Characterization of a Programmable Argonaute Nuclease from the Mesophilic Bacterium <i>Rummeliibacillus suwonensis</i>

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