An innovative programmable tool for targeting nucleic acids has been created, utilizing a prokaryotic immune defense system—and it is not CRISPR-Cas. Russian Academy of Sciences researchers have successfully re-engineered prokaryotic Argonautes (pAgos) to utilize RNA guides for locating nucleic acid sequences. These systems have been modified to form a complex with effector nucleases.

The researchers employed a two-component system known as SPARDA (short prokaryotic Argonaute, DNase, and RNase-associated) to effectively identify DNA sequences with a notable level of sensitivity and induce collateral nuclease activity. SPARDA and other concise pAgos systems that encode diverse effectors have the potential to offer a novel programmable tool for the field of biotechnology.

The research article “DNA-targeting short Argonautes complex with effector proteins for collateral nuclease activity and bacterial population immunity” was published in Nature Microbiology.

A second prokaryotic defense system

Argonautes are a group of proteins that are widely found and can be designed to recognize specific nucleic acid targets using short guide oligonucleotides. Argonautes were initially identified in eukaryotes because of their pivotal involvement in RNA interference. However, further examination revealed their extensive presence in prokaryotes.

Similar to CRISPR-Cas, pAgos employ complementary guides and the nuclease activities of pAgo or Cas proteins to identify and cleave foreign nucleic acids. Not all pAgos exhibit enzymatic activity as nucleases. Due to the lack of research on most short pAgo systems, the mechanisms by which invader DNA is differentiated and the diversity of effector domains are not well understood.

SPARDA = short pAgo protein + effector nuclease

Maria Prostova and Anna Kanevskaya, as co-lead authors, conducted a study with a team of researchers to examine short pAgos that are linked to potential effector nucleases. Two previously unexplored pAgos with potential effector nucleases were identified through phylogenetic analysis. It was shown that the pAgos can form a heterodimeric complex with co-encoded effector nucleases called SPARDA. Like CRISPR-Cas, SPARDA can be activated to cleave collateral ssDNA, dsDNA, ssRNA, and DNA-RNA substrates.

In this study, the researchers devised a technique to activate SPARDA using plasmids or phages. This activation led to the breakdown of cellular DNA and the subsequent death or dormancy of cells. As a result, the researchers were able to provide targeted protection to specific populations and broaden the scope of recognized immune systems in prokaryotes. Further investigation is required to determine whether activated SPARDA can permanently impede phage replication or merely postpone phage release for different phages.

In addition, the researchers utilized SPARDA to detect single-stranded DNA (ssDNA) targets using a fluorescent beacon assay, which enhances the existing Cas12- or Cas13-based techniques. The assay’s sensitivity can be improved by incorporating a polymerase chain reaction (PCR) step before detection.

With its physiological temperature range, low background activity, and ability to recognize specific motifs in target DNA, SPARDA may find use in a wide range of applications, from in vitro nucleic acid detection to programmable removal of bacterial or eukaryotic cells for microbiome engineering and therapy.

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