Evolution, role and mechanism of prokaryotic Argonaute proteins

cum laude graduatio

Bacteriophage DNA glucosylation impairs target DNA binding by type I and II but not by type V CRISPR-Cas effector complexes

Prokaryotes encode various host defense systems that provide protection against mobile genetic elements. Restriction-modification (R-M) and CRISPR-Cas systems mediate host defense by sequence specific targeting of invasive DNA. T-even bacteriophages employ covalent modifications of nucleobases to avoid binding and therefore cleavage of their DNA by restriction endonucleases. Here, we describe that DNA glucosylation of bacteriophage genomes affects interference of some but not all CRISPR-Cas systems. We show that glucosyl modification of 5-hydroxymethylated cytosines in the DNA of bacteriophage T4 interferes with type I-E and type II-A CRISPR-Cas systems by lowering the affinity of the Cascade and Cas9-crRNA complexes for their target DNA. On the contrary, the type V-A nuclease Cas12a (also known as Cpf1) is not impaired in binding and cleavage of glucosylated target DNA, likely due to a more open structural architecture of the protein. Our results suggest that CRISPR-Cas systems have contributed to the selective pressure on phages to develop more generic solutions to escape sequence specific host defense systems

MOLECULAR-CELL-D-16-01367 Swarts et al

This data contains the raw imaging data and sequencing data of the manuscript "Autonomous generation and loading of DNA guides by bacterial Argonaute" written by Swarts, Szczepaniak, Sheng et al

Stirring Up the Type V Alphabet Soup.

Type II and type V CRISPR-Cas effectors are multidomain enzymes that utilize a guide RNA to bind and cleave complementary DNA targets.1 The facile programmability of these effectors allows for their exploitation as genome editing tools. While the type II effector Cas9 is undoubtedly the best-known genome editing tool, the more recently characterized type V effectors (i.e., Cas12a and Cas12b) have also been repurposed for genome editing.1–3Despite their highly similar functionality, Cas9 and Cas12 have distinct domain architectures and mechanisms, making them complementary tools for genome editing.1 Strikingly, there is a high diversity in sequence and domain organization among the different type V effectors.4,5 Therefore, characterization of these functionally diverse type V effectors could lead to the expansion of the CRISPR toolbox. Writing in Science,6 Yan et al. from Arbor Biotechnologies and the National Center for Biotechnology Information have extended the type V alphabet by identifying and characterizing four novel type V effectors: type V-C Cas12c type V-G Cas12g, type V-H Cas12h, and type V-I Cas12i

DNA-guided DNA interference by a prokaryotic Argonaute

This disclosure provides for compositions and methods for the use of designed nucleic acid-targeting nucleic acids, Argonautes, and complexes thereof. In one aspect, the disclosure provides for a composition comprising: a complex comprising: an Argonaute and a designed nucleic acid-targeting nucleic acid; and a target nucleic acid, wherein the designed nucleic acid-targeting nucleic acid is hybridized to the target nucleic acid. In some embodiments, the target nucleic acid is double-stranded. In some embodiments, the Argonaute comprises at least 30% amino acid identity to a prokaryotic Argonaute. In some embodiments, the Argonaute comprises at least 30% amino acid identity to a bacterial Argonaute. In some embodiments, the Argonaute comprises at least 30% amino acid identity to an archaeal Argonaute. In some embodiments, the Argonaute comprises at least 30% amino acid identity to an Argonaute from a mesophile. In some embodiments, the Argonaute comprises at least 30% amino acid identity to an Argonaute from a thermophile

Planting the seed: target recognition of short guide RNAs

Small guide RNAs play important roles in cellular processes such as regulation of gene expression and host defense against invading nucleic acids. The mode of action of small RNAs relies on protein-assisted base pairing of the guide RNA with target mRNA or DNA to interfere with their transcription, translation, or replication. Several unrelated classes of small noncoding RNAs have been identified including eukaryotic RNA silencing-associated small RNAs, prokaryotic small regulatory RNAs (sRNAs), and prokaryotic CRISPR (clustered regularly interspaced short palindromic repeats) RNAs (crRNAs). All three groups identify their target sequence by base pairing after finding it in a pool of millions of other nucleotide sequences in the cell. In this complicated target search process, a region of 612 nucleotides (nt) of the small RNA termed the seed plays a critical role. We review the concept of seed sequences and discuss its importance for initial target recognition and interferenc

