Prokaryotic argonautes are a unique class of nucleic acid-guided endonucleases putatively involved in cellular defense against foreign genetic elements. While their eukaryotic homologs and Cas protein counterparts require single-stranded RNAs as guides， some prokaryotic argonautes are able to utilize short single-stranded DNAs as guides for sequence-specific endonuclease activity. Many complications currently prevent the use of prokaryotic argonautes for in vivo gene-editing applications; however， they do exhibit potential as a new class of in vitro molecular tools if certain challenges can be overcome， specifically the limitations on substrate accessibility which leads to unequal levels of activity acros... More
Prokaryotic argonautes are a unique class of nucleic acid-guided endonucleases putatively involved in cellular defense against foreign genetic elements. While their eukaryotic homologs and Cas protein counterparts require single-stranded RNAs as guides， some prokaryotic argonautes are able to utilize short single-stranded DNAs as guides for sequence-specific endonuclease activity. Many complications currently prevent the use of prokaryotic argonautes for in vivo gene-editing applications; however， they do exhibit potential as a new class of in vitro molecular tools if certain challenges can be overcome， specifically the limitations on substrate accessibility which leads to unequal levels of activity across a broad palate of substrates and the inability to act on double-stranded DNA substrates. Here we demonstrate the use of accessory factors， including thermostable single-stranded DNA binding proteins and UvrD-like helicase， in conjunction with prokaryotic argonautes to significantly improve enzymatic activity and enable functionality with a broader range of substrates， including linear double-stranded DNA substrates. We also demonstrate the use of Thermus thermophilus argonaute with accessory factors as a programmable restriction enzyme to generate long， unique single-stranded overhangs from linear double-stranded substrates compatible with downstream ligation.
