Programmable site-specific nucleases promise to unlock myriad applications in basic biology research, biotechnology and gene therapy. Gene-editing systems have revolutionized our ability to engineer genomes across diverse eukaryotic species. However, key challenges, including delivery, specificity and targeting organellar genomes, pose barriers to translational applications. Here, we use peptide nucleic acids (PNAs) to facilitate precise DNA strand invasion and unwinding, enabling prokaryotic Argonaute (pAgo) proteins to specifically bind displaced single-stranded DNA and introduce site-specific double-strand breaks (DSBs) independent of the target sequence. We named this technology PNA-assisted pAgo editing (PNP editing) and determined key parameters for designing PNP editors to efficiently generate programable site-specific DSBs. Our design allows the simultaneous use of multiple PNP editors to generate multiple site-specific DSBs, thereby informing design considerations for potential in vitro and in vivo applications, including genome editing.
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