Natronobacterium Research Articles

Long-term monitoring of ultratrace nucleic acids using tetrahedral nanostructure-based NgAgo on wearable microneedles

Real-time and continuous monitoring of nucleic acid biomarkers with wearable devices holds potential for personal health management, especially in the context of pandemic surveillance or intensive care unit disease. However, achieving high sensitivity and long-term stability remains challenging. Here, we report a tetrahedral nanostructure-based Natronobacterium gregoryi Argonaute (NgAgo) for long-term stable monitoring of ultratrace unamplified nucleic acids (cell-free DNAs and RNAs) in vivo for sepsis on wearable device. This integrated wireless wearable consists of a flexible circuit board, a microneedle biosensor, and a stretchable epidermis patch with enrichment capability. We comprehensively investigate the recognition mechanism of nucleic acids by NgAgo/guide DNA and signal transformation within the Debye distance. In vivo experiments demonstrate the suitability for real-time monitoring of cell-free DNA and RNA with a sensitivity of 0.3 fM up to 14 days. These results provide a strategy for highly sensitive molecular recognition in vivo and for on-body detection of nucleic acid.

Prokaryotic Argonaute Protein from Natronobacterium gregoryi Requires RNAs To Activate for DNA Interference In Vivo.

ABSTRACTThe Argonaute proteins are present in all three domains of life, which are archaea, bacteria, and eukarya. Unlike the eukaryotic Argonaute proteins, which use small RNA guides to target mRNAs, some prokaryotic Argonaute proteins (pAgos) use a small DNA guide to interfere with DNA and/or RNA targets. However, the mechanisms of pAgo natural function remain unknown. Here, we investigate the mechanism by which pAgo from Natronobacterium gregoryi (NgAgo) targets plasmid and bacteriophage T7 DNA using a heterologous Escherichia coli-based model system. We show that NgAgo expressed from a plasmid linearizes its expression vector. Cotransformation assays demonstrate that NgAgo requires an RNA in trans that is transcribed from the bacteriophage T7 promoter to activate cleavage of a cotransformed plasmid, reminiscent of the trans-RNA function in CRISPR/Cas9. We propose a mechanism to explain how NgAgo eliminates invading foreign DNA and bacteriophage. By leveraging this discovery, we show that NgAgo can be programmed to target a plasmid or a chromosome locus.

Characterization of endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2 cells

As nucleic acid-guided endonucleases, some prokaryotic Argonautes have been used as programmable nucleases. Natronobacterium gregoryi Argonaute (NgAgo) has also been proposed for gene editing, but this remains very controversial. Until now, the endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2 (N. gregoryi sp2) have not been characterized. We expressed the conserved PIWI domain of NgAgo and used it to induce anti-PIWI antibody. We also cultured the N. gregoryi sp2 strain and performed immunoprecipitation, chromatin immunoprecipitation (ChIP), and RNA immunoprecipitation (RIP) assays. The nucleic acids that endogenously bound NgAgo in N. gregoryi sp2 cells were sequenced and analyzed. The results showed that NgAgo endogenously bound RNA rather than DNA. NgAgo-associated RNAs were mainly transcripts of genes that encoded tRNA, transcriptional regulators, RNA polymerases, and RNA-binding proteins. NgAgo mainly binds to the transcripts inside genes or in their upstream sequences. Interestingly, the top enriched motif of peaks was the same as that of miR-1289, suggesting that NgAgo may regulate gene expression post-transcriptionally. GO enrichment analysis showed that the peak-associated genes were enriched in transmembrane transport processes. These results revealed that NgAgo binds RNA and may function in post-transcriptional regulation in vivo.

Characterization of endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2 cells

As nucleic acid-guided endonucleases, some prokaryotic Argonautes have been used as programmable nucleases. Natronobacterium gregoryi Argonaute (NgAgo) has also been proposed for gene editing, but this remains very controversial. Until now, the endogenous nucleic acids that bind to NgAgo in Natronobacterium gregoryi sp2 (N. gregoryi sp2) have not been characterized. We expressed the conserved PIWI domain of NgAgo and used it to induce anti-PIWI antibody. We also cultured the N. gregoryi sp2 strain and performed immunoprecipitation, chromatin immunoprecipitation (ChIP), and RNA immunoprecipitation (RIP) assays. The nucleic acids that endogenously bound NgAgo in N. gregoryi sp2 cells were sequenced and analyzed. The results showed that NgAgo endogenously bound RNA rather than DNA. NgAgo-associated RNAs were mainly transcripts of genes that encoded tRNA, transcriptional regulators, RNA polymerases, and RNA-binding proteins. NgAgo mainly binds to the transcripts inside genes or in their upstream sequences. Interestingly, the top enriched motif of peaks was the same as that of miR-1289, suggesting that NgAgo may regulate gene expression post-transcriptionally. GO enrichment analysis showed that the peak-associated genes were enriched in transmembrane transport processes. These results revealed that NgAgo binds RNA and may function in post-transcriptional regulation in vivo.

