRISPR/Cas9, they might hard to switch their direction to this new stuff. But I strongly believe that there will be more and more scientists harbor this new emerging tool in next few months. 

LET'S SEE!

[Shouguang Huang]

Me too.

[tmcmor...@gmail.com]

I read this paper, and attracted by this method. if the detailed mechanism uncovered, probably, more applications will be published.

[Jiabiao Hu]

Combination of Cas9 and NgAgo would greatly expand the tools for study biology. Cas9 still has its own advantage, as it is easy to repurpose for Imaging (Cas9-GFP), transcriptional regulation (CRISPRi & CRISPRa). Does anyone want know how to express a ~24 ssDNA in mammalian cells?

[SKim]

Jiabiao,

I agree with you in that repurposing could be easier with CRIPSR. Lots of Cas9 engineering have been reported. In addition, gRNA has rooms to accommodate different functional domains.
As you pointed out, if short ssDNA can not be stably expressed in the cells, this could be limitation with NgAgo for further application.
However, people always find a way to overcome limitations. Hope to see further validation and how this field is moving with this new genome editing tool!

[pei...@gmail.com]

If this technology can be easily repeated in other labs, it will be very useful for editing genome.  I couldn't access and read the full text of the paper.  Do you know if a synthetic oligo can be used as guide-DNA?  How did the authors perform transfection, use plasmid or lentivirus? 

On Monday, May 9, 2016 at 11:02:30 AM UTC-4, SKim wrote:

[Fatwa Adikusuma]

I am wondering whther the plasmid will be available on Addgene? Would love to give this a try.

Cheers,

Fatwa

[Harrys Kishore Charles Jacob]

Hi Fatwa,

We checked with the authors and they said that it has been submitted to Addgene and should soon be available.

CJ

[Aaron Zhang]

Please let people know if one finds it available on Addgene. 

[Nam]

If this works efficiently, at least similar level to the CRISPR/Cas9 system, I am willing to change my method from the CRISPR/Cas9 to the Argo method.

Some company already stop providing TALEN service because there is no new order. The CRISPR/Cas9 might have same destiny.

[Manuel Kaulich]

Just wondering if anybody is concerned about the fact that ssDNA can easily be incorporated into the host genome, generating unpredictable off-target effects.

On Monday, May 9, 2016 at 5:02:30 PM UTC+2, SKim wrote:

[Joe Miano]

This has my full attention as they targeted multiple genes, including one (HBA2) that was inaccessible to Cas9.  We need the protein/mRNA so we can generate some mice and compare with Cas9  That there is no PAM requirement opens up the sequence landscape considerably though their testing of some 40-odd ssDNAs revealed a low efficiency (20-40 percent versus ~80% for CRISPR gRNAs). Also need to think about secondary editing after HDR integration though it sounds like this may not be an issue with the NgAgo-ssDNA since the editing is transient and once NgAgo is loaded with a ssDNA, it does not exchange.  The big question is how transient?  Will we see secondary editing?  

On Monday, May 9, 2016 at 11:02:30 AM UTC-4, SKim wrote:

[YIng Dang]

HI Joe,

Would you mind point out which 40-odd ssDNA you are talking about in the paper. I don't see they comparing NgAgo with Cas9 side by side except in figure 3c and figure 4e 4f. and supplemental figure 10. None of these showed a lower efficiency. Thanks for help.

Ying

[Joe Miano]

Hi Ying,

I was referring to Suppl FIgure 5 and the T7E1 assay.  They tested 47 ssDNA guides across multiple genes.  The efficiency of cutting was 20-40%.  This is quite good and yes, they did not compare with Cas9 in that specific case.  WHat I meant to indicate, which we all recognize, is that there looks to be superior ssDNA accessibility to the genome for DSB versus Cas9 where the latter did not appear to occur at the HBA2 locus   SOrry for the confusion.

My question, which we won't know definitively until we all try it out, is the extent of secondary editing and mosaicism in the zygote.  One thing revealed to me, not surprisingly, is need for 5' utr in NgAgo for mouse zygote injections.

I want badly to compare head to head with Cas9 in mouse zygote!

Joe

On Monday, May 9, 2016 at 11:02:30 AM UTC-4, SKim wrote:

[Yuting]

Me too! Has anybody asked the correspondence author for the availability of the Ng-Ago plasmid?

