NgAgo possesses guided DNA nicking activity.

Prokaryotic Argonautes (pAgos) have been proposed as more flexible tools for gene-editing as they do not require sequence motifs adjacent to their targets for function, unlike popular CRISPR/Cas systems. One promising pAgo candidate, from the halophilic archaeon Natronobacterium gregoryi (NgAgo), has been the subject of debate regarding its potential in eukaryotic systems. Here, we revisit this enzyme and characterize its function in prokaryotes. NgAgo expresses poorly in non-halophilic hosts with most of the protein being insoluble and inactive even after refolding. However, we report that the soluble fraction does indeed act as a nicking DNA endonuclease. NgAgo shares canonical domains with other catalytically active pAgos but also contains a previously unrecognized single-stranded DNA binding domain (repA). Both repA and the canonical PIWI domains participate in DNA cleavage activities of NgAgo. NgAgo can be programmed with guides to nick targeted DNA in Escherichia coli and in vitro 1 nt outside the 3' end of the guide sequence. We also found that these endonuclease activities are essential for enhanced NgAgo-guided homologous recombination, or gene-editing, in E. coli. Collectively, our results demonstrate the potential of NgAgo for gene-editing and provide new insight into seemingly contradictory reports.

The therapeutic landscape of HIV-1 via genome editing.

Current treatment for HIV-1 largely relies on chemotherapy through the administration of antiretroviral drugs. While the search for anti-HIV-1 vaccine remain elusive, the use of highly active antiretroviral therapies (HAART) have been far-reaching and has changed HIV-1 into a manageable chronic infection. There is compelling evidence, including several side-effects of ARTs, suggesting that eradication of HIV-1 cannot depend solely on antiretrovirals. Gene therapy, an expanding treatment strategy, using RNA interference (RNAi) and programmable nucleases such as meganuclease, zinc finger nuclease (ZFN), transcription activator-like effector nuclease (TALEN), and clustered regularly interspaced short palindromic repeats/CRISPR-associated proteins (CRISPR-Cas9) are transforming the therapeutic landscape of HIV-1. TALENS and ZFNS are structurally similar modular systems, which consist of a FokI endonuclease fused to custom-designed effector proteins but have been largely limited, particularly ZFNs, due to their complexity and cost of protein engineering. However, the newly developed CRISPR-Cas9 system, consists of a single guide RNA (sgRNA), which directs a Cas9 endonuclease to complementary target sites, and serves as a superior alternative to the previous protein-based systems. The techniques have been successfully applied to the development of better HIV-1 models, generation of protective mutations in endogenous/host cells, disruption of HIV-1 genomes and even reactivating latent viruses for better detection and clearance by host immune response. Here, we focus on gene editing-based HIV-1 treatment and research in addition to providing  perspectives for refining these techniques.

No evidence for genome editing in mouse zygotes and HEK293T human cell line using the DNA-guided Natronobacterium gregoryi Argonaute (NgAgo).

A recently published research article reported that the extreme halophile archaebacterium Natronobacterium gregoryi Argonaute enzyme (NgAgo) could cleave the cellular DNA under physiological temperature conditions in cell line and be implemented as an alternative to CRISPR/Cas9 genome editing technology. We assessed this claim in mouse zygotes for four loci (Sptb, Tet-1, Tet-2 and Tet-3) and in the human HEK293T cell line for the EMX1 locus. Over 100 zygotes were microinjected with nls-NgAgo-GK plasmid provided from Addgene and various concentrations of 5'-phosphorylated guide DNA (gDNA) from 2.5 ng/µl to 50 ng/µl and cultured to blastocyst stage of development. The presence of indels was verified using T7 endonuclease 1 assay (T7E1) and Sanger sequencing. We reported no evidence of successful editing of the mouse genome. We then assessed the lack of editing efficiency in HEK293T cell line for the EMX1 endogenous locus by monitoring the NgAgo protein expression level and the editing efficiency by T7E1 assay and Sanger sequencing. We reported that the NgAgo protein was expressed from 8 hours to a maximum expression at 48 hours post-transfection, confirming the efficient delivery of the plasmid and the gDNA but no evidence of successful editing of EMX1 target in all transfected samples. Together our findings indicate that we failed to edit using NgAgo.

TO EDIT OR NOT: THE NGAGO STORY.

New genome-editing approaches always receive widespread attention. But in the case of a novel Argonaute-based technique published last spring, attention has been particularly intense.

DNA-guided genome editing using the Natronobacterium gregoryi Argonaute.

