NgAgo possesses guided DNA nicking activity.

Area of Science:

• Molecular Biology
• Biochemistry
• Genetics

Background:

• Prokaryotic Argonautes (pAgos) offer potential gene-editing flexibility compared to CRISPR/Cas systems.
• Natronobacterium gregoryi Argonaute (NgAgo) has shown debated efficacy in eukaryotic systems.
• This study focuses on characterizing NgAgo function within prokaryotic systems.

Purpose of the Study:

• To characterize the prokaryotic function of Natronobacterium gregoryi Argonaute (NgAgo).
• To investigate the DNA cleavage activity and gene-editing capabilities of NgAgo in prokaryotes.
• To reconcile conflicting previous reports on NgAgo's functionality.

Main Methods:

• Expression and refolding of NgAgo in non-halophilic hosts.
• Biochemical assays to determine DNA nicking endonuclease activity.
• In vitro and in vivo (Escherichia coli) gene-editing experiments using guide DNA.
• Analysis of NgAgo domains, including PIWI and the novel repA domain.

Main Results:

• NgAgo exhibits poor expression and solubility in non-halophilic hosts, but the soluble fraction is a nicking DNA endonuclease.
• NgAgo possesses both canonical PIWI and a novel repA single-stranded DNA binding domain, both contributing to DNA cleavage.
• NgAgo can be programmed to nick targeted DNA in E. coli and in vitro, 1 nt from the 3' end of the guide sequence.
• NgAgo-guided homologous recombination, a gene-editing process, was enhanced by its endonuclease activities.

Conclusions:

• The soluble fraction of NgAgo acts as a nicking DNA endonuclease in prokaryotes.
• NgAgo's unique domain structure contributes to its DNA cleavage activity.
• NgAgo demonstrates potential for gene-editing applications in prokaryotes, clarifying previous findings.