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.

Inhibition of KU70 and KU80 by CRISPR interference, not NgAgo interference, increases the efficiency of homologous recombination in pig fetal fibroblasts

Non-homologous end-joining (NHEJ) is a predominant pathway for the repair of DNA double-strand breaks (DSB). It inhibits the efficiency of homologous recombination (HR) by competing for DSB targets. To improve the efficiency of HR, multiple CRISPR interference (CRISPRi) and Natronobacterium gregoryi Argonaute (NgAgo) interference (NgAgoi) systems have been designed for the knockdown of NHEJ key molecules, KU70, KU80, polynucleotide kinase/phosphatase (PNKP), DNA ligase IV (LIG4), and NHEJ1. Suppression of KU70 and KU80 by CRISPRi dramatically promoted (P<0.05) the efficiency of HR to 1.85- and 1.58-fold, respectively, whereas knockdown of PNKP, LIG4, and NHEJ1 repair factors did not significantly increase (P>0.05) HR efficiency. Interestingly, although the NgAgoi system significantly suppressed (P<0.05) KU70, KU80, PNKP, LIG4, and NHEJ1 expression, it did not improve (P>0.05) HR efficiency in primary fetal fibroblasts. Our result showed that both NgAgo and catalytically inactive Cas9 (dCas9) could interfere with the expression of target genes, but the downstream factors appear to be more active following CRISPR-mediated interference than that of NgAgo.

The prokaryotic Argonaute proteins enhance homology sequence-directed recombination in bacteria.

Argonaute proteins are present and conserved in all domains of life. Recently characterized prokaryotic Argonaute proteins (pAgos) participates in host defense by DNA interference. Here, we report that the Natronobacterium gregoryi Argonaute (NgAgo) enhances gene insertions or deletions in Pasteurella multocida and Escherichia coli at efficiencies of 80–100%. Additionally, the effects are in a homologous arms-dependent but guide DNA- and potential enzyme activity-independent manner. Interestingly, such effects were also observed in other pAgos fragments including Thermus thermophilus Argonaute (TtAgo), Aquifex aeolicus Argonaute (AaAgo) and Pyrococcus furiosus Argonaute (PfAgo). The underlying mechanism of the NgAgo system is a positive selection process mainly through its PIWI-like domain interacting with recombinase A (recA) to enhance recA-mediated DNA strand exchange. Our study reveals a novel system for enhancing homologous sequence-guided gene editing in bacteria.

When the research is not reproducible: the importance of author-initiated and institution-driven responses and investigations

ABSTRACTImportant and potentially useful findings in the sciences are under more intense public scrutiny now more than ever. Other researchers in the field dive into replicating and expanding the findings while the media swamps the community and the public with peripheral reporting and analyses. How should authors and the hosting/funding institutions respond when other workers in the field could not reproduce or replicate their published results? To illustrate the importance of author-initiated and institution-driven investigations in response to outcries of research irreproducibility, I draw on comparisons between three recent and well-publicized cases in the life sciences: betatrophin, Stimulus-Triggered Acquisition of Pluripotency (STAP) cells, and Natronobacterium gregoryi Argonaute (NgAgo). Swift, transparent responses and investigations facilitate activation of the self-correcting mechanism of science and are likely also critical in preserving the community’s resources, public trust, and the reputation of the institutions and individuals concerned. Operational guidelines for “author and institutional responses” towards external reports of irreproducibility should therefore be in place for all research intensive institutions.

HPV Oncogene Manipulation Using Nonvirally Delivered CRISPR/Cas9 or Natronobacterium gregoryi Argonaute.