The RNA-guided endonuclease Cas9 has made genome editing a widely accessible technique. Similar to Cas9, endonucleases from the Argonaute protein family also use oligonucleotides as guides to degrade invasive genomes. Here we report that the Natronobacterium gregoryi Argonaute (NgAgo) is a DNA-guided endonuclease suitable for genome editing in human cells. NgAgo binds 5' phosphorylated single-stranded guide DNA (gDNA) of ∼24 nucleotides, efficiently creates site-specific DNA double-strand breaks when loaded with the gDNA. The NgAgo-gDNA system does not require a protospacer-adjacent motif (PAM), as does Cas9, and preliminary characterization suggests a low tolerance to guide-target mismatches and high efficiency in editing (G+C)-rich genomic targets.

A physiologically based kinetic model for bacterial sulfide oxidation.

In the biotechnological process for hydrogen sulfide removal from gas streams, a variety of oxidation products can be formed. Under natron-alkaline conditions, sulfide is oxidized by haloalkaliphilic sulfide oxidizing bacteria via flavocytochrome c oxidoreductase. From previous studies, it was concluded that the oxidation-reduction state of cytochrome c is a direct measure for the bacterial end-product formation. Given this physiological feature, incorporation of the oxidation state of cytochrome c in a mathematical model for the bacterial oxidation kinetics will yield a physiologically based model structure. This paper presents a physiologically based model, describing the dynamic formation of the various end-products in the biodesulfurization process. It consists of three elements: 1) Michaelis-Menten kinetics combined with 2) a cytochrome c driven mechanism describing 3) the rate determining enzymes of the respiratory system of haloalkaliphilic sulfide oxidizing bacteria. The proposed model is successfully validated against independent data obtained from biological respiration tests and bench scale gas-lift reactor experiments. The results demonstrate that the model is a powerful tool to describe product formation for haloalkaliphilic biomass under dynamic conditions. The model predicts a maximum S° formation of about 98 mol%. A future challenge is the optimization of this bioprocess by improving the dissolved oxygen control strategy and reactor design.

Isolation of a chymotrypsinogen B-like enzyme from the archaeon Natronomonas pharaonis and other halobacteria.

A protease of a molecular mass of approximately 30kDa was isolated and purified from the haloalkaliphilic archaeon Natronomonas (formerly Natronobacterium) pharaonis. The enzyme hydrolyzed synthetic peptides, preferentially at the carboxyl terminus of phenylalanine or leucine, as well as large proteins. Hydrolysis occurred over the range of pH from 6 to 12, with an optimum at pH 10. The temperature optimum was 61 degrees C. The enzyme was nearly equally active over the range of salt concentration from 0.5 to 4M (NaCl or KCl). A strong cross-reaction with a polyclonal antiserum against human chymotrypsin was observed. Enzymatic activity was inhibited by typical serine protease inhibitors. There was significant homology between N-terminal and internal sequences from autolytic fragments and the sequence of bovine chymotrypsinogen B; the overall amino acid composition was similar to that of vertebrate chymotrypsinogens. Evidence for a zymogen-like processing of the protease was obtained. Cell extracts from other halobacteria exhibited similar proteolytic activity and immunoreactivity. The data suggested a widespread distribution of a chymotrypsinogen B-like protease among halo- and haloalkaliphilic Archaea.

Nucleoside diphosphate kinase from haloalkaliphilic archaeon Natronobacterium magadii: purification and characterization.

An ATP-binding protein from the haloalkaliphilic archaeon Natronobacterium magadii was purified and characterized by affinity chromatography on ATP-agarose and by fast protein liquid chromatography (FPLC) on a Mono Q column. The N-terminal 20 amino acid sequence of the kinase showed a strong sequence similarity of this protein with nucleoside diphosphate (NDP) kinases from different organisms and, accordingly, we believe that this protein is a nucleoside diphosphate kinase, an enzyme whose main function is to exchange gamma-phosphates between nucleoside triphosphates and diphosphates. Comparison of the molecular weights of the NDP kinase monomer determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) (23,000) and of the oligomer determined by sedimentation equilibrium experiments (125,000) indicated that the oligomer is a hexamer. The enzyme was autophosphorylated in the presence of [gamma-32P]ATP, and Mg2+ was required for the incorporation of phosphate. The kinase preserved the ability to transfer gamma-phosphate from ATP to GDP in the range of NaCl concentration from 90 mM to 3.5 M and in the range of pH from 5 to 12. It was found and confirmed by Western blotting that this kinase is one of the proteins that bind specifically to natronobacterial flagellins. NDP kinase from haloalkaliphiles appeared to be simple to purify and to be a suitable enzyme for studies of structure and stability compared with NDP kinases from mesophilic organisms.