NgAgo

NgAgo is a single-stranded DNA (ssDNA)-guided Argonaute endonuclease, an acronym for Natronobacterium gregoryi Argonaute. NgAgo binds 5′ phosphorylated ssDNA of ~24 nucleotides (gDNA) to guide it to its target site and will make DNA double-strand breaks at the gDNA site. Like the CRISPR/Cas system, NgAgo was reported to be suitable for genome editing,[1] but this has not been replicated. In contrast to Cas9, the NgAgo–gDNA system does not require a protospacer adjacent motif (PAM).

Role

NgAgo was proposed to be useful for genome editing in May 2016 because of the system’s high accuracy and efficiency, which was said to minimize off-target effects. The specificity of the gDNA is essential, as cleavage efficiency is impaired by a single nucleotide mismatch between the guide and target molecules. Using 5’ phosphorylated ssDNAs as guide molecules reduces the possibility of cellular oligonucleotides misleading NgAgo. A guide molecule can only be attached to NgAgo during the expression of the protein. Once the guide is loaded, NgAgo cannot swap free floating ssDNA for its gDNA. Designing, synthesizing, and adjusting the concentration of ssDNAs is easier compared to systems using sgRNA. The required dosage of ssDNA is less than that of a sgRNA expression plasmid.[1]

Controversy

Doubts about the technique were raised on gene editing forums as early as June and have persisted.[2] There have been several allegations that this procedure is impossible to reproduce. Nature Biotechnology, which originally published the research, is investigating.[3][4] In November 2016, a letter was published in Protein & Cell questioning the research and the lead author's claim that replication requires "superb experimental skill".[5] The same month, Nature Biotechnology published a critical correspondence article by three groups[6] and an accompanying expression of concern by the editors on the original article.[7] The authors retracted the study in a statement published in Nature Biotechnology on 3 August 2017, citing the continued inability of the research community to replicate their results.[8] In 2018, an investigation led by Han's university concluded that while Han's findings were flawed, he and his team did not intend to deceive the scientific community.[9] In April 2019, a preprint article found that NgAgo does have the ability to edit genes and implied that previous results might have been difficult to reproduce due to difficulties related to purification of the active protein.[10][11]

gollark: What, really? Weird. What is this *based* on?
gollark: t!top
gollark: curse the accursed rate limit
gollark: t!top
gollark: t!top global

References
  1. Gao, Feng; Shen, Xiao Z; Jiang, Feng; Wu, Yongqiang; Han, Chunyu (2016). "DNA-guided genome editing using the Natronobacterium gregoryi Argonaute". Nature Biotechnology. 34 (7): 768–773. doi:10.1038/nbt.3547. PMID 27136078.
  2. Blow, Nathan (October 4, 2016). "To Edit or Not:The NgAgo Story". BioTechniques. Retrieved November 29, 2016.
  3. Cyranoski, David (2016). "Replications, ridicule and a recluse: the controversy over NgAgo gene-editing intensifies". Nature. 536 (7615): 136–137. doi:10.1038/536136a. PMID 27510204. Retrieved August 17, 2016.
  4. Cyranoski, David (2016). "NgAgo gene-editing controversy escalates in peer-reviewed papers". Nature. 540 (7631): 20–21. doi:10.1038/nature.2016.21023. PMID 27905463. Retrieved November 25, 2016.
  5. Burgess, Shawn; Cheng, Linzhao; Gu, Feng; Huang, Junjiu; Huang, Zhiwei; Lin, Shuo; Li, Jinsong; Li, Wei; Qin, Wei; Sun, Yujie; Songyang, Zhou; Wei, Wensheng; Wu, Qiang; Wang, Haoyi; Wang, Xiaoqun; Xiong, Jing-Wei; Xi, Jianzhong; Yang, Hui; Zhou, Bin; Zhang, Bo (November 15, 2016). "Questions about NgAgo". Protein & Cell. 7 (12): 913–915. doi:10.1007/s13238-016-0343-9. PMC 5205665. PMID 27848216.
  6. Lee, Seung Hwan; Turchiano, Giandomenico; Ata, Hirotaka; Nowsheen, Somaira; Romito, Marianna; Lou, Zhenkun; Ryu, Seuk-Min; Ekker, Stephen C; Cathomen, Toni; Kim, Jin-Soo (November 28, 2016). "Failure to detect DNA-guided genome editing using Natronobacterium gregoryi Argonaute". Nature Biotechnology. 35 (1): 17–18. doi:10.1038/nbt.3753. PMC 5662444. PMID 27893702.
  7. Gao, Feng; Shen, Xiao Z; Jiang, Feng; Wu, Yongqiang; Han, Chunyu (November 28, 2016). "Corrected online 28 November 2016". Nature Biotechnology. 34 (7): 768–773. doi:10.1038/nbt.3547. PMID 27136078.
  8. Cyranoski, David (2017). "Authors retract controversial Ng Ago gene-editing study". Nature. doi:10.1038/nature.2017.22412.
  9. Cyranoski, David (2018). "University clears NgAgo gene-editing study authors of deception". Nature. doi:10.1038/d41586-018-06163-0.
  10. "New protein for gene editing may improve disease treatment, sustainable manufacturing". April 3, 2019.
  11. Solomon, Kevin (2019). "NgAgo-enhanced homologous recombination in E. coli is mediated by DNA endonuclease activity". bioRxiv. doi:10.1101/597237.
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