In vitro programmable DNA cleavage by a eukaryotic Argonaute

Abstract

Eukaryotic Argonautes (eAgos) have traditionally been characterized by their ability to utilize RNA guides to identify RNA targets, thereby engaging in post-transcriptional gene silencing pathways. While some eAgos have been demonstrated to use DNA guides for RNA cleavage, the ability of eAgos to cleave DNA targets remains unclear. In this study, we characterized CsAgo, an eAgo protein derived from thermophilic eukaryote Chaetomiumsp. MPI-CAGE-AT-0009, demonstrating a novel ability to cleave both DNA and RNA targets in vitro. Guided by short single-stranded DNA (ssDNA) or RNA, CsAgo exhibits robust RNA cleavage activity at 20-90°C in vitro. CsAgo can effectively cleave ssDNA guided by RNA guides at 20-50°C in vitro. Notably, CsAgo can utilize DNA guides to effectively cleave ssDNA, plasmid double-stranded DNA (dsDNA), and linear dsDNA at ≥80°C in vitro. Based on its ability to cleave dsDNA at high temperatures, CsAgo demonstrates versatility and efficacy in simplifying routine cloning workflows. Additionally, we have developed a CsAgo-based nucleic acid detection method based on a Pyrococcus furiosus Ago-mediated nucleic acid detection method, which exhibits a high sensitivity of six copies/reaction. These results suggest that eAgos include that, in theory, can be utilized for potential DNA-targeting applications. This not only enhances our understanding of eAgos but also expands the toolkit for DNA manipulation.

Graphical Abstract

Figure 1.

CsAgo utilizes four types of guides for target nucleic acid cleavage. (A) A maximum likelihood phylogenetic tree comparing CsAgo with other previously characterized Agos. (B) A diagram illustrating the guide (18-nt DNA or 21-nt RNA) and target nucleic acids (45-nt DNA or RNA), with cleavage sites indicated by red and blue triangles. (C) A cleavage activity assay of CsAgo and its variant (CsAgo_DM, which has substitutions of two catalytic tetrad residues, D817A/D899A) using FAM-labeled T-RNA. (D) A cleavage activity assay of CsAgo and CsAgo_DM using FAM-labeled T-DNA. CsAgo or CsAgo_DM, along with guides and targets, were mixed in a 5:1:1 molar ratio and incubated at 37°C for 30 min in a reaction buffer containing 10 mM Mn²⁺ for RNA cleavage. For ssDNA cleavage, reactions utilizing gRNAs were conducted at 37°C in a buffer containing 40 mM Mn²⁺ for 1 h, whereas those employing gDNAs were performed at 85°C in a buffer containing 30 mM Mg²⁺ for 1 h. M1, M2, and M3 represent synthesized 5′-FAM-labeled product markers for 32-nt, 33-nt, and 34-nt RNA, respectively. M1′, M2′, and M3′ denote synthesized 5′-FAM-labeled product markers for 32-nt, 33-nt, and 34-nt ssDNA, respectively.

Figure 2.

Impact of temperature on the cleavage activity of CsAgo. (A) Temperature effects on DNA-guided DNA cleavage activity. (B) Temperature effects on RNA-guided DNA cleavage activity. (C) Temperature effects on DNA-guided RNA cleavage activity. (D) Temperature effects on RNA-guided RNA cleavage activity. CsAgo, guides, and targets were mixed in a 5:1:1 molar ratio and incubated in a reaction buffer with 30 mM Mg²⁺. Cleavage of target DNA (A, B) and RNA (C, D) was performed for 1 h and 30 min, respectively, at the specified temperatures. The data are presented as mean ± standard deviation (SD) from three independent experiments. Refer to Supplementary Fig. S3.

Figure 3.

Impact of guide length (nt) on the cleavage activity of CsAgo. Representative denaturing PAGE (polyacrylamide gel electrophoresis) results (one of three independent experiments) demonstrating the impact of guide length on CsAgo cleavage activity. (A) CsAgo cleavage of DNA guided by 12–25-nt 5′P-gDNAs. (B) CsAgo cleavage of DNA guided by 5′OH-gDNAs. (C) Histogram illustrating conversion of DNA cleavage products mediated by two types of gDNAs. (D) CsAgo cleavage of DNA guided by 12–25-nt 5′P-gRNAs. (E) CsAgo cleavage of DNA guided by 5′OH-gRNAs. (F) Histogram illustrating conversion of DNA cleavage products mediated by two types of gRNAs. (G) CsAgo cleavage of RNA guided by 12–25-nt 5′P-gDNAs. (H) CsAgo cleavage of RNA guided by 5′OH-gDNAs. (I) Histogram illustrating conversion of RNA cleavage products mediated by two types of gDNAs. (J) CsAgo cleavage of RNA guided by 12–25-nt 5′P-gRNAs. (K) CsAgo cleavage of RNA guided by 5′OH-gRNAs. (L) Histogram illustrating conversion of RNA cleavage products mediated by two types of gRNAs. RNA cleavage was conducted at 37°C for 30 min, whereas DNA cleavage was conducted at 37°C for 1 h with gRNAs and at 85°C for 1 h with gDNAs. Refer to Supplementary Fig. S9 for additional information.

Figure 4.

