NgAgo is a single-stranded DNA (ssDNA)-guided

Argonaute The Argonaute protein family, first discovered for its evolutionarily conserved stem cell function, plays a central role in RNA silencing processes as essential components of the RNA-induced silencing complex (RISC). RISC is responsible for the g ...

endonuclease Endonucleases are enzymes that cleave the phosphodiester bond within a polynucleotide chain. Some, such as deoxyribonuclease I, cut DNA relatively nonspecifically (without regard to sequence), while many, typically called restriction endonucleases ...

, 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 by Chunyu Han et al. to be suitable for genome editing, but this has not been replicated. In contrast to

Cas9 Cas9 (CRISPR associated protein 9, formerly called Cas5, Csn1, or Csx12) is a 160 kilodalton protein which plays a vital role in the immunological defense of certain bacteria against DNA viruses and plasmids, and is heavily utilized in genetic e ...

, 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.

Controversy

Doubts about the technique were raised on gene editing forums as early as June and have persisted. There have been several allegations that this procedure is impossible to reproduce. '' Nature Biotechnology'', which originally published the research, is investigating. 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". The same month, ''Nature Biotechnology'' published a critical correspondence article by three groups and an accompanying expression of concern by the editors on the original article. 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. 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. In April 2019, a

preprint In academic publishing, a preprint is a version of a scholarly or scientific paper that precedes formal peer review and publication in a peer-reviewed scholarly or scientific journal. The preprint may be available, often as a non-typeset versio ...

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.

References

Bacterial enzymes Discovery and invention controversies DNA EC 3.1 Genome editing {{Genetics-stub