Transposition plays a key role in reshaping genomes of all living organisms1. Insertion sequences (ISs) of IS200/IS605 and IS607 families2 are among the simplest mobile genetic elements and contain only the genes required for their transposition and its regulation. These elements encode tnpA transposase that is essential for mobilization and often carry an accessory tnpB gene which is dispensable for transposition. Although the role of TnpA in IS200/IS605 transposon mobilization is well documented, the function of TnpB had remained largely unknown. It had been suggested that TnpB plays a role in transposition regulation though no mechanism for this was established3–5. Intriguingly, a bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR–Cas9/Cas12 nucleases6–8. However, no biochemical activities had been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by RE-derived RNA (reRNA) to cleave DNA next to the 5′ TTGAT transposon associated motif (TAM). We also show that TnpB could be reprogrammed to cleave DNA target sites in human cells. Together, this study expands our understanding of transposition mechanisms by highlighting the role of TnpB in transposition, experimentally confirms that TnpB is a functional progenitor of CRISPR-Cas nucleases and establishes TnpB as a prototype of a new system for genome editing.
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