Prokaryotic Argonautes – variations on the RNA interference theme

The discovery of RNA interference (RNAi) has been a major scientific breakthrough. This RNA-guided RNA interference system plays a crucial role in a wide range of regulatory and defense mechanisms in eukaryotes. The key enzyme of the RNAi system is Argonaute (Ago), an endo-ribonuclease that uses a small RNA guide molecule to specifically target a complementary RNA transcript. Two functional classes of eukaryotic Ago have been described: catalytically active Ago that cleaves RNA targets complementary to its guide, and inactive Ago that uses its guide to bind target RNA to down-regulate translation efficiency. A recent comparative genomics study has revealed that Argonaute-like proteins are also encoded by prokaryotic genomes. Interestingly, there is a lot of variation among these prokaryotic Argonaute (pAgo) proteins with respect to domain architecture: some resemble the eukaryotic Ago (long pAgo) containing a complete or disrupted catalytic site, while others are truncated versions (short pAgo) that generally contain an incomplete catalytic site. Prokaryotic Agos with an incomplete catalytic site often co-occur with (predicted) nucleases. Based on this diversity, and on the fact that homologs of other RNAi-related protein components (such as Dicer nucleases) have never been identified in prokaryotes, it has been predicted that variations on the eukaryotic RNAi theme may occur in prokaryotes

Purification and Sequencing of DNA Guides from Prokaryotic Argonaute

Some proteins utilize nucleic acids to guide them to complementary nucleic acid targets. One example is prokaryotic Argonaute protein, which, binds small single stranded DNA molecules as guides (Swarts et al., 2014). This protocol describes a method to purify DNA guides from these proteins. It also describes a PCR-based method to enrich the guides by PCR amplification. This methods relies on addition of a poly-A tail at the 3’-end of the ssDNA molecules by Terminal Deoxynucleotidyl Transferase (TdT), followed by ligation of a oligonucleotide to the 5’-end of the ssDNA molecule using T4 RNA ligase, and amplification by PCR. The generated dsDNA products are suitable for traditional cloning and sequencing and high-throughput sequencing. Importantly, the information which strand matches the ssDNA molecule is not lost during this process

Purification and Sequencing of DNA Guides from Prokaryotic Argonaute

Some proteins utilize nucleic acids to guide them to complementary nucleic acid targets. One example is prokaryotic Argonaute protein, which, binds small single stranded DNA molecules as guides (Swarts et al., 2014). This protocol describes a method to purify DNA guides from these proteins. It also describes a PCR-based method to enrich the guides by PCR amplification. This methods relies on addition of a poly-A tail at the 3’-end of the ssDNA molecules by Terminal Deoxynucleotidyl Transferase (TdT), followed by ligation of a oligonucleotide to the 5’-end of the ssDNA molecule using T4 RNA ligase, and amplification by PCR. The generated dsDNA products are suitable for traditional cloning and sequencing and high-throughput sequencing. Importantly, the information which strand matches the ssDNA molecule is not lost during this process

Effects of Argonaute on gene expression in Thermus thermophilus

To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. Background Eukaryotic Argonaute proteins mediate RNA-guided RNA interference, allowing both regulation of host gene expression and defense against invading mobile genetic elements. Recently, it has become evident that prokaryotic Argonaute homologs mediate DNA-guided DNA interference, and play a role in host defense. Argonaute of the bacterium Thermus thermophilus (TtAgo) targets invading plasmid DNA during and after transformation. Using small interfering DNA guides, TtAgo can cleave single and double stranded DNAs. Although TtAgo additionally has been demonstrated to cleave RNA targets complementary to its DNA guide in vitro, RNA targeting by TtAgo has not been demonstrated in vivo. Methods To investigate if TtAgo also has the potential to control RNA levels, we analyzed RNA-seq data derived from cultures of four T. thermophilus strain HB27 variants: wild type, TtAgo knockout (Δago), and either strain transformed with a plasmid. Additionally we determined the effect of TtAgo on expression of plasmid-encoded RNA and plasmid DNA levels. Results In the absence of exogenous DNA (plasmid), TtAgo presence or absence had no effect on gene expression levels. When plasmid DNA is present, TtAgo reduces plasmid DNA levels 4-fold, and a corresponding reduction of plasmid gene transcript levels was observed. We therefore conclude that TtAgo interferes with plasmid DNA, but not with plasmid RNA. Interestingly, TtAgo presence stimulates expression of specific endogenous genes, but only when exogenous plasmid DNA was present. Specifically, the presence of TtAgo directly or indirectly stimulates expression of CRISPR loci and associated genes, some of which are involved in CRISPR adaptation. This suggests that TtAgo-mediated interference with plasmid DNA stimulates CRISPR adaptation