在 2016年5月9日星期一 UTC-7下午3:46:32，YIng Dang写道：

[Martin]

NgAgo plasmid is currently undergoing quality control at Addgene according to their twitter

On Monday, May 9, 2016 at 5:02:30 PM UTC+2, SKim wrote:

[Joe Miano]

Cool.  SInce my kids tell me I am too old for Twitter Ha! I have no info on progress of QC.  Can you find out about the presence of a 5'utr which will be needed for efficient translation in context of mouse zygote injections?

I hear though from my postdocs that through Social Media in CHina, the data are reproducible and so it will be just a matter of time before this new innovation spreads quickly.'

The Sr author has not responded to my email requests for protein etc.  He probably is getting 1000s of emails each day!

On Monday, May 9, 2016 at 11:02:30 AM UTC-4, SKim wrote:

[Martin]

https://www.addgene.org/78253/

On Monday, May 9, 2016 at 5:02:30 PM UTC+2, SKim wrote:

[Aaron Zhang]

Thanks for posting, Martin!

Cannot wait for people's result!

[Genome-editing]

This is very exciting and promising. Could be a game changer if it turns out to be better than CRISPR-Cas9 method.

https://dnascissors.wordpress.com/2016/06/05/genome-editing-by-a-dna-guided-enzyme/

[Joe Miano]

By now, many of you have obtained the NgAgo plasmid from addgene.  We are gearing up for some zygote injections and would like to hear from others who plan on testing this in mice as well

It is tempting to try injecting the plasmid from addgene with 5'phospho-ssDNA oligo and HDR template though we did not have any luck with this approach using the pX330 plasmid containing 

Further, the amino acid sequence is not codon optimized for mice.

So......we plan on making a new version with optimal codons for the 887 aa protein with 5' utr and 3' polyA signal 

Does anyone have other ideas etc?

Thanks

Joe

On Monday, May 9, 2016 at 11:02:30 AM UTC-4, SKim wrote:

[dirk....@monash.edu]

Hi Joe

 I also tried injecting pX330 early on with no luck.  Are you planning to make protein from the NgAgo plasmid for your zygote injections?  if so any hints would be appreciated.

Many thanks

Dirk

[AT]

Hi all,

Just curious what's known about the 5'phosphorylated short ssDNA and if this is only rare in mammalian system.

Wonder if the DNA guided editing can be adapted for use in other organisms such as plant, fungus etc...

[Joe Miano]

Dirk,

We are codon optimizing for mouse and having the entire RNA (with 5' and 3' utr plus polyA for stability) generated for injection with the ssDNA plus HDR template (same strand as the ssDNA).

Looking forward to hearing from the forum on the use of NgAgo for mouse studies.

JOe

[Aidan]

Hi all,

I am trying out the NgAgo system in hard to transfect cells in which the CRISPR-Cas9 system never produced any edits. I'm hoping for a non-coding large deletion mediated by 2 cuts. As I am relatively inexperienced in transfection I wanted to seek some advice on the amounts of ssDNA to transfect etc.

I used electroporation (Nucleofector IIB) and pMaxGFP looks very good, >75% transfected. However, I wasn't sure how much 5' P-ssDNA to transfect. Since this nucleofector kit indicated 2 microg plasmid DNA, I tried various amounts and ratios:
     2 microg total, 1:1 ratio of plasmid to ssDNA --> 1 microg NgAgo plasmid + 1 microg 5' P-ssDNA (500 ng each 24 mer)
     2 microg total, 2:1 ratio of plasmid to ssDNA --> 1.333 migrog NgAgo plasmid + 0.667 migrog 5' P-ssDNA (0.333 microg each 24 mer)
     2.6 microg total, 1:1 ratio of plasmid to ssDNA --> 1.333 migrog NgAgo plasmid + 1.333 migrog 5' P-ssDNA (0.667 microg each 24 mer)

Attached is the gel where I expected 579 bp Wt band and ~172 bp deletion band.  As you can see its all Wt.
Lane 1 - 100 bp ladder
Lane 2, 3 - PCR +ve controls
Lane 4, 5, 7 - NgAgo transfected with above DNA
Lane 6 - NgAgo transfected with just 5' P-ssDNA
Lane 8 - PCR -ve control

I will sequence the wt bands I got to see if there are small indels at each 24mer site - it could be that NgAgo won't create large deletions via 2 cut sites? Also to double check that there are no SNPs overlapping the 24mers that could affect targeting/cutting.

