Prokaryotic Argonaute Function and Application

Prokaryotic Argonautes (pAgos) are programmable endonucleases capable of recognizing and cleaving nucleic acid targets with no need for a PAM sequence, distinguishing them from CRISPR-Cas systems. This feature enables pAgos to target a wider range of sequences, making them promising tools in synthetic biology. In the Lohman lab, we are interested in utilizing DNA-guided pAgos for rapid, scarless, and sequence-independent DNA assembly. pAgos allow for creating overhangs of arbitrary sequences and lengths across multiple fragments in a single reaction. These fragments can then be joined via ligases in a predetermined order dictated by the overhangs. This methodology enables the construction of large DNA molecules, up to the size of full bacteriophage genomes, in a sequence-independent and scarless manner.