A programmable pAgo nuclease with universal guide and target specificity from the mesophilic bacterium Kurthia massiliensis

Abstract

Argonaute proteins are programmable nucleases that are found in both eukaryotes and prokaryotes and provide defense against invading genetic elements. Although some prokaryotic argonautes (pAgos) were shown to recognize RNA targets in vitro, the majority of studied pAgos have strict specificity toward DNA, which limits their practical use in RNA-centric applications. Here, we describe a unique pAgo nuclease, KmAgo, from the mesophilic bacterium Kurthia massiliensis that can be programmed with either DNA or RNA guides and can precisely cleave both DNA and RNA targets. KmAgo binds 16–20 nt long 5′-phosphorylated guide molecules with no strict specificity for their sequence and is active in a wide range of temperatures. In bacterial cells, KmAgo is loaded with small DNAs with no obvious sequence preferences suggesting that it can uniformly target genomic sequences. Mismatches between the guide and target sequences greatly affect the efficiency and precision of target cleavage, depending on the mismatch position and the nature of the reacting nucleic acids. Target RNA cleavage by KmAgo depends on the formation of secondary structure indicating that KmAgo can be used for structural probing of RNA. These properties of KmAgo open the way for its use for highly specific nucleic acid detection and cleavage.

A programmable pAgo nuclease with RNA target preference from the psychrotolerant bacteria Mucilaginibacter paludis

“…To determine if Mbp Ago has specificity for the first nucleotide of the guide, we also tested four guide variants with different first nucleotides but otherwise identical sequences. Similar to that of Cb Ago and Km Ago, we observed no changes in the cleavage efficiency and rate when Mbp Ago was loaded with 5’phosphorylated DNA guides with different first nucleotides (Figure 3C and Supplementary Figure S2E) (17,21).…”

“…In contrast, when the reaction was performed at 50°C almost all RNA was cleaved during 60 min, indicative of multiple-turnover reaction (Figure 3D, right panel; Supplementary Figure S2G). In conclusion, while Mbp Ago function as a multiple-turnover enzyme, its steady-state rate may be limited by product release and exchange of target molecules like that of Cb Ago and Km Ago (18,21).…”

“…The copyright holder for this preprint this version posted June 8, 2021. ; https://doi.org/10.1101/2021.06.08.447469 doi: bioRxiv preprint Finally, we have demonstrated that MbpAgo can efficiently cleave highly-structured RNA targets using both 5'P-gDNAs and 5'OH-gDNAs in the presence of Mg 2+ or Mn 2+ . Previously, eAgo from the budding yeast Kluyveromyces polysporus and pAgo from the mesophilic bacteria Kurthia masiliensis were used for highly structured RNA cleavage (21,38). Different from their findings, the cleavage efficiency of MbpAgo does not greatly depend on the secondary structure at 37°C.…”

“…We further investigated MbpAgo cleavage efficiency using DNA guides of different lengths, revealing that MbpAgo was most active with 15-20 nt DNA guides, with a lower efficiency observed with longer or shorter guides (Figure 3A). Similar to that of KmAgo, the cleavage position was shifted if shorter (11-13 nt) and longer (21-40) 5'P-gDNAs were used (20,21). Interestingly, the cleavage only occurred between the 10th and 11th guide positions when using 15-17 nt long 5'OH-gDNA, while the cleavage position was shifted if shorter (10-14 nt) or longer (18-30 nt) 5'OH-gDNA were used (Figure 3A).…”

“…Initially, pAgos were characterized from thermophilic prokaryotes, including Tt Ago ( Thermus thermophilus ) (3), Pf Ago ( Pyrococcus furiosus ) (4), Mp Ago ( Marinitoga piezophile ) (13), and Mj Ago ( Methanocaldococcus jannaschii ) (15,16), which can be programmed with DNA guides or RNA guides to cleave DNA targets or RNA targets effectively in elevated temperatures but not in moderate temperatures. Recently, active pAgos from mesophilic prokaryotes including Cb Ago ( Clostridium butyricum ) (17,18), Lr Ago ( Limnothrix rosea ) (17), Se Ago ( Synechococcus elongatus ) (19), Km Ago ( Kurthia massiliensis ) (20,21), Cp Ago ( Clostridium perfringens ) and Ib Ago ( Intestinibacter bartlettii ) (22) were characterized to search for a pAgo that can cleave double-stranded DNA (dsDNA) at moderate temperatures and be used for genome editing. Besides, some pAgos perform guide-independent target DNA processing activity, including Tt Ago, Mj Ago, Cb Ago, Lr Ago, and Se Ago (15,17,19,23).…”