Abstract
Transposition has a key role in reshaping genomes of all living organisms1. Insertion sequences of IS200/IS605 and IS607 families2 are among the simplest mobile genetic elements and contain only the genes that are required for their transposition and its regulation. These elements encode tnpA transposase, which is essential for mobilization, and often carry an accessory tnpB gene, which is dispensable for transposition. Although the role of TnpA in transposon mobilization of IS200/IS605 is well documented, the function of TnpB has remained largely unknown. It had been suggested that TnpB has a role in the regulation of transposition, although no mechanism for this has been established3–5. A bioinformatic analysis indicated that TnpB might be a predecessor of the CRISPR–Cas9/Cas12 nucleases6–8. However, no biochemical activities have been ascribed to TnpB. Here we show that TnpB of Deinococcus radiodurans ISDra2 is an RNA-directed nuclease that is guided by an RNA, derived from the right-end element of a transposon, to cleave DNA next to the 5′-TTGAT transposon-associated motif. 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.
Highlights
The function of TnpA in transposition is well established, the role of TnpB remains elusive
The enrichment of non-coding RNAs associated with tnpB-encoding transposons from the IS200/IS605 family has been previously reported; their function remains elusive . 17,18 Here we provide experimental evidence that TnpB forms a ribonucleoprotein (RNP) complex with right end element RNAs (reRNAs) derived from the 3′ end of the transposon
We suggest that TnpB guided by reRNA makes a double-stranded break (DSB) at the 5′-TTGAT flanked donor joint site that is formed in DNA when the transposon circle is excised during replication
Summary
The function of TnpA in transposition is well established, the role of TnpB remains elusive. Guided by the similarities of TnpB to the CRISPR–Cas12f effector complexes that function as RNA-guided double-stranded DNA (dsDNA) nucleases[19], we hypothesized that the approximately 16-nt 3′ terminal of reRNA, which are derived from the DNA adjacent to the transposon and would be variable per se (Fig. 1d), might function as a guide sequence that directs TnpB to its target and activates DNA cleavage. To test this hypothesis, we adopted the previously developed protospacer adjacent motif (PAM) identification assay for Cas9/Cas[12] nucleases[19].
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