Any advice on how much 5' P-ssDNA to transfect would be great,

Thanks,
Aidan

[Isaac]

Hi all,

Here's a NgAgo specific group to allow topical discussion. 

https://groups.google.com/forum/#!forum/ngago 

Mine is in the mail, can't wait.

Isaac

[1872n...@gmail.com]

Hi all,

Does anyone have some exciting result using NgAgo. I have transfected the 293 cells with NgAgo and phosphorylated ssDNA targeted two genes, however, there is no Indels at all. Even I did multiple transfections of ssDNA, no positive result. 

[Aidan]

Me again,

Tried a different region again in my cell type that I had edited successfully with CRISPR-Cas9 in HEK293 (note that these are not the cells I'm trying now). The guides are essentially the same except for 2 extra bp on either side of the sgRNA sequence. Tried a number of different combinations for nucleofection but no deletions. The first lane is PCR from edited HEK293 gDNA that I had from before, the rest of the lanes are various combinations of NgAgo plasmid with two 24mer 5'P guides.

Will do a side by side comparison with the sgRNAS in HEK293 cells but from the other posts around here I'm seeing it doesn't look like it is going to work.

So what is it? did they send the wrong plasmid? is there no nls even though addgene has one annotated on their plasmid map? I've also sequenced the whole Ago coding region in the plasmid and it matched the sequence from addgene. double digest diagnostic gave the appropriate sized bands.

so what's the deal, are we missing something in the methods?? has anyone had success with NgAgo? if so please let us know....

Aidan

[1872n...@gmail.com]

I do not think it is because of the plasmid or nls signals. I think it might be very difficult to assemble the NgAgo-ssODN complex in cells after transfection.

[SKim]

Thanks Aidan for sharing your results with us.

It seems that the NgAgo system is not cooperating well for now. It would be a great idea to share successful results whoever gets there first.
I believe we are still in a validation stage. Whether it is a reliable method compatible to CRISPR or not would be answered in the near future.

[Francisco J Sanchez Rivera]

Hmmm... This is definitely not looking promising at all...

I guess for reproducibility purposes, one would need to carry out the experiments pretty much identically to the way the authors performed them in the paper. That said, a robust system should be very straightforward to validate, so the fact that it hasn't worked properly for 2/2 testers here is a bit worrisome.

I remember seeing a previous post from someone saying that the system had been used efficiently in China?

It would be great if this system works.

F

[Zhiqi Sun]

Has anybody reproduce the NgAgo-ssODN assembly experiment?

在 2016年6月27日星期一 UTC+2下午10:26:33，1872n...@gmail.com写道：

[Debojyoti Chakraborty]

Hello everyone,

we have successfully used NgAgo in HeLa cells following a protocol where we used 700ng NgAgo plasmid and 500 ng gDNA (non purified, heat inactivated PNK). We confirmed knockout of GFP from a plasmid and could see the effect even after 96h. About 40% reduction in Western blot could be seen although there is scope for more optimization.

cheers
debo

[Chikai Zhou]

Hi Debo

do you have some sequencing results? are you sure it is not RNAi?

thanks.

[Debojyoti Chakraborty]

we are currently sequencing it.. will be able to let you know once the results come

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[crisp...@gmail.com]

Dear Debo,

Very exciting news.

However, I'm a little worrying about the PNK treating. How do you exclude the effects of RNAi activated by non-phosphorylated ssODN? Do you have the controls with the ssOND not treated by PNK?

Best Regards

On Wednesday, June 29, 2016 at 7:17:02 AM UTC+2, Debojyoti Chakraborty wrote:

[Debojyoti Chakraborty]

Hi,

No way to know yet actually since we have done this once just to check if there is a reduction in gfp which we observed both by facs and western. We are currently sequencing the products to rule out Rnai if any. Will let you know if that's confirmed

Best
Debo

Dr. Debojyoti Chakraborty

Room 129, CSIR-Institute of Genomics and Integrative Biology

Mathura Road, 

New Delhi - 110025

www.rnabiologylab.com

   

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