Efficient manipulation of gene expression using Natronobacterium gregoryi Argonaute in zebrafish

Abstract

Background Natronobacterium gregoryi Argonaute (NgAgo) was found to reduce mRNA without generating detectable DNA double-strand breaks in a couple of endogenous genes in zebrafish, suggesting its potential as a tool for gene knockdown. However, little is known about how it interacts with nucleic acid molecules to interfere with gene expression. Results In this study, we first confirmed that coinjection of NgAgo and gDNA downregulated target genes, generated gene-specific phenotypes and verified some factors (including 5’ phosphorylation, GC ratio, and target positions) of gDNAs affecting gene downregulation. Therein, the sense and antisense gDNAs were equally effective, suggesting that NgAgo possibly binds to DNA. NgAgo-VP64 with gDNAs targeting promoters upregulated the target genes, further providing evidence that NgAgo interacts with genomic DNA and controls gene transcription. Finally, we explain the downregulation of NgAgo/gDNA target genes by interference with the process of gene transcription, which differs from that of morpholino oligonucleotides. Conclusions The present study provides conclusions that NgAgo may target genomic DNA and that target positions and the gDNA GC ratio influence its regulation efficiency.

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

“…According to previous research 45 , 46 , the NgAgo-gDNA complex has been reported to have the ability to knock down genes and induce transcriptional silencing. Thus, we suspect that RNA might also be recognized by our TDN-Ng system.…”

“…In addition to the four domains mentioned above, NgAgo was confirmed to have a fifth domain, that is, the RepA domain 39 . More recently, it was reported that NgAgo not only might bind with target DNA via the RepA domain 39 but also knockdown the fabp11a gene and block transcription through binding to the coding sequence 45 , 46 . Similar to other pAgo systems, a 5’-phosphorylated 24-nt single-stranded oligonucleotide as the guide DNA (gDNA) is essential to form the NgAgo-gDNA complex.…”

Characterization of Argonaute Nucleases from Mesophilic Bacteria Pseudobutyrivibrio ruminis

“…Recently, significant advancements have been made in the study of mesophilic Ago. Natronobacterium gregory Ago (NgAgo) has demonstrated the ability to use 3 RNA as a guide to modulate gene expression in mammalian cells or selectively bind to specific genomic DNA loci to disrupt gene transcription in zebrafish (Dong et al, 2023;Qi et al, 2016). Furthermore, both NgAgo and Clostridium butyricum Ago (CbAgo) have been observed to enhance homology recombination in bacteria (Fu et al, 2019;Huang, Wang, & Mayo, 2023).…”