CRISPR/Cas9 technology enables targeted gene editing; yet, the efficiency and specificity remain unsatisfactory, particularly for the nonvirally delivered, plasmid‐based CRISPR/Cas9 system. To tackle this, a self‐assembled micelle is developed and evaluated for human papillomavirus (HPV) E7 oncogene disruption. The optimized micelle enables effective delivery of Cas9 plasmid with a transient transgene expression profile, benefiting the specificity of Cas9 recognition. Furthermore, the feasibility of using the micelle is explored for another nucleic acid‐guided nuclease system, Natronobacterium gregoryi Argonaute (NgAgo). Both systems are tested in vitro and in vivo to evaluate their therapeutic potential. Cas9‐mediated E7 knockout leads to significant inhibition of HPV‐induced cancerous activity both in vitro and in vivo, while NgAgo does not show significant E7 inhibition on the xenograft mouse model. Collectively, this micelle represents an efficient delivery system for nonviral gene editing, adding to the armamentarium of gene editing tools to advance safe and effective precision medicine‐based therapeutics.

Unexpected binding behaviors of bacterial Argonautes in human cells cast doubts on their use as targetable gene regulators.

Prokaryotic Argonaute proteins (pAgos) have been proposed as an alternative to the CRISPR/Cas9 platform for gene editing. Although Argonaute from Natronobacterium gregoryi (NgAgo) was recently shown unable to cleave genomic DNA in mammalian cells, the utility of NgAgo or other pAgos as a targetable DNA-binding platform for epigenetic editing has not been explored. In this report, we evaluated the utility of two prokaryotic Argonautes (NgAgo and TtAgo) as DNA-guided DNA-binding proteins. NgAgo showed no meaningful binding to chromosomal targets, while TtAgo displayed seemingly non-specific binding to chromosomal DNA even in the absence of guide DNA. The observed lack of DNA-guided targeting and unexpected guide-independent genome sampling under the conditions in this study provide evidence that these pAgos might be suitable for neither gene nor epigenome editing in mammalian cells.

Structural Evolution of a Retinal Chromophore in the Photocycle of Halorhodopsin from Natronobacterium pharaonis.

We revealed the chloride ion pumping mechanism in halorhodopsin from Natronobacterium pharaonis ( pHR) by exploring sequential structural changes in the retinal chromophore during its photocycle using time-resolved resonance Raman (RR) spectroscopy on the nanosecond to millisecond time scales. A series of RR spectra of the retinal chromophore in the unphotolyzed state and of the three intermediates of pHR were obtained. Using singular value decomposition analysis of the C═C and C-C stretch bands in the time-resolved RR spectra, we identified the spectra of the K, L, and N intermediates. We focused on structural markers of the RR bands to explore the structure of the retinal chromophore. In the unphotolyzed state, the retinal chromophore is in the planar all- trans, 15- anti geometry. The bound ion affects the polyene chain but does not interact with the protonated Schiff base. In the observed intermediates, the chromophore is in the 13- cis configuration. The chromophore in the K intermediate is distorted due to the photoisomerization of retinal. The hydrogen bond is weak in the unphotolyzed state and in the K intermediate, resulting in exchange of the hydrogen-bond acceptor to a water molecule in the K-to-L transition, relaxation of the polyene chain distortion, and generation of an alternative distortion near the Schiff base. The bound halide ion interacts with the protonated Schiff base through the water molecule bound to the protonated Schiff base. In the L-to-N transition, the hydrogen acceptor of the protonated Schiff base switches from the water molecule to another species, although the strong hydrogen bond of the protonated Schiff base remains. This paper reports the first observation of sequential changes in the RR spectra in the pHR photocycle, provides information on the structural evolution of the retinal chromophore, and proposes a model for chloride ion translocation in pHR.

Zebrafish Embryonic Slow Muscle Is a Rapid System for Genetic Analysis of Sarcomere Organization by CRISPR/Cas9, but Not NgAgo.

