Cryo-EM structure of the transposon-associated TnpB enzyme

The class 2 type V CRISPR effector Cas12 is thought to have evolved from the IS200/ IS605 superfamily of transposon-associated TnpB proteins1. Recent studies have identified TnpB proteins as miniature RNA-guided DNA endonucleases2,3. TnpB associates with a single, long RNA (omega RNA) and cleaves double-stranded DNA targets complementary to the.RNA guide. However, the RNA-guided DNA cleavage mechanism of TnpB and its evolutionary relationship with Cas12 enzymes remain unknown. Here we report the cryo-electron microscopy (cryo-EM) structure of Deinococcus radiodurans ISDra2 TnpB in complex with its cognate.RNA and target DNA. In the structure, the.RNA adopts an unexpected architecture and forms a pseudoknot, which is conserved among all guide RNAs of Cas12 enzymes. Furthermore, the structure, along with our functional analysis, reveals how the compact TnpB recognizes the omega RNA and cleaves target DNA complementary to the guide. A structural comparison of TnpB with Cas12 enzymes suggests that CRISPR-Cas12 effectors acquired an ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, by either asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Collectively, our findings provide mechanistic insights into TnpB function and advance our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.

Automated summary

Researchers used cryo-electron microscopy to determine the structure of the complex formed by ISDra2 TnpB, its cognate.RNA, and target DNA. They found that the.RNA had an unexpected structure and formed a pseudoknot, which is a common feature among all guide RNAs of Cas12 enzymes. Furthermore, the structure revealed how the compact TnpB recognized the omega RNA and cleaved the target DNA complementary to the guide. Comparison with Cas12 enzymes suggested that CRISPR-Cas12 effectors acquired the ability to recognize the protospacer-adjacent motif-distal end of the guide RNA-target DNA heteroduplex, possibly through asymmetric dimer formation or diverse REC2 insertions, enabling engagement in CRISPR-Cas adaptive immunity. Overall, this study provides mechanistic insights into TnpB function and advances our understanding of the evolution from transposon-encoded TnpB proteins to CRISPR-Cas12 effectors.