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Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea.

ABSTRACT: Argonaute (Ago) proteins are key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleavage of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures, which limits their potential use as a tool in genomic applications. Here, we describe two pAgos from mesophilic bacteria, Clostridium butyricum (CbAgo) and Limnothrix rosea (LrAgo), that act as DNA-guided DNA nucleases at physiological temperatures. In comparison with previously studied pAgos, CbAgo and LrAgo do not show strong preferences for the 5'-nucleotide in guide DNA and can use not only 5'-phosphorylated but also 5'-hydroxyl DNA guides. Both CbAgo and LrAgo can tolerate guide/target mismatches in the seed region, but are sensitive to mismatches in the 3'-guide region. Both pAgos can perform programmable endonucleolytic cleavage of double-stranded DNA substrates, showing enhanced activity at AT-rich regions and at elevated temperatures. The biochemical characterization of mesophilic pAgo proteins paves the way for their use for DNA manipulations both in vitro and in vivo.

SUBMITTER: Kuzmenko A

PROVIDER: S-EPMC6582412 | biostudies-literature | 2019 Jun

REPOSITORIES: biostudies-literature

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Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea.

Kuzmenko Anton A Yudin Denis D Ryazansky Sergei S Kulbachinskiy Andrey A Aravin Alexei A AA

Nucleic acids research 20190601 11

Argonaute (Ago) proteins are key players in RNA interference in eukaryotes, where they function as RNA-guided RNA endonucleases. Prokaryotic Argonautes (pAgos) are much more diverse than their eukaryotic counterparts but their cellular functions and mechanisms of action remain largely unknown. Some pAgos were shown to use small DNA guides for endonucleolytic cleavage of complementary DNA in vitro. However, previously studied pAgos from thermophilic prokaryotes function at elevated temperatures, ...[more]

PMID: 31114878

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Project description:Genome engineering with programmable nucleases depends on cellular responses to a targeted double-strand break (DSB). The first truly targetable reagents were the zinc finger nucleases (ZFNs) showing that arbitrary DNA sequences could be addressed for cleavage by protein engineering, ushering in the breakthrough in genome manipulation. ZFNs resulted from basic research on zinc finger proteins and the FokI restriction enzyme (which revealed a bipartite structure with a separable DNA-binding domain and a non-specific cleavage domain). Studies on the mechanism of cleavage by 3-finger ZFNs established that the preferred substrates were paired binding sites, which doubled the size of the target sequence recognition from 9 to 18bp, long enough to specify a unique genomic locus in plant and mammalian cells. Soon afterwards, a ZFN-induced DSB was shown to stimulate homologous recombination in cells. Transcription activator-like effector nucleases (TALENs) that are based on bacterial TALEs fused to the FokI cleavage domain expanded this capability. The fact that ZFNs and TALENs have been used for genome modification of more than 40 different organisms and cell types attests to the success of protein engineering. The most recent technology platform for delivering a targeted DSB to cellular genomes is that of the RNA-guided nucleases, which are based on the naturally occurring Type II prokaryotic CRISPR-Cas9 system. Unlike ZFNs and TALENs that use protein motifs for DNA sequence recognition, CRISPR-Cas9 depends on RNA-DNA recognition. The advantages of the CRISPR-Cas9 system-the ease of RNA design for new targets and the dependence on a single, constant Cas9 protein-have led to its wide adoption by research laboratories around the world. These technology platforms have equipped scientists with an unprecedented ability to modify cells and organisms almost at will, with wide-ranging implications across biology and medicine. However, these nucleases have also been shown to cut at off-target sites with mutagenic consequences. Therefore, issues such as efficacy, specificity and delivery are likely to drive selection of reagents for particular purposes. Human therapeutic applications of these technologies will ultimately depend on risk versus benefit analysis and informed consent.

Dataset Information

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea.

Publications

Programmable DNA cleavage by Ago nucleases from mesophilic bacteria Clostridium butyricum and Limnothrix rosea.

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