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New Genome Editing Tool Elicits Interest, Concern About Reproducibility


This story has been updated to reflect recent comments made by a scientist who has attempted gene editing with NgAgo.

NEW YORK (GenomeWeb) – A newly proposed genome-editing platform based on a bacterial Argonaute protein is drawing significant interest from researchers, even as some struggle to get it to work.

In May, scientists from China's Hebei University of Science and Technology, led by Chunyu Han, published a study in Nature Biotechnology suggesting that the argonaute protein from Natronobacterium gregoryi (NgAgo) could be deployed as a programmable, DNA-guided, DNA-targeting genome editing tool.

According to the plasmid repository Addgene, which began shipping plasmids containing the NgAgo system deposited by Han's lab in June, over 300 labs in more than 30 countries have requested NgAgo since it became available. "I would say that's a lot, for one plasmid," Addgene Executive Director Joanne Kamens told GenomeWeb. "That's pretty impressive."

While there has been significant interest, many scientists are saying they can't reproduce the results presented in the Nature Biotechnology paper. While some scientists have reported success, in both English- and Chinese-language Internet forums, other scientists have aired their inability to detect indel mutations in gene-editing experiments using NgAgo.

Argonaute proteins, essential to RNA interference in higher-order eukaryotes, have been known to use short single-stranded nucleic acids to target other nucleic acids. Often, Argonaute proteins bind single-stranded RNAs to target other RNAs; however, Han's group described how NgAgo binds short DNA strands to cleave DNAs. Guides for NgAgo were only 24 nucleotides long and did not require a protospacer-adjacent motif — a sort of target recognition sequence, key areas of difference with CRISPR/Cas9.

The scientists said they were able to create double-stranded DNA breaks in vivo in several cell lines transfected with a plasmid, reporting genome editing efficiencies of between 21 and 41 percent.

But on a Google Group titled "Genome Engineering using CRISPSR/Cas Systems," several threads have sprung up where users, some anonymous, have reported failure to create indels using NgAgo. It's a place where scientists go to look for advice when their genome editing experiments fail or underperform. Many of the discussions are about CRISPR/Cas9, and it's routine to find questions asking for help troubleshooting or optimizing experiments. 

Regarding NgAgo, some have simply stated their failure, some have posted protocols looking for help in where they may have gone wrong, and some have questioned whether reductions in the targeted protein (usually GFP) are due to genome editing or to repressed transcription.

As reported by University of California, Davis stem cell scientist Paul Knoepfler on his blog, The Niche, some scientists are beginning to question whether NgAgo works as advertised.

On the Google Group, it's no different.

"I will echo others here that there is a discrepancy between what the authors have shown and what others have been able to repeat," a commenter who said he attempted to use NgAgo posted on July 15.

While some have struggled to replicate Han's results, others have reported success. On Twitter, Australian National University researcher Gaetan Burgio reported successful editing with NgAgo in mice. On the Google Group, Debojyoti Chakrabory, a scientist at India's Institute of Genomics and Integrative Biology, said a plasmid supplied by a Chinese biotech company yielded successful gene editing in HeLa cells.

Burgio's initial optimisim, based on a PCR analysis, has waned, however. Last week, he tweeted to Knoepfler that analysis by Sanger sequencing was "very messy." He added in a follow-up tweet, "I think there are tricks we are not aware of."

If nothing else, NgAgo has raised a lot of questions in the field, some routine and others contentious.

Even in the ultra-competitive, fast-moving world of programmable genome editing, two months is too early to make a call on whether it works as touted or not. "I think you'll see things in three to four months," Addgene's Kamens said.

But the early questions about reproducibility are a challenge to the notion that these gene editing systems are simple to work with.

"There are a lot of devils in the details," a researcher at a US laboratory familiar with CRISPR, and who requested not to be named, told GenomeWeb. "A lot of technologies aren't as robust as one might hope."