Transposon-associated TnpB is a programmable RNA-guided DNA endonuclease

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.

Recruitment of Mobile Genetic Elements for Diverse Cellular Functions in Prokaryotes

“…Similarly to Cas9 and Cas12, the nucleases of IS200/IS605-like transposons form a complex with a distinct guide RNA encoded within the same transposon ( Altae-Tran et al, 2021 ). However, in these elements, the guide RNAs are not responsible for cleaving foreign DNA, but rather might direct the transposons to specific integration sites in the host chromosomal DNA; the details of the functions of these nucleases in transposons remain to be studied ( Altae-Tran et al, 2021 ; Karvelis et al, 2021 ). Collectively, these observations indicate that most if not all major components of CRISPR-Cas systems originated via exaptation of MGE genes.…”

Microbial defenses against mobile genetic elements and viruses: Who defends whom from what?

“…While successful functional innovations by co-option of these systems may be unlikely, the very frequent transfer of systems and their rapid evolution may result in such a high rate of novel combinations of domains that some will eventually evolve to become novel defense systems. Such processes of co-option may have been at the independent origins of both Cas-9 and Cas-12 proteins from transposon-encoded RNA-guided endonucleases [ 75 , 76 ].…”

Evolutionary plasticity and functional versatility of CRISPR systems

“…The role of IscB and TnpB in the transposon life cycle remains unknown. Recently, however, it has been shown that these proteins are RNA-guided nucleases that utilize a small RNA encoded by an adjacent gene (denoted ωRNA, after Obligate Mobile Element Guided Activity, OMEGA) to target specific sequences in a manner resembling the CRISPR mechanism [ 49 , 50 ]. All these proteins contain homologous RuvC-like nuclease domains, but, likely, specific evolutionary relationships can be traced.…”