Impact of single mismatches in 5′-phosphorylated guide–target on CsAgo cleavage activity. (A) Influence of guide–target mismatches on DNA cleavage mediated by 5′P-gDNA. (B) Influence of guide–target mismatches on RNA cleavage with 5′P-gDNA. (C) Influence of guide–target mismatches on DNA cleavage mediated by 5′P-gRNA. (D) Influence of guide–target mismatches on RNA cleavage activity with 5′P-gRNA. In panels (A) and (C), the samples were incubated at 85°C and 37°C, respectively, for 60 min. In panels (B) and (D), the reactions were conducted at 37°C for 30 min. The data are presented as mean ± SD from three independent experiments. Statistical significance was assessed using Student’s t-test, with significance levels indicated as follows: *P < .05, **P < .01, ***P < .001, and ^****P < .0001, compared to control reactions using guides without mismatches. Refer to Supplementary Fig. S11 for additional information.

Figure 5.

Cleavage of plasmid DNA by the CsAgo–gDNA complex. (A) Schematic overview of the pUC19 target plasmid. Black lines indicate the target region, with percentages denoting the GC content of the 80-bp segments encompassing these sites. (B) CsAgo and pairs of 5′P-gDNAs targeting a 41% GC content in the pUC19 plasmid. CsAgo was pre-incubated with both F guide and R guide targeting each strand of pUC19 plasmid at 37°C for 20 min. The complexes with single guide and guide pairs were used to cleave plasmid DNA at 88°C for 10 min. (C) Examination of Mg²⁺ concentration and temperature effects on pUC19 plasmid cleavage. (D) Effects of GC content on plasmid DNA cleavage. CsAgo and pairs of 5′P-gDNAs targeting regions with different GC contents on pUC19 were mixed and incubated with target plasmid at 88°C for 10 min. (E) CsAgo and two pairs of 5′P-gDNAs targeting pUC19 plasmid. The positions of the cleavage products are indicated by asterisks. In all plasmid cleavage experiments, CsAgo and guides were mixed in a 1:1 molar ratio and pre-incubated at 37°C for 20 min to form complexes in a reaction buffer containing 2 mM Mg²⁺ and 100 mM betaine. The products were treated with proteinase K for 20 min and then subjected to 1% agarose gel electrophoresis. M represents the 1-kb DNA ladder. T indicates plasmid DNA of pUC19; NC denotes a negative control using noncomplementary guide nucleic acid; No-Ago represents a negative control without CsAgo; and No-guide represents a negative control without a guide. Lin represents the linearized plasmid; SC denotes supercoiled plasmid; and OC signifies the open circular plasmid. Refer to Supplementary Figs S16–S21 for further information.

Figure 6.

CsAgo-mediated linear dsDNA cleavage and molecular cloning. (A) Effects of various SSB proteins (CaSSB, TaSSB, TthSSB, and ETSSB) on the cleavage of 2.7-kb linear dsDNA. The CsAgo and pairs of 5′P-gDNAs, designed to target a region with 41% GC content, were incubated with linear dsDNA at 88°C for 10 min. (B) Effects of reaction temperature on the cleavage of 2.7-kb linear dsDNA. The CsAgo and pairs of 5′P-gDNAs targeting a region with 41% GC content were incubated with linear dsDNA at the specified temperature for 10 min. (C) Effects of GC content on the cleavage of 1-kb linear dsDNA. The reactions were incubated with dsDNA at 88°C for 10 min. For these experiments, the reaction buffer contained 20 mM Mg²⁺ and 100 mM betaine. In panels (B) and (C), the reaction buffer contained 20 μM CaSSB. M represents the 1-kb DNA ladder in panels (A) and (B), while DL2000 DNA ladder represents the M in panel (C). T2 denotes the purified 1-kb dsDNA (PCR products). T3 denotes the purified 2.7-kb dsDNA linearized by XbaI from pUC19 plasmid. For additional information, see Supplementary Figs S22 and S23. (D) A schematic diagram illustrates two pairs of gDNAs bound to CsAgo, cleaving plasmids to produce the vector backbone and the target gene fragment for cloning. (E) A pie chart displays the sequencing accuracy of recombinants from a DNA cloning experiment utilizing the vector backbone and a gene fragment, as depicted in panel (D). (F) A schematic diagram depicts three pairs of gDNAs bound to CsAgo, cleaving plasmids to generate the vector backbone and two unique target gene fragments for DNA cloning. The three pairs of gDNAs are differentiated by color. (G) A pie chart displays the sequencing accuracy of recombinants from a DNA cloning experiment involving the vector backbone and two gene fragments, as depicted in panel (F). Refer to Supplementary Figs S24 and S25 for additional information.

Figure 7.

CsAgo-mediated nucleic acid detection. (A) Schematic diagram of CsAgo-mediated nucleic acid detection. MB represents the molecular beacon; gN signifies newly synthesized gDNAs resulting from PCR product cleavage by the CsAgo–guide complex. The green cluster signifies the fluorescent group FAM, while the gray sphere represents the quenching group BHQ. The red wedge arrow marks the specific cleavage site of the CsAgo–guide complex target. (B) Normalized FL intensity after PCR and CsAgo cleavage, plotted against varying concentrations of HPV16E6 over 1 h, in a reaction buffer containing 10 mM MgCl₂ and 20 μM CaSSB. (C) Schematic representation of selected sequence information with complete match and double base mismatch for HPV16E6. (D) Comparison of normalized FL intensity after PCR and CsAgo cleavage using gDNA with mismatches at various positions versus control reactions with guides without mismatches. The “wt” denotes gDNA complementary to HPV16E6. The data are presented as mean ± SD, based on three technical replicates. Statistical significance denoted as *P < .05, **P < .01, ***P < .001, and ^****P < .0001. Also refer to Supplementary Figs S26 and S27.