Abstract
Prokaryotic Argonautes (pAgos) from mesophilic bacteria are attracting increasing attention for their genome editing potential. So far, it has been reported that KmAgo from Kurthia massiliensis can utilize DNA and RNA guide of any sequence to effectively cleave DNA and RNA targets. Here we find that three active pAgos, which have about 50% sequence identity with KmAgo, possess typical DNA-guided DNA target cleavage ability. Among them, RsuAgo from Rummeliibacillus suwonensis is mainly explored for which can cleave both DNA and RNA targets. Interestingly, RsuAgo-mediated RNA target cleavage occurs only with short guide DNAs in a narrow length range (16–20 nt), and mismatches between the guide and target sequence greatly affect the efficiency of RNA target cleavage. RsuAgo-mediated target cleavage shows a preference for a guide strand with a 5′-terminal A residue. Furthermore, we have found that RsuAgo can cleave double-stranded DNA in a low-salt buffer at 37 °C. These properties of RsuAgo provide a new tool for DNA and RNA manipulation at moderate temperatures.
Highlights
Eukaryotic Argonautes are key players in all small-RNA-guided gene-silencing processes, which have the function of loading small RNA, and play an important role in transcription, alternative splicing and even DNA repair [1]
To test whether the four prokaryotic Agos (pAgos) act uniformly on DNA and RNA targets mediated by guides in vitro, we used a set of synthetic guides and targets to analyze cleavage activity. gDNA or gRNA was loaded onto pAgos, and Ago-guide complex was incubated with DNA target or RNA target
In vitro cleavage assays showed that P-gDNA or P-gRNA containing a 50 -C, 50 -T, or 50 -G nucleotide resulted in substantial tDNA cleavage activity (Figure 3B,D)
Summary
Eukaryotic Argonautes (eAgos) are key players in all small-RNA-guided gene-silencing processes, which have the function of loading small RNA, and play an important role in transcription, alternative splicing and even DNA repair [1]. Cleavage at 37 ◦ C, including CbAgo, LrAgo, SeAgo, CpAgo, IbAgo and KmAgo [14,15,16,17,18] These pAgos exhibit low levels of nuclease activity on dsDNA, and preferentially cleave negatively supercoiled plasmids and/or DNA fragments with low GCcontent [14,19]. Considering that pAgos have been implicated in the cell’s defense against mobile genetic elements [9,21] and pAgos can cleave the singlestranded nucleic acid targets and dsDNA in vitro, they have the powerful potential for genome manipulation. Considering the potential application of mesophilic pAgos in genome editing and RNA manipulation, we proceed here to identify mesophilic pAgos with desired biochemical cleavage function and high specificity. DNA and RNA nuclease activity of RsuAgo broadens our understanding of pAgos and can promote the development of pAgos-based applications at moderate temperatures
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