NgAgo-gDNA Editing RNA And Its Diversity Cleavage Site Observation

Objective:The most widely used RNA-guide gene editing system is CRISPR/Cas9 which is an endonuclease.The Ago2 protein also has the same function too when under the guidance of 5' phosphorylated or hydroxylated guide DNA.However,the Ago proteins only works at high temperatures which limits its use in mammalian.Recently there are reports indicate that the NgAgo protein belongs to Ago protein family may degrade RNA at 37?.our study aimed to investigate the editing ability of NgAgo-gDNA gene editing technology in vitro or intracellular experiments and to identify its new cleavage site.Methods:1.In vitro cleavage assay: The pET-24a(+)-NgAgo prokaryotic expression plasmid was constructed by double endonuclease cleavage methods and then transformed it into E.coli strain for expression.The expression of the pET-24a(+)-NgAgo positive strain was induced by IPTG,and the NgAgo protein was purified by ammonium sulfate precipitation.The gDNA is designed to be inversely complementary to the cleaved target RNA,and the gDNA is modified by phosphorylation or hydroxylation at the 5' end.Target RNAs are synthesized by in vitro transcription: such as GFP-RNA,HCV-RNA1 and HCV-RNA2.The cleavage reaction was designed to perform an experiment in which NgAgo-gDNA cleaves the target RNA in vitro.The RNA after the completion of the cleavage reaction is collected,and then the RNA is purified.The purified RNA was reverse transcribed into cDNA and subjected to PCR.The PCR product was verified by sequencing.2.Intracellular cleavage assay: pcDNA3.1-NLS-NgAgo eukaryotic expression plasmid was constructed.The pcDNA3.1-NLS-NgAgo recombinant plasmid and the corresponding gDNA were co-transfected into MCF-7/GFP cells or 5-8F/AKR1B10 cells.Observe the changes in the corresponding target m RNA and protein.3.Statistical analysis: Statistical analysis was performed using GraphPad Prism 7.00 statistical software.The measurement data is expressed as?x±s.One-way analysis of variance(One-way ANOVA)was used for comparison between two groups.P<0.05 was considered statistically significant.Results:When NgAgo and gDNA were added simultaneously there are two cleavage bands were formed below the corresponding target RNA,in vitro experiments.As the cleavage time prolonged,the original band became weaker and the cleavage bands became more and more obvious until the original bands completely disappear.It is speculated that these two cleavage bands may be cut by NgAgo-gDNA.Sequencing results showed that the RNA was cleaved behind the gDNA.In vivo cleavage experiments showed that mRNA transfected with pcDNA3.1-NLS-NgAgo and gDNA groups was reduced and the protein expression was also reduced compared to the control group.Conclusion:1.NgAgo works on RNA rather than DNA in a 5'-terminal phosphorylation or hydroxylation-dependent manner.2.The cleavage site of the NgAgo-gDNA cleavage target RNA is located at the 5' end of the gDNA.