Zebrafish embryonic slow muscle cells, with their superficial localization and clear sarcomere organization, provide a useful model system for genetic analysis of muscle cell differentiation and sarcomere assembly. To develop a quick assay for testing CRISPR-mediated gene editing in slow muscles of zebrafish embryos, we targeted a red fluorescence protein (RFP) reporter gene specifically expressed in slow muscles of myomesin-3-RFP (Myom3-RFP) zebrafish embryos. We demonstrated that microinjection of RFP-sgRNA with Cas9 protein or Cas9 mRNA resulted in a mosaic pattern in loss of RFP expression in slow muscle fibers of the injected zebrafish embryos. To uncover gene functions in sarcomere organization, we targeted two endogenous genes, slow myosin heavy chain-1 (smyhc1) and heat shock protein 90 α1 (hsp90α1), which are specifically expressed in zebrafish muscle cells. We demonstrated that injection of Cas9 protein or mRNA with respective sgRNAs targeted to smyhc1 or hsp90a1 resulted in a mosaic pattern of myosin thick filament disruption in slow myofibers of the injected zebrafish embryos. Moreover, Myom3-RFP expression and M-line localization were also abolished in these defective myofibers. Given that zebrafish embryonic slow muscles are a rapid in vivo system for testing genome editing and uncovering gene functions in muscle cell differentiation, we investigated whether microinjection of Natronobacterium gregoryi Argonaute (NgAgo) system could induce genetic mutations and muscle defects in zebrafish embryos. Single-strand guide DNAs targeted to RFP, Smyhc1, or Hsp90α1 were injected with NgAgo mRNA into Myom3-RFP zebrafish embryos. Myom3-RFP expression was analyzed in the injected embryos. The results showed that, in contrast to the CRISPR/Cas9 system, injection of the NgAgo-gDNA system did not affect Myom3-RFP expression and sarcomere organization in myofibers of the injected embryos. Sequence analysis failed to detect genetic mutations at the target genes. Together, our studies demonstrate that zebrafish embryonic slow muscle is a rapid model for testing gene editing technologies in vivo and uncovering gene functions in muscle cell differentiation.

NgAgo-gDNA system efficiently suppresses hepatitis B virus replication through accelerating decay of pregenomic RNA

Covalently closed circular DNA (cccDNA) in the hepatocytes nucleus is responsible for persistent infection of Hepatitis B virus (HBV). Current antiviral therapy drugs nucleos(t)ide analogs or interferon fail to eradicate HBV cccDNA. Genome editing technique provides an effective approach for HBV treatment through targeting viral cccDNA. Natronobacterium gregoryi Argonaute (NgAgo)-guide DNA (gDNA) system with powerful genome editing prompts us to explore its application in inhibiting HBV replication. Preliminary function verification indicated that NgAgo/EGFP-gDNA obviously inhibited EGFP expression. To further explore the potential role of NgAgo in restricting HBV replication, 10 of gDNAs targeting the critical region of viral genome were designed, only S-142, P-263 and P-2166 gDNAs led to significant inhibition on HBsAg, HBeAg and pregenomic RNA (pgRNA) level in Huh7 and HepG2 cells transfected with pcDNA-HBV1.1 plasmid. Similar results were also found in HBV infected HLCZ01 cells and Huh7-NTCP cells. However, we failed to detect any DNA editing in S-142, P-263 and P-2166 targeting region through T7E1 assay and Sanger sequencing. Remarkably, we found that NgAgo/P-2166 significantly accelerated the decay of viral pgRNA. Taken together, our results firstly demonstrate the potential of NgAgo/gDNA in inhibiting HBV replication through accelerating pgRNA degradation, but not DNA editing.

Abstract 5367: Ngago: no evidence of targeted endonuclease activity

Abstract The development of a versatile and easy to use genome editing tool based on the bacterial CRISPR/Cas9 system has led to a vast increase in applications of genome editing in laboratories around the world. As an extremely powerful and versatile tool it has facilitated important discoveries in both basic and translational research as well as in a potentially wide range of therapeutic applications. Natronobacterium gregoryi Argonaute (NgAgo) protein was described as a favorable alternative to Cas9 due to its simplicity and higher endonuclease specificity. Thus far, there are no formal reports documenting the success of NgAgo in other laboratory settings and cellular systems. Utilizing the identical cellular background and protocol that was made publicly available by the authors of the initial study, we were unable to reproduce the endonuclease function of NgAgo. These results do not support the role of NgAgo in gene editing experiments. Note: This abstract was not presented at the meeting. Citation Format: Sabine Topka, Sara Kazzaz, Kenneth Offit, Vijai Joseph. Ngago: no evidence of targeted endonuclease activity [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 5367. doi:10.1158/1538-7445.AM2017-5367

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.

No evidence of genome editing activity from Natronobacterium gregoryi Argonaute (NgAgo) in human cells.

