Prokaryotic argonaute proteins: From ancient defense mechanisms to modern biosensing applications

Prokaryotic Argonaute (pAgo) proteins constitute an evolutionarily ancient nuclease family that is rapidly maturing into a versatile molecular toolkit rivaling CRISPR-Cas. This review synthesizes recent advances in pAgo biology and biotechnology, tracing their phylogeny across thermophilic, mesophilic, and psychrotolerant lineages and highlighting temperature-adapted catalytic signatures that diverge from eukaryotic Agos. In vivo studies reveal pAgo roles in gDNA-guided host defense, transcriptional silencing and recombination, all executed through programmable DNA- or RNA-guided nuclease activity. We detail how guide length, 5' nucleotide identity, divalent cations and accessory factors modulate cleavage efficiency, enabling rational optimization. The review then maps the explosion of pAgo-based biosensing platforms, including selective nucleic acid enrichment platforms, ultrasensitive pathogen detection methods, programmable DNA cloning systems, and high-resolution imaging techniques. Their independence from protospacer adjacent motifs (PAMs), stable DNA guides, multi-turnover kinetics, and broad thermal tolerance position pAgos as ideal complements to CRISPR systems. Finally, we outline current limitations and future directions, including the discovery and engineering of novel variants, elucidation of guide-generation mechanisms, and development of next-generation gene-editing tools, aiming to accelerate translation of these versatile enzymes into practical biotechnological and therapeutic translation.