Discovery of the Widespread Site-Specific Single-Stranded Nuclease Family Ssn

Site-specific endonucleases that exclusively cut single-stranded DNA have hitherto never been described and constitute a barrier to the development of ssDNA-based technologies. Here, we identify and characterize one such novel family of widely distributed site-specific single-stranded nucleases (Ssn) exhibiting unique ssDNA cleavage properties. This family is a member of the GIY-YIG nuclease superfamily that encompasses restriction enzymes, homing endonucleases, and transposases involved in important biological processes. Unlike other families of the GIY-YIG superfamily that typically possess additional functional domains that dictate their various specificities, Ssn are small proteins only consisting of the GIY-YIG domain. By first comprehensively studying the Ssn homolog from Neisseria meningitidis, we demonstrate that it interacts specifically with a sequence element (called NTS) present in hundreds of copies and surrounding important genes in pathogenic Neisseria. In this species, NTS/Ssn interactions modulate horizontal gene transfers through natural transformation and thus constitute a novel mechanism shaping genome dynamics. We further identified thousands of Ssn homologs throughout the bacterial kingdom and demonstrate, in vitro, a range of Ssn nuclease specificities for their corresponding NTS. We demonstrate proofs of concept for applications including ssDNA detection and digestion of ssDNA from Rolling Circle Amplification. This discovery and its applications set the stage for the development of innovative ssDNA-based molecular tools and technologies.