Functions of prokaryotic Argonautes (pAgo) have long remained elusive. Recently, Argonautes of the bacteria Rhodobacter sphaeroides and Thermus thermophilus were demonstrated to be involved in host defense. The Argonaute of the archaeon Pyrococcus furiosus (PfAgo) belongs to a different branch in the phylogenetic tree, which is most closely related to that of RNA interference-mediating eukaryotic Argonautes. Here we describe a functional and mechanistic characterization of PfAgo. Like the bacterial counterparts, archaeal PfAgo contributes to host defense by interfering with the uptake of plasmid DNA. PfAgo utilizes small 5′-phosphorylated DNA guides to cleave both single stranded and double stranded DNA targets, and does not utilize RNA as guide or target. Thus, with respect to function and specificity, the archaeal PfAgo resembles bacterial Argonautes much more than eukaryotic Argonautes. These findings demonstrate that the role of Argonautes is conserved through the bacterial and archaeal domains of life and suggests that eukaryotic Argonautes are derived from DNA-guided DNA-interfering host defense systems.

Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave complementary invading DNA. This activity can be repurposed for programmable DNA cleavage. However, currently characterized DNA-cleaving pAgos require elevated temperatures (≥65°C) for their activity, making them less suitable for applications that require moderate temperatures, such as genome editing. Here, we report the functional and structural characterization of the siDNA-guided DNA-targeting pAgo from the mesophilic bacterium Clostridium butyricum ( Cb Ago). Cb Ago displays a preference for siDNAs that have a deoxyadenosine at the 5′-end and thymidines at nucleotides 2–4. Furthermore, Cb Ago mediates DNA-guided DNA cleavage of AT-rich double stranded DNA at moderate temperatures (37°C). This study demonstrates that certain pAgos are capable of programmable DNA cleavage at moderate temperatures and thereby expands the scope of the potential pAgo–based applications.

Argonaute proteins constitute a highly diverse family of nucleic acid-guided proteins. They were first discovered in eukaryotes as key proteins in RNA interference systems, but homologous prokaryotic Argonaute proteins (pAgos) have also been found in archaea and bacteria. In this Progress article, we focus on long pAgo variants, a class of pAgos that are involved in nucleic acid-guided host defence against invading nucleic acids, and discuss the potential of pAgos in genome editing.

Detection of disease-associated, cell-free nucleic acids in body fluids enables early diagnostics, genotyping and personalized therapy, but is challenged by the low concentrations of clinically significant nucleic acids and their sequence homology with abundant wild-type nucleic acids. We describe a novel approach, dubbed NAVIGATER, for increasing the fractions of Nucleic Acids of clinical interest Via DNA-Guided Argonaute from Thermus thermophilus (TtAgo). TtAgo cleaves specifically guide-complementary DNA and RNA with single nucleotide precision, greatly increasing the fractions of rare alleles and, enhancing the sensitivity of downstream detection methods such as ddPCR, sequencing, and clamped enzymatic amplification. We demonstrated 60-fold enrichment of the cancer biomarker KRAS G12D and ∼100-fold increased sensitivity of Peptide Nucleic Acid (PNA) and Xenonucleic Acid (XNA) clamp PCR, enabling detection of low-frequency (<0.01%) mutant alleles (∼1 copy) in blood samples of pancreatic cancer patients. NAVIGATER surpasses Cas9-based assays (e.g. DASH, Depletion of Abundant Sequences by Hybridization), identifying more mutation-positive samples when combined with XNA-PCR. Moreover, TtAgo does not require targets to contain any specific protospacer-adjacent motifs (PAM); is a multi-turnover enzyme; cleaves ssDNA, dsDNA and RNA targets in a single assay; and operates at elevated temperatures, providing high selectivity and compatibility with polymerases.

16 Prokaryotic Argonaute proteins (pAgos) constitute a diverse group of endonucleases of which 17 some mediate host defense by utilizing small interfering DNA guides (siDNA) to cleave 18 complementary invading DNA. This activity has been repurposed for programmable DNA 19 cleavage in vitro. However, currently characterized DNA-cleaving pAgos require elevated 20 temperatures (≥65°C) for their activity, making them less suitable for in vivo applications at 21 moderate temperatures. Here, using biochemistry, X-ray crystallography, and single-molecule 22 fluorescence methods, we report the functional and structural characterization of the pAgo from 23 the mesophilic bacterium Clostridium butyricum (CbAgo). CbAgo can be reprogrammed with 24 siDNAs to cleave complementary DNA, but not RNA. CbAgo displays a preference for siDNAs 25 that have a deoxyadenosine at the 5'-end and thymidines in the sub-seed segment (siDNA 26 nucleotides 2-4). Furthermore, CbAgo mediates DNA-guided DNA cleavage of AT-rich double 27 stranded DNA at moderate temperatures (37°C). Taken together, this study provides an important 28 step towards the development of pAgos for genetic engineering applications. 29 77 development of pAgo proteins as genome editing tools. 78 79 80 81 82 83 84 85 86 87 88 4 Results 89CbAgo mediates siDNA-guided ssDNA cleavage 90 CbAgo was successfully expressed in E. coli from a codon-optimized gene using a T7-based pET 91 expression system and purified ( Supplementary Fig. 1a). To determine the guide and target binding 92 characteristics of CbAgo, we performed single-molecule experiments using Förster resonance energy 93 transfer (FRET). We immobilized either Cy5-labeled single stranded RNA or DNA targets (FRET 94 acceptor) on a polymer-coated quartz surface ( Fig. 1a). Next we introduced CbAgo in complex with 95 either a Cy3-labeled siRNA or siDNA guide (FRET donor) and recorded the interactions. Strikingly, 96CbAgo could utilize both siRNAs and siDNAs to bind DNA or RNA targets ( Fig. 1b). To test which guide 97 is preferentially bound by CbAgo we performed a competition assay in which CbAgo was immobilized 98 into the microfluidic chamber, and an equimolar mixture of siDNA and siRNAs was introduced. While 99 only short-lived interactions were observed for siRNA, siDNA was strongly bound by CbAgo (Fig. 1c). 100This results suggests that CbAgo utilizes siDNA rather than siRNA as a guide. 101CbAgo is phylogenetically closest related to the clade of halobacterial pAgos, among which also 102 pAgo from Natronobacterium gregoryi (NgAgo) can be found ( Fig. 1d and Supplementary Fig. 2). A 103 multiple sequence alignment of CbAgo with other pAgos ( Supplementary Fig. 1b) suggests that CbAgo 104 contains the conserved DEDX catalytic residues (where X can be an D, H or N) which are essential for 105 nuclease activity in 'slicing' Agos 31 . In the case of CbAgo, this concerns residues D541, E577, D611 and 106 D722. 107To confirm whether CbAgo indeed is an active nuclease, we performed in vitro activity assays 108 in which CbA...