Characterization of a Programmable Argonaute Nuclease from the Mesophilic Bacterium Rummeliibacillus suwonensis

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.

Keywords: DNA cleavage; RNA cleavage; argonaute; mesophilic; nuclease.

Figure 1

Sequence analysis and phylogenetic tree visualizations. (A) Maximum-likelihood phylogenetic tree analysis of RsuAgo based on amino acid sequences. The numbers at the nodes indicate the bootstrap values for the maximum likelihood analysis of 1000 resampled data sets. RsuAgo is indicated as a black triangle, BsAgo, DeAgo, and PmAgo are indicated as a black rectangle. (B) Multiple sequence alignment of RsuAgo, DeAgo, BsAgo, and PmAgo with several other characterized pAgo proteins. The catalytically dead variant of the RsuAgo protein (RsuAgo_DM) with amino acid substitutions in the catalytic tetrad is shown.

Figure 2

Analysis of single-stranded nucleic acid cleavage activity of pAgos. (A) Cleavage by RsuAgo with FAM-labeled DNA and RNA targets. (B) The sequence of the synthetic miRNA-based guide and target sequences that were used for the in vitro cleavage assays. The black triangle indicates the cleavage site. The vertical lines indicate contiguous Watson-Crick pairing. F-M1 (FAM-M1) and F-M2 (FAM-M2) are chemically synthesized, 34 nt long ssDNA (F-M1) and ssRNA (F-M2) oligonucleotides with a FAM-label at the 5′-end that were loaded on gels as size markers corresponding to the length of the DNA and RNA cleavage products, respectively. All experiments were performed at the 4:2:1 RsuAgo: guide: target molar ratio in reaction buffer containing Mn²⁺ ions for 1 h at 37 °C. All cleavage experiments were carried out in triplicates.

Figure 3

Effects of gDNA modification and 5′-end nucleotide on RsuAgo cleavage activity. (A) Cleavage kinetics on DNA and RNA targets using DNA-guided and RNA-guided RsuAgo. (B) Cleavage kinetics on DNA target using different 5′-end nucleotides of DNA guide. (C) Cleavage kinetics on RNA target using different 5′-end nucleotides of DNA guide. (D) Cleavage kinetics on DNA target using different 5′-end nucleotides of RNA guide. All experiments were performed at the 4:2:1 RsuAgo: guide: target molar ratio in reaction buffer containing 5 mM Mn²⁺ ions at 37 °C. All cleavage experiments were carried out in triplicates and quantified. Error bars represent the SD based on three independent experiments.

Figure 4

Effects of different divalent metal ions on RsuAgo cleavage activity. (A) Effects of different divalent metal cations on DNA target cleavage activity mediated by P-gDNA. (B) Effects of different divalent metal cations on RNA target cleavage activity mediated by P-gDNA. (C) Cleavage kinetics efficiency of DNA target mediated by Mn²⁺ and Mg²⁺. (D) Cleavage kinetics efficiency of RNA target mediated by Mn²⁺ and Mg²⁺. All experiments were performed at the 4:2:1 RsuAgo: guide: target molar ratio in reaction buffer containing different divalent metal ions for 1 h at 37 °C. All cleavage experiments were carried out in triplicates and quantified. Error bars represent the SD based on three independent experiments.

Figure 5

Effects of temperature and guide length on RsuAgo activity. (A) Effects of temperature on DNA target cleavage guided by P-gDNA. (B) Effects of temperature on RNA target cleavage guided by P-gDNA. (C) Effects of P-gDNA length on DNA cleavage activity. (D) Effects of P-gDNA length on RNA cleavage activity. All experiments were performed at the 4:2:1 RsuAgo: guide: target molar ratio in reaction buffer containing 5 mM Mn²⁺ ions for 1 h at 37 °C. All cleavage experiments were carried out in triplicates.

Figure 6

Effects of mismatches in the guide-target duplex on the cleavage activity of RsuAgo. (A) Schematic diagram of guide single nucleotide mismatch sites, mismatched positions are indicated in red. (B) Effects of single nucleotide mismatches in the P-gDNA: tDNA duplex on the slicing activity of RsuAgo. (C) Sequences of the gDNA (blue) and DNA/RNA target (grey). The different functional gDNA elements are indicated above the gDNA; sup., supplementary region. (D) Effects of single nucleotide mismatches in the P-gDNA: tRNA duplex on the slicing activity of RsuAgo. Control, gDNA with full complementarity to the target. All experiments were performed at the 4:2:1 RsuAgo: guide: target molar ratio in reaction buffer containing 5 mM Mn²⁺ ions for 1 h at 37 °C. All cleavage experiments were carried out in triplicates and quantified. Error bars represent the SD based on three independent experiments.

Figure 7

Cleavage of double-stranded plasmid DNA by RsuAgo. (A) RsuAgo-mediated cleavage of plasmid pUC19 at different NaCl concentrations. RsuAgo and a gDNA with 29% GC-content were preincubated for 30 min at 37 °C, followed by addition of pUC19 dsDNA, incubation for 2 h at 37 °C in reaction buffer containing 0.5 mM Mn²⁺ and cleavage analysis by 1% agarose gel electrophoresis. (B) RsuAgo-mediated plasmid pUC19 cleavage with gDNAs differing in their GC-content in reaction buffer containing 0.5 mM Mn²⁺ and 55 mM NaCl for 2 h at 37 °C. ScaI lane: the isolated plasmid was digested with ScaI for 2 h. FW/RV-gDNA, forward and reverse gDNA corresponding to a specific target site in plasmid pUC19; NC-gDNA, a non-complementary gDNA control; M, molecular weight marker; LIN, linearized plasmid; SC, supercoiled plasmid; OC, open circular plasmid. All cleavage experiments were carried out in triplicates.