The argonaute protein from the thermophilic bacterium Thermus thermophilus shows DNA-guided DNA interfering activity at high temperatures, complicating its application in mammalian cells. A recent work reported that the argonaute protein from Natronobacterium gregoryi (NgAgo) had DNA-guided genome editing activity in mammalian cells. We compared the genome editing activities of NgAgo and Staphylococcus aureus Cas9 (SaCas9) in human HEK293T cells side by side. EGFP reporter assays and DNA sequencing consistently revealed high genome editing activity from SaCas9. However, these assays did not demonstrate genome editing activity by NgAgo. We confirmed that the conditions allowed simultaneous transfection of the NgAgo expressing plasmid DNA and DNA guides, as well as heterologous expression of NgAgo in the HEK293T cells. Our data show that NgAgo is not a robust genome editing tool, although it may have such activity under other conditions.

SsDNA and the Argonautes: The Quest for the Next Golden Editor.

Genome engineering has gone mainstream because of breakthroughs in defining and harnessing naturally occurring, customizable DNA recognition cursors (protein or RNA-guided). At present, most gene editing relies on these cursors to direct custom DNA endonucleases to a specific genomic sequence to induce a double-strand break. New tools for genome engineering are continuously being explored, and another advance in DNA targeting has recently been described. Argonaute isolated from Natronobacterium gregoryi (NgAgo) is an ssDNA-based cursor that thus far has no known limitations in sequence recognition, shows promise for high specificity, and for many applications may represent a potentially more accessible genome-editing system over prior tools as it requires only a single, 24-base, 5' phosphorylated ssDNA for DNA targeting. Genome engineering is in a remarkable moment of unprecedented growth with exponential reduction in costs reminiscent of Moore's law in electronics. Many questions remain with regard to Argonaute utility in specific systems, but there is no doubt that genome engineering is expanding into new and exciting areas from synthetic biology to gene therapy.

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.

Biosorption and Bio-Kinetic Properties of Solar Saltern Halobacterial Strains for Managing Zn2+, As2+ and Cd2+ Metals

Halobacteria are extreme halophilic archaeobacteria belonging to the family Halobacteriaceae. Geomicrobiologists are concerned about saline tolerant organisms with metal toxicity and bioremediation. Soil samples of Cape comorin coast were analyzed and the zinc, arsenic and cadmium biosorbent halobacterial strains were isolated. Among the fifty-six halobacterial strains isolated, Halobacterium saccharovorum, Hb. salinarium and Natronobacterium gregoryi showed maximum resistance up to 0.01, 0.001 and 0.001 mM against zinc, arsenic and cadmium, respectively. Biokinetic feature such as maximum specific growth rate (μmax. d−1), the rate of trace metal removal [s], and half velocity constant [Ks] were calculated. The SEM-EDS pictures show the metal uptake of the halobacterial isolates. It assumes that Hb. saccharovorum, Hb. salinarium and Natronobacterium gregoryi isolated from the salt pan of Rajakkamangalam coast Tamilnadu in India can be used as metals bioremident.

Thermal and Spectroscopic Characterization of a Proton Pumping Rhodopsin from an Extreme Thermophile

So far retinylidene proteins (∼rhodopsin) have not been discovered in thermophilic organisms. In this study we investigated and characterized a microbial rhodopsin derived from the extreme thermophilic bacterium Thermus thermophilus, which lives in a hot spring at around 75 °C. The gene for the retinylidene protein, named thermophilic rhodopsin (TR), was chemically synthesized with codon optimization. The codon optimized TR protein was functionally expressed in the cell membranes of Escherichia coli cells and showed active proton transport upon photoillumination. Spectroscopic measurements revealed that the purified TR bound only all-trans-retinal as a chromophore and showed an absorption maximum at 530 nm. In addition, TR exhibited both photocycle kinetics and pH-dependent absorption changes, which are characteristic of rhodopsins. Of note, time-dependent thermal denaturation experiments revealed that TR maintained its absorption even at 75 °C, and the denaturation rate constant of TR was much lower than those of other proton pumping rhodopsins such as archaerhodopsin-3 (200 ×), Haloquadratum walsbyi bacteriorhodopsin (by 10-times), and Gloeobacter rhodopsin (100 ×). Thus, these results suggest that microbial rhodopsins are also distributed among thermophilic organisms and have high stability. TR should allow the investigation of the molecular mechanisms of ion